Citation
Analysis of a putative cleavage motif in rice receptor-like kinases

Material Information

Title:
Analysis of a putative cleavage motif in rice receptor-like kinases
Creator:
Cory, Gina Marie ( Dissertant )
Song, Wen-Yuan ( Thesis advisor )
Harmon, Alice ( Reviewer )
Rollins, Jeff ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
2006
Language:
English

Subjects

Subjects / Keywords:
Cells ( jstor )
Chromosomes ( jstor )
Disease resistance ( jstor )
Gene therapy ( jstor )
Genomes ( jstor )
Pathogens ( jstor )
Phosphorylation ( jstor )
Proteins ( jstor )
Receptors ( jstor )
Rice ( jstor )
Plant Pathology thesis, M.S
Dissertations, Academic -- UF -- Plant Pathology
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )
theses ( marcgt )

Notes

Abstract:
Plant receptor-like kinases (RLKs) are a large group of proteins found in many different species. XA21 is a rice RLK conferring resistance to bacterial blight disease. Previous findings demonstrate that phosphorylated residues in XA21s juxtamembrane domain assist in the stabilization of XA21. These residues exist within a motif identical to the proteolytic cleavage motif, P/GX₅-₇P/G, originally identified in the animal EGFR protein family. Here, it is shown that approximately 47 percent of 800 rice kinases contain this motif within their juxtamembrane domains. Additionally, a second putative cleavage motif in XA21, P₉₂₂TDSTFRP, was identified within its intracellular kinase domain. Similar to previous findings, mutation of the phosphorylated residues within this second putative motif leads to degradation of XA21.
General Note:
Title from title page of source document.
General Note:
Document formatted into pages; contains 105 pages.
General Note:
Includes vita.
Thesis:
Thesis (M.S.)--University of Florida, 2006.
Bibliography:
Includes bibliographical references.
General Note:
Text (Electronic thesis) in PDF format.

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Source Institution:
University of Florida
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University of Florida
Rights Management:
Copyright Cory, Gina Marie. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
7/24/2006
Resource Identifier:
003589313 ( aleph )
496181280 ( OCLC )

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Full Text











ANALYSIS OF A PUTATIVE CLEAVAGE MOTIF IN RICE RECEPTOR-LIKE
KINASES














By

GINA MARIE CORY


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006
































Copyright 2006

By

GINA MARIE CORY






















This thesis is dedicated to all those who have left footprints in my life.












ACKNOWLEDGMENTS

I must first thank my high school AP Biology and Independent Science teacher,

Anthony Arico. He introduced me to the joys of science and is a true diamond in the

rough.

I also thank Dr. William Zettler, who reeled me as he did many others into the field

of plant pathology with his enthusiasm and his ability to make a student feel like a

student and not a social security number. Also at the Plant Pathology Department of UF,

I thank Dr. Gail Wisler and all the faculty and staff who guided me along the way.

I thank the members of my advisory committee, Dr. Alice Harmon and Dr. Jeff

Rollins, for their assistance and patience. I give special thanks to my advisory chair, Dr.

Wen-Yuan Song, for giving me a chance as an undergraduate and then as a graduate

student. He never stopped raising the bar of accomplishment.

Next, I must thank all the members of the Song lab, past and present: Dr. Guozhen

Liu, Dr. Pranjib Chakrabarty, Anita Snyder-Pineda, Dr. Porntip Tinjuanjun, Dr. Weihui

Xu, Dr. Yongsheng Wang, Dr. Xiuhua Chen, Dr. Yan Zhang, Dr. Xiadong Ding,

Yingnan Jiang, and especially Lisa Nodzon and Terry Davoli. I also thank Chris Dardick

of the University of California at Davis. All of these people graciously provided support

and necessary laughter.

I thank all of my family, in particular my parents, whose generosity and caring

never cease.

Finally, I thank my husband, Dr. Juan Jose Suarez. I am so happy to enter the real

world with him at my side.




















TABLE OF CONTENTS


IM Le

ACKNOWLEDGMENT S .............. .................... iv

LI ST OF T ABLE S ................. ................. vii........ ....


LIST OF FIGURES ................. ..............viii...............


AB S TRAC T ................. ...............x........ .....


CHAPTERS

1 LITERATURE REVIEW .............. ...............1.....


Receptor Kinases ..........._...__........ ...............1.......
Plant RLKs ......... ...............2.......
RLK Structure ..... ... ........... ........ ...............2
Comparisons of Animal Receptors and Plant RLKs ............ .. ........._ ....4
RLKs in Growth and Development............... ...............
RLKs in Disease Resistance .............. .........___ ...............9...
Phosphorylation and Protein Stability in XA21 ......___ ............. ..............15
Receptor Shedding .............. ...............17....

2 SEQUENCE ANALYSIS OF RICE RECEPTOR-LIKE KINASES ................... .........21

Introduction ........... ...............21......
Materials and Methods ................... .. ......._. .......... ..... .................2
Identifying Putative Proteolytic Cleavage Motifs in the JM Domain of Rice
RLK s ................. ........ .............2
Average Length of JM Domains of Rice RLKs .............. ...... .._.._..........23
Creating Rice Chromosome Maps Depicting the Locations of Rice RLK
Genes............... ...............23.
Results and Discussion................. ......... ...... ... ..... .. .........2
A Significant Number of Rice RLK's Contain the Putative Cleavage Motif.24
A Significant Number of Select Plant Proteins Contain the Putative Cleavage
M otif. ................._ ............. ...............24.......
Average Length of JM Domains of Rice RLKs ........._.. .... ........._...26
Rice Chromosome Maps Depicting the Locations of Rice RLKs ........._.......26

3 MOLECULAR CHARACTERIZATION OF THE XA21T923A/S925/T926A MUTANT ...72














Introducti on ............ ............72......... ......
Materials and Methods ................. ...............72........... ....
Mutant Rice Plants ............... ............72.. .......... ....
XA2 1 Detecti on ............... ............73.. ..............
X A2 1 Tran script Level s............... ...............74
Results and Discussion............... ...............7


4 DIRECT MUTATION OF THE PUTATIVE CLEAVAGE MOTIF IN THE
JUXTAMEMBRANE DOMAIN OF XA21 ................ .............. ......... .....78


Introduction ........... ...............78......
Materials and Methods ................. ...............78........... ....
Results and Discussion............... ...............8


5 CLONING OF SELECTED ARABIDOPSIS RLKS .................. ................8


Introduction ........... ...............82......
M materials and M ethods............._ ....... ........._._.............._._.......... .. .......8

Amplification of the At~g0 1890, Atlg48480, At2g36570 and At3g28040
ORFs ................... ...............83..
Molecular Cloning of the PCR Products ............ ..... ._ ................84
Results and Discussion............... ...............8


LIST OF REFERENCE S ............. ...... ._ ...............87..


BIOGRAPHICAL SKETCH .............. ...............95....

















LIST OF TABLES

Table pg

2-1. Distribution of the P/GX5-7P/G motif in the JM domains of plant RLKs............... .25

2-2. The P/GX5.,P/G motif in the JM domains of predicted rice RLKs............._.._.. ........27

4-1. Primers used in site-directed mutagenesis............... ..............7

5-1. Sequence alignment of XA21 and selected Arabidopsis proteins of the DE group..83


















LIST OF FIGURES


figure pg

1-1. Generalized structure of a receptor-like kinase protein with major domains labeled.4

1-2. Proposed model of evolution of the receptor kinase family ................. ................. .6

1-3. Model of XA21 mediated resistance through binding proteins ................ .. .............10

1-4. Components of signaling pathways of innate immunity in Drosophila, mammals,
and Arabidopsis ................. ...............13.__._.......

1-5. A c-Myc-tagged version of XA21 ............... ............... ....___..16

1-6. Intracellular juxtamembrane domain of XA21 ................ ................ ......... .17

1-7. A conserved motif in the juxtamembrane domain of the EGFR family....................20

2-3. Rice RLKs selected for study mapped on the twelve rice chromosomes. ........._......61

2-4. Motif-positive rice RLKs mapped on the twelve rice chromosomes. .......................62

2-5. Motif-like rice RLKs mapped on the twelve rice chromosomes. .........._... .............63

2-6. Motif-negative rice RLKs mapped on the twelve rice chromosomes. ......................64

2-7. Rice RLKs in subfamily CrRLKIL-1 selected for study mapped on the twelve rice
chromosomes. ........._.._.. ...._... ...............65....

2-8. Rice RLKs in subfamily DUF261c selected for study mapped on the twelve rice
chromosomes. ................. ...............66....... ......

2-9. Rice RLKs in subfamily L-LEC selected for study mapped on the twelve rice
chromosomes. ........._.._.. ...._... ...............67....

2-10. Rice RLKs in subfamily SD-2a selected for study mapped on the twelve rice
chromosomes. ........._.._.. ...._... ...............68....

2-11. Rice RLKs in subfamily WAK selected for study mapped on the twelve rice
chromosomes. ........._.__...... ._ __ ...............69....











2-12. Rice RLKs in subfamily LRR-la selected for study mapped on the twelve rice
chromosomes .........____...... .____ ...............70.....

2-13. Rice RLKs in subfamily SD-2b selected for study mapped on the twelve rice
chromosomes ........... ..... ._.._ ...............71.....

3-1. Analyses of XA2 1T923A/S925A/T926A mutant .................... ............... 7

3-2. Schematic representation of XA21 indicating putative cleavage motifs (A and B)..77

3-3. Amino acid sequence of the intracellular domain of XA21 ........._._. ..........._......77

4-1. The intracellular domain of XA21 .........____.....__ .......___ .........8

5-1. Sequence alignment of XA21 and selected Arabidopsis proteins of the DE group..83

5-2. Identification of recombinants carrying the ORFs of At~g0 1890, Atlg48480,
At2g36570 and At3 g28040 .............. ...............86....
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ANALYSIS OF A PUTATIVE CLEAVAGE MOTIF IN RICE RECEPTOR-LIKE
KINASES

By

Gina Marie Cory

May 2006

Chair: Wen-Yuan Song
Major Department: Plant Pathology

Plant receptor-like kinases (RLKs) are a large group of proteins found in many

different species. XA21 is a rice RLK conferring resistance to bacterial blight disease.

Previous findings demonstrate that phosphorylated residues in XA21s juxtamembrane

domain assist in the stabilization of XA21. These residues exist within a motif identical

to the proteolytic cleavage motif, P/GX5.,P/G, originally identified in the animal EGFR

protein family. Here, it is shown that approximately 47% of 800 rice kinases contain this

motif within their juxtamembrane domains. Additionally, a second putative cleavage

motif in XA21, P922TDSTFRP, was identified within its intracellular kinase domain.

Similar to previous findings, mutation of the phosphorylated residues within this second

putative motif leads to degradation of XA21.


















CHAPTER 1
LITERATURE REVIEW

Receptor Kinases

Perception of signal at the surface in animal cell is known to be regulated by

receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSKs) (Hubbard

and Till, 2000). Many receptor kinases have a signal-receiving extracellular domain, a

transmembrane region, and an intracellular kinase domain. Based on their greatly

different extracellular domains, the animal receptor kinases can be placed into 20

subgroups (Robinson et al., 2000). Recognition of ligands, mostly soluble though some

are membrane-associated, often leads to dimerization of the receptors and results in

phosphorylation of either tyrosine or serine and threonine residues of the kinase (Hubbard

and Till, 2000). While one function of this phosphorylation is activation of the kinase,

the other is to provide a binding surface for interacting proteins (Burgess et al., 2003; ten

Dijke and Hill, 2004; Johnson and Ingram, 2005).

Receptor kinases function in numerous important processes in the cell, such as

embryonic development, metabolism, and immune system function (Hubbard and Till,

2000). For instance, effects of insulin on the body are regulated by the insulin receptor,

an RTK (White, 1998). Once insulin is bound to the receptor, phosphorylation of the

receptor and recruitment of downstream proteins occurs. Those recruited downstream

signaling proteins are the IRS adaptor proteins, which are phosphorylated on several










tyrosine residues by the receptor. IRS proteins then bind and activate PI-3k, which is

involved in the movement of glucose transporters to the cell membrane (Cheatham et al.,

1994; Okada et al., 1994).

Nonreceptor tyrosine kinases (NRTKs), lacking extracellular and transmembrane

domains, are components within the signal transduction pathways triggered by receptor

kinases (Hubbard and Till, 2000). Most NRTKs are located in the cytoplasm, though

some are actually bound to the membrane. The largest subfamily of the NRTKs is the

Src family, whose mutation can cause human cancers (Biscardi et al., 1999).

Plant RL~Ks

Plants contain a large number of predicted proteins called RLKS (receptor-like

kinases) with structures similar to animal receptor kinases. Over 600 and 1000 RLKs

have been reported in the model plant Arabidopsis thaliana and rice (Oryza sativa),

respectively (Shiu and Bleecker, 2001a; Shiu et al., 2004). Much knowledge about the

exact function of these kinases is lacking, with relatively few being described (Shiu and

Bleecker, 2001a). It is clear however that RLKs are involved in a variety of processes,

including disease resistance, self-incompatibility, hormone perception and development.

RL~K Structure

Like animal receptor kinases, many plant RLKs are composed of an extracellular

domain, a transmembrane domain, and an intracellular kinase that contains twelve

subdomains with fifteen invariant residues (Figure 1-1) (Walker, 1994; Hanks et al.,

1988). Some of the invariant residues, such as the lysine residue corresponding to

Lys736 of XA21, are important for catalysis. The extracellular domain is thought to

perceive signals (also known as ligands) through binding. The transmembrane domain

anchors the protein on the cell's membrane, while the kinase domain juts into the










cytoplasm of the cell. It is important to note the juxtamembrane (JM) domain, which is

located just after the transmembrane domain. A corresponding extracellular region is

termed the extracellular JM domain. Phosphorylation of residues in the JM domain often

acts as binding sites for other signaling proteins in animal systems. In plants, such

phosphorylation has also been observed in XA21 in vitro and BRI1 in vivo (discussed in

more detail below).

Plant RLKs can be classified into 45 subgroups based on their extracellular

structures (Shiu and Bleecker, 2001a). Some major subgroups are described below. The

leucine-rich repeat (LRR) subgroup contains proteins with a core tandem sequence of

Leu-x-x-Leu-x-Leu-x-x-Asn-x-Leu. LRRs have been implicated in protein-protein

interactions. The S-domain RLKs, the first class ofRLKs to be described in plants, share

similarities with the S-locus glycoproteins involved in incompatibility of Brassica. One

distinct characteristic of the S-domain RLKs thought to be important for folding of the

extracellular domain is a group of ten cysteine residues found next to the transmembrane

region. Another subgroup is the lectin receptor kinases, which was defined by the

Arabidopsis kinase Ath.lecRK1 which has an extracellular domain homologous to the

legume lectin family (Herve et al., 1996). Lectin receptor kinases may interact with

oligosaccharides or cell wall fragments (Buchanan et al., 2000). Epidermal growth

factor-like RLKs share repeats in the extracellular domain with their animal protein

namesake, and may perceive signals at the cell wall-plasma membrane interface (Braun

and Walker, 1996; Buchanan et al., 2000). There are several other groups of RLKs

including thaumatins and WAKs (summarized in Shiu and Bleecker, 2001b).









In a phylogenetic study of RLKs, a group of RLKs lacking extracellular and

transmembrane domains existed within the same monophyletic group as other RLKs

(Shiu and Bleecker, 2001a). These were so termed the receptor-like cytoplasmic kinases

(RLCKs). PBS 1 is a representative of this class (Swiderski and Innes, 2001). This

Arabidopsis serine/threonine kinase, along with another protein RPS5, recognizes the

avirulence gene (avrPphB) product from Pseudomona~s syringe. More recently, PBS1

was shown to be cleaved by a bacterial type III effector molecule, and that this cleavage

was necessary for the induction of defense against the pathogen (Shao et al., 2003). It is

thought the PBS1 cleavage product binds with RPS5 and subsequently activates

resistance pathways. Animal NRTKs and plant RLCKs can be considered corresponding

soluble forms of their respective groups, as some members of both have been shown to

form complexes with receptors and help in the transduction of signals downstream.



Signal Extracellular





N-terrninuse C-terrninus



Tra nsmembra ne


Figure 1-1. Generalized structure of a receptor-like kinase protein with major domains
labeled.

Comparisons of Animal Receptor Kinases and Plant RL~Ks

Despite similarities between RLKs and animal receptors, maj or differences can be

seen (Johnson and Ingram, 2005). It could be possible that plant and animal receptors









evolved independently, and their similar structure organization being due to their inherent

suitability to the processes they regulate. To date, most plant RLKs appear to be

serine/threonine kinases, while animal receptors, except the mammalian TGF-P receptors,

are tyrosine kinases. And while both RLKs and animal receptors show a great variety of

extracellular domains, the sequence and structure of the extracellular domains vary

greatly between them. Thirdly, increasing evidence shows that downstream interacting

proteins of plant RLKs differ from those of animal receptor kinases.

Shiu and Bleecker (2001a) phylogenetically analyzed Arabidopsis RLKs in relation

to animal receptor kinases using the conserved kinase domains. This analysis included

more than 900 candidate RLKs and related kinases from plants. Among the animal

receptors, which included representatives from 16 subfamilies, data supported a strong

monophyletic origin for the RTKs. Raf kinases, a family of kinases which can elicit or

arrest the cell cycle in mouse fibroblasts (Woods et al., 1997), were more closely related

to the animal RTKs; the plant RLKs formed a separate monophyletic group within the

eukaryotic protein kinase superfamily (previously, RLKs were placed into a separate

family based on their serine/threonine specificity). This indicates that RLKs have a

distinct origin from Raf kinases and RTKs. In contrast, plant RLKs were found to be

related to the Drosophila and Caenorhabditis Pelle kinases, and to three human IRAKs.

Based on these results, a separate group was defined as the RLK/Pelle kinases gene

family.

Shiu and Bleecker then proposed a model for the evolution of receptor kinases

(Figure 1-2) chronologically beginning with (1) divergence of RLK/Pelle from RTK/Raf.

Next (2) the divergence of RTK from Raf occurs followed by (3) the divergence of plant









and animal lineages. The split of Raf and RTK from RLK/Pelle is attributed to an early

gene duplication event which probably occurred before the divergence of plants and

animals. The fact that only five RLK homologs were found in animals supports this

notion.








Raf Pleat




RLK



Figure 1-2. Proposed model of evolution of the receptor kinase family. (1) Divergence
of RLK/Pelle from RTK/Raf. (2) Divergence of RTK from Raf. (3)
Divergence of plant and animals. Reprinted with permission. Shiu, SH and
Bleecker A (2001a). Receptor-like kinases from Arabidopsis form a
monophyletic gene family related to animal receptor kinases. PNAS.
98:10763-10768.

RL~Ks in Growth and Development

A number of RLKs have been implicated in plant growth and development.

Several examples are briefly highlighted here.

CLAVATAl (CLV1) is an Arabidopsis RLK with twenty-one LRRs in its

extracellular domain (Clark et al., 1997). CLV1 mutants show a surplus of

undifferentiated cells (Clark et al., 1993). Cell proliferation and differentiation is

regulated within the shoot apical meristem of plants. Consequently, CLV1 mutant plants

have enlarged floral meristems, as well as inflorescence and vegetative meristems. Also,

mutant plants show an increase in the number of floral organs and additional whorls in









their flowers. In the CLV1 pathway, additional components, CLV2 and CLV3, have

been identified (Jeong et al. 1999; Fletcher et al., 1999). CLV2 is a receptor-like protein

with LRRs, whereas CLV3 appears to be a small protein that is hypothesized to function

as a ligand for the CLV1 receptor.

HAESA, previously known as RLK5, is an LRR-RLK from Arabidopsis (Horne

and Walker, 1994; Jinn et al., 2000). Antisense strategy produced HAESA mutants

which exhibit delayed floral organ abscission. Jinn et al. (2000) also demonstrate that the

intensity of this phenotype is directly related to the level of HAESA within a particular

plant; that is, the less amount of HAESA, the greater delay in floral organ abscission.

Therefore, HAESA may be involved in floral organ abscission in Arabidopsis.

The gene Crinkly4 (cr4) encodes an RLK whose extracellular domain has a

cysteine-rich region quite similar to mammalian tumor necrosis factor receptors' ligand

binding domains (Becraft et al. 1996). Maize mutants of this gene show affected leaf

epidermis, which in turn compromises those cells' functioning. It has also been

implicated in the differentiation of internal tissues as well as the epidermal tissues (Jin et

al. 2000). It is suggested in the same study that cr4 is also involved in regulatory

pathways of cell proliferation, fate, and pattern. The Arabidopsis CRINKLY4 is a

involved in the development of sepals and ovuoles (Gifford et al. 2003).

The SRK (S Receptor Kinase) gene is located at the S locus of Bra~ssica oleracea

(Stein et al., 1991). This locus controls self-recognition of pollen and stigma in Bra~ssica

oleracea. Pollen germination is halted if both pollen and stigma come from plants having

identical S-locus genotypes, preventing self-fertilization. SRK is capable of in vitro

autophosphorylation (Stein and Nasrallah, 1993). Confirmation of SRKs involvement in









self-incompatibility was shown through studying self-compatible plants. These plants

were determined to have mutated SRK genes (Goring et al., 1993). Later, an SRK-related

protein SFR2, an S-locus family member, was shown to be rapidly induced by wounding

and bacterial infection ofXanthomona~s campestris py. campestris (Xcc) (Pastuglia et al.,

1997). SFR2 mRNA accumulates after bacterial infection by Xcc and Escherichia coli.

This evidence points towards a role in plant defense.

BRI1 is an Arabidopsis LRR-RLK and is involved in the perception of

brassinosteroid, a growth-promoting hormone (Li and Chory, 1997). DwarfArabidopsis

mutants, unresponsive to exogenous brassinosteroid treatments, mapped to the same

region as a previously-described brassinosteroid mutant (Clouse et al., 1996). BRI1 is

autophosphorylated, which has uniquely been shown in vivo, in response to

brassinosteroid; six phosphorylation sites of BRI1 are located in the JM domain (Wang et

al., 2001; Wang et al., 2005). Also, BRI1 is one of few RLKs with a known ligand

(Kinoshita et al., 2005). Recently it has been shown that BRI1 exists as a homodimer

even in the absence of applied steroids; this state of the protein is thought to autoregulate

the activity of the protein (Wang et al., 2005). The proposed model of BRI1 activation

begins with this homodimer going through a conformational change upon ligand binding.

The subsequent transphophorylation of the kinase domain separates the homodimer,

leading to activation of BRI1 through further phosphorylation. Afterwards, BRI1 likely

then forms a complex with BAK1 (BRI1-associated receptor kinase 1) and transduces the

brassinolide signal (Nam and Li, 2002; Li et al., 2002). The tomato BRI1, also known as

SR160, was shown to bind systemin in transformed tobacco (Montoya et al., 2002;

Scheer and Ryan, 2002). Systemin is a molecule which triggers defense responses










against insects after wounding. Additionally, a mutant tomato line with non-functional

BRI1 showed reduced production of protease inhibitor in response to systemin.

RL~Ks in Disease Resistance

Plant RLKs also function in disease resistance. Flor (1971) first described the

gene-for-gene theory of disease resistance. This states that two corresponding genes are

required for the onset of disease resistance responses in the plant: one avrirulence gene

(avr gene) from the pathogen and one disease resistance gene (R gene) from the plant. If

either of those genes is absent in a particular host-pathogen interaction, disease resistance

does not occur. Instead, disease of the plant begins. R gene products, a number of which

are RLKs, recognize directly or indirectly Avr gene products (Jones and Dangl, 2001).

Reviewed below are RLKs to date implicated in pathogen or insect resistance.

The rice gene Xa21 confers resistance to the bacterial pathogen Xanthomona~s

oryzae py. oryzae (Xoo) and is a member of a small gene family (Ronald, 1992; Song et

al., 1995; Song et al. 1997). The protein XA21 is a serine/threonine transmembrane

kinase with an extracellular domain of twenty-three LRRs (Figure 1-3). The cloning and

characterization of this gene was a milestone in the understanding plant disease resistance

genes (R genes) because at the time of its discovery, it represented a new class ofR genes

as well as a possible evolution between previously described classes of R genes. The

LRRs of the extracellular domain of XA21 is similar to tomato' s CF-9, while its kinase

domain is reminiscent of another serine/threonine tomato kinase, PTO (Song et al., 1995).

XA21 is thought to bind a polypeptide signal from Xoo, activating the kinase

through autophosphorylation (Ronald, 1997). It is hypothesized that XA21 forms a

homodimer which activates the intramolecular phosphorylation upon pathogen infection,

after which it binds with several other proteins (Figure 1-3) (Liu et al., 2002). The










resulting protein interactions are believed to be the beginning of possibly several defense

responses.







mocipathogen ~ 0
produced

















Resistance Response

Figure 1-3. Model of XA21 mediated resistance through binding proteins (courtesy of
Dr. Wen-Yuan Song, University of Florida). Upon ligand binding, it is
believed XA21 forms a homodimer. Subsequent phosphorylation recruits
binding proteins (blue spheres, purple squares, and green ovals). This then
may activate several pathways leading to resistance responses. P:
autophosphorylated residues.



XA21 binds with several proteins in the yeast-two hybrid system (Wang et al.,

unpublished). One XA21 binding protein, XB3, has been studied in depth to date. XB3,

whose interaction with XA21 has been confirmed through co-immunoprecipitati on

assays, contains a RING finger domain capable of autoubiquitination. Downregulation of

XB3 in vivo yields greater susceptibility to Xoo as well as lower steady states of XA21.









ERECTA is an LRR-RLK which was originally implicated in developmental

control of aerial organs (Torii et al., 1996). Plants carrying a mutated ERECTA gene

show compact flowers, shorter internodes, and shorter floral organs. More recently,

ERECTA has been implicated in resistance against bacterial wilt disease caused by

Ralstonia solan2acearum (Godiard et al., 2003). Arabidopsis thaliana Landsberg, an

accession susceptible to bacterial wilt, transformed with ERECTA exhibited an increased

resistance to R. solan2acaearum. ERECTA, more specifically its LRR and kinase

domains, is also implicated the defense against the fungal pathogen Plectosphaerella

cucumerina (Llorente et al., 2005).

The Xa26(t) gene confers resistance to Chinese Xoo strain JL691 (Yang et al.,

2003). This gene was cloned by using a map-based strategy. Xa26(t) encodes an LRR-

RLK and shares the most homology with XA21 out of reported LRR-RLKs (Sun et al.,

2004). Their different resistance spectra are suggested to be attributed to their different

structure and variations in the LRR region. Interestingly, Xa26 confers resistance to Xoo

from seedling to adult stages, which differs from the Xa21-controlled resistance that is

only present in adult plants (Xu et al., 2006).

Resistance to Puccinia gramninis f. sp. tritici, causing stem rust of barley, is

conferred by RPG1 (Brueggeman et al., 2002). The Rpgl gene was cloned using a map-

based cloning strategy and subsequent characterization identified it as an RLK. RPG1

has two predicted tandem intracellular kinase domains, representing a novel structure of

disease resistance genes. One of these tandem kinase domains does not contain all of the

highly conserved residues seen in most kinases, suggesting it may not be functional. The

membrane-targeting sequence of RPG1 is not clear and a ligand binding sequence is not









identifiable. Additionally, Thre204 ofPTO, which has been implicated in the interaction

of PTO and AvrPto, is conserved in both predicted kinase domains of RPG1 (Frederick et

al., 1998).

FLS2 is an LRR-RLK which is involved in the perception of bacterial flagellin.

Flagellin is a surface protein on numerous bacterial propellants, also known as flagella

(Moens and Vanderleyden, 1996), has been implicated in the pathogenicity of bacteria

(Tans-Kersten et al. 2001). The FLS2 gene was identified by screening flagellin-

insensitive Arabidopsis thaliana ecotype Ws-0 plants (G6mez-G6mez and Boller, 2000).

The Ws-0 plants did not respond to the inj section of fig22, a synthetic peptide mimicking

the conserved N-terminus of flagellin. The ability of the plant to perceive flagellin was

further illuminated by Zipfel et al. (2004). In their study, fis2 mutant plants not only

showed susceptibility to the infiltrated bacterial pathogen Pseudoomonas syringae py.

tomato, but showed a greater susceptibility when the bacteria was just sprayed on the

leaves, which bypasses natural infection supported by flagella functioning. Flagellin

perception then could possibly inhibit bacterial infiltration of the plant.

FLS2 signaling is often compared to innate immunity of animals and insects

(Figure 1-4) (Gomez-Gomez and Boller, 2002). In animal and insect innate immunity

systems, the perception of a pathogen leads to rapid responses, describing a first line of

disease defense. In Drosophila, the Toll receptor, an LRR transmembrane protein with

an intracellular domain of high similarity to the interleukin-1 receptor, mediates innate

immune responses (Hoffmann and Reichhart, 2002); in animals, innate immunity is

mediated by the Toll-like Receptors (TLRs), of which TLR5 recognizes flagellin.



















Plasma membrane


Drosophila



TOLL TLR5


Tu My8 P LS2 kinase
dMyD88 Cytop


TRAF2 TRAF6
AtMEKK1

DmlKK ~ IKK L' AtMKK4/5
kinases kinasesAtK3

C Degraded
Degraded -B~ IrB
Cactus-



~mPL~~a~J ~~ 'jNu(
Gene expression



Development: Innate Development?
dorsoventral pattern immunity
TRENJDS in Phant


lasm


cleus


Science


Figure 1-4. Components of signaling pathways of innate immunity in Drosophila,
mammals, and Arabidopsis. Reprinted with permission. Gomez-Gomez, L
and T Boller (2002). Flagellin perception:a paradigm for innate immunity.
7RENDS in Planzt Science. 7(6):251-256.

The perception of pathogens in innate immunity is dependent upon the recognition

of pathogen-associated molecular patterns (PAMPs). PAMPs are highly conserved,

general elicitor molecules including chitin, lipopolysaccharide, glycoproteins, as well as

flagellin (Lamaitre et al., 1997; McDermott et al., 2000). After pathogen perception in

Toll and Toll-like systems, receptor dimers then form and signaling proceeds through

adapter molecules (dMyD88 and Tube in Drosophila, and MyD88 in animals) (Gomez-


Mammals A



FLS2


,rabidopsis



Apoplast









Gomez and Boller, 2002). This leads to the activation of downstream kinases Pelle in

Drosophila and IRAK in animals. Eventually, the pathway results in the degradation of

Cactus in Drosophila and IKB (inhibitor of nuclear factor xB (NF-KB)) in animals, which

in turn leads to the release of Dif (dorsal-related immunity factor) in Drosophila and NF-

xB in animals. The released molecules are transported to the nucleus, where gene

expression results. The mitogen- activated-p rotei n ki na se (MAPK) p athway i s activated

in animals also.

There is no direct evidence in plants for a pathway like Toll and Toll-like systems,

but many parallels can be seen. It is known that the MAPK cascade is activated in plants

in response to elicitors as well as gene-for-gene interactions (Ligterink and Hert, 2001;

Romeis, 2001). This supports the notion of a common system for the recognition of and

response to elicitors. Additionally, Toll from Drosophila, now implicated in innate

immunity, was originally implicated in dorsoventral development. This is much like the

duality of plant RLKs ERECTA, BRI1, and SRK described earlier.

A proposed model for FLS2 signaling in Arabidopsis begins with the recognition of

flagellin, or degraded forms of flagellin (like the fig22 synthetic peptide), by the

extracellular domain of FLS2. The activation of the FLS2 kinase possibly occurs via

dimerization and autophosphorylation. Subsequently, an unknown pathway proceeds to

the activation of ion channels and the NADPH oxidase complex, while simultaneously

the activation of AtMEKKl1, which activates AtMAKK4/5, which then activates

MAPK3/6. This activation may in turn activate the WRKY type transcription factors

involved in expression of defense genes.









Molecular cloning of plant disease resistance genes and thorough understanding of

the molecular mechanisms of plant defense can have global impacts. Historically, plant

pathogens have greatly affected humans, especially with reference to our food sources.

The late blight of potato caused by the oomycete fungal pathogen Phytophthora infestans

led to the Irish potato famine of 1845 (Ristaino et al., 2001). Identification of molecular

components involved in plant disease defense would allows us to manipulate them and

create transgenic plants with broad spectrum and durable resistance, thereby reducing the

usage of pesticides, fungicides, and the like.

Phosphorylation and Protein Stability in XA21

Protein stability has emerged as a common regulatory step of plant R gene

products. As known in Arabidopsis, the disease resistance protein RPM1 (recognizing

Psuedoomonas syringae py. tomato carrying avrRPM1 or avrB) requires the presence of

RIN4 and HSP90 to accumulate (Mackey et al, 2002; Hubert et al., 2003). RPM1

interacts with both RIN4 and HSP90. Additionally, RAR1 regulates the accumulation of

MLAl and MLA6, which provides resistance to powdery mildew, although their direct

interactions with RAR1 have not yet been shown (Bieri et al., 2004).

In addition to the proteins that directly or indirectly interact with resistance gene

products, protein phosphorylation has also been implicated in the stabilization of plant

resistance proteins (Xu et al., 2006). An XA21 dead kinase mutant, XA21K736E, iS

unstable in rice extracts. In XA21K736E, glutamic acid was substituted for Lys736, which

corresponds to a highly conserved lysine required for the kinase activity XA21.

Additionally, the simultaneous mutation of the phosphorylated Ser686, Thr688, and

Ser689 to alanine implicates these residues in the stability of XA21. Both

XA21S686A/T688A/S689A and XA21K736E transgenic plants exhibit lower steady-state protein









levels than plants containing wild-type XA21. Because wild-type XA21 is stable for at

least six hours in plant extract, this suggests phosphorylation is a key factor in the

stability of this protein.

Western blot analysis illustrates that the 140 kD XA21 protein can be cleaved to a

100 kD product in the microsomal fraction. Because XA21 is tagged at its N-terminus

with c-Myc for convenience of antibody detection and the calculated weight of the

extracellular domain of XA21 is approximately 100 kD, this detected 100 kD cleavage

product should represent the extracellular domain and transmembrane domains.

Subsequently, it is hypothesized that the missing 40 kD corresponds with the intracellular

portion of the kinase, which was calculated at approximately 36.6 kD (Figure 1-5).

Western blot analysis showed a significantly increases accumulation of the 100 kD

cleavage product for both XA21 S686A/T688A/S689A and XA21K736E, further supporting that

autophosphorylation of these three residues plays a role in the stabilization of XA21.

40 kD
140 kD






N-terminus C-terminus




Transmemb~rane



Figure 1-5. A c-Myc tagged version of XA21. A blue triangle marks the N-terminal tag
c-Myc used for immunodetection of XA21. At the top of the figure, bars
indicate the portion of the protein of the corresponding kD weight: the full-









length protein is predicted to weigh 140 kD, and the intracellular portion is 40
kD.

Further investigation illustrates that XA21 stability is developmentally controlled,

with younger plants containing higher steady states of the protein. This suggests that an

unknown proteolytic activity increases with the age of the plants. Finally, inoculation

with Xoo demonstrated that XA21 S686A/T688A/S689A and XA21K736E mutations lead to

decreased disease resistance in rice, indicating that accumulation of XA21 is required for

resistance against Xoo.

Ser686, Thr688, and Ser689 are located in the JM domain of XA21, more

specifically within a region identical to a cleavage motif identified in the epidermal

growth factor receptor (EGFR) family of animal receptor kinases (Figure 1-6).


XA21 Juxtamembrane Domain

677HKRTKKGAPS RT SMKGHPLVSYSQZLVKATDG





|Cleavage motif found in the EGFR family
P/GX,,P/G



Figure 1-6. Intracellular juxtamembrane domain of XA21. The putative cleavage motif
is underlined, and the autophosphorylated residues are shown in red.

Receptor Shedding

Many receptors in animal systemscan be cleaved in the extracellular JM or

transmembrane domain. This phenomenon is known as receptor shedding or ectodomain

shedding (Hooper et al, 1997; Dello Sbarba and Rovida, 2002). Shedding of the









ectodomain is often regulated by a protease, also known as a sheddase, acting upon a

regulatory protein(s) at the surface (Dello Sbarba and Rovida, 2002). These proteases are

usually metalloproteinases belonging to the groups known as MMPs (matrix

metalloproteinases) or ADAMs (a disintegrin and metalloproteinase domain). One

example is the regulation of Met, a hepatocyte growth factor receptor, which is involved

in cell growth amongst other processes (Nath et al., 2001). Shedding of Met by a

metalloproteinase is activated through EGF (epidermal growth factor) and LPA

(lysophosphatidic acid) binding to their receptors.

Possible biological purposes of receptor shedding include cessation of signaling

after the initial signal transduction resulting from a ligand-receptor interaction (Dello

Sbarba and Rovida, 2002). Overexpression of receptors has been shown to be deleterious

to the organism. Another significance of receptor shedding is that the resulting

transmembrane-cytoplasmic domains of the protein may act differently in signaling than

their full-length precursors, and are not controlled by ligand-binding (Dello Sbarba and

Rovida, 2002).

Much is still unknown about the regulation of receptor shedding. In the case of

Her-2 however, a particular cleavage site was identified and implicated in its receptor

shedding (Yuan et al. 2003).

Her-2 is a receptor tyrosine kinase of great sequence homology to the EGFR family of

proteins (Coussens et al., 1985). The cleavage product of Her-2 has been suggested to

be involved in the aggressiveness, metastasis, and morbidity of breast tumors and it is

suggested that the ectodomain released during shedding of Her-2 may act as a decoy for









treatments (Christianson et al., 1998). Recent advances have led to the use of EGFR

inhibitors as treatments (Shelton et al. 2005).

Yuan et al. (2003) identified a cleavage site in the extracellular JM domain of Her-

2 at which the receptor portion of the protein is released. The cleaved extracellular

domain of Her-2 was detected in supernatants of the cell culture through Western blot

analysis. The Her-2 extracellular domain was then immuno-purified from soluble

fraction of a conditioned cell culture. Identification of the cleavage site was performed

via C-terminal sequencing and MALDI-TOF mass spectroscopy.

Sequence analysis of the JM domains of eight other EGFR family members led to

the hypothesis that this cleavage site rests within a particular motif, P/GX5.7P/G (Figure

1-7) (Yuan et al., 2003). Proteases may recognize this short, unstructured motif flanked

by proline and glycine. The P/GX5.7P/G motif is seen in EGFR family members

originating from very different species suggesting the motif is highly conserved. In

contrast, Her-4, which does not carry a complete motif, is not cleavable. Proline and

glycine are commonly associated with proteolyitc in cleavage sites because they can

locally disrupt the secondary structure of the protein, possibly leaving that portion

exposed to cleavage (Hooper et al., 1997). Therefore, it cannot be assumed that

protease(s) recognizing P/GX5.7P/G in EGFRs are identical to the suggested protease(s)

recognizing P/GX5.7P/G in XA21.











N530 q5


PDLsYMPIWKF~PDeEGACOPCOPTNCT;IWISML 9 S TSIIVSAVQO2YVLYV922KRQKIRYTRRLQT
Nos Cys-rich JM TM

Humana Her-2: CPA 1EQR3ASPLTS
nat fner-2: cPA EQR ALSPVTs Cleavable
IHuman Her-4 (JM-a) : CITYYPwwanswtonans
numan ser-4 an-b)1) :ClGLM6DRT NOncleavable
BuIhanP Her-3 r CLOQTLrVLI~Kxano
Ruman Her-1: CPTNGPKIPs
Mouse Her-1: asvwPSGIPKIPs
Rat Brer-1: QCOOPEPKIPS
Chick HETr-1: CPRGi~SKTPS
Motif : P/Gx s-7 PIG


Figure 1-7. A conserved motif in the juxtamembrane domain of the EGFR family.
Several EGFR family members from different organisms are represented.
Reprinted with permission. Yuan CX, Lasut AL, Wynn R, Neff NT, Hollis
GF, Ramaker ML, Rupar MJ, Liu P, and Meade R (2003). Purification of
Her-2 extracellular domain and identification of its cleavage site. Protein
Expr Purif: 29(2):217-22.
















CHAPTER 2

SEQUENCE ANALYSIS OF RICE RECEPTOR-LIKE KINASES

Introduction

Bioinformatics studies have become crucial in modern molecular biology,

particularly when the genomes of a number of organisms, including rice and Arabidopsis,

have been sequenced. Through analyzing the rice genomes sequences, 1429 protein

kinases have been predicted (Rice Kinase Database, http:.//rkd.ucdavis.edu). Genome-

wide comparative analysis suggests that rice has significantly more numbers of RLKs

that are involved in resistance and defense than Arabidopsis (Shiu et al., 2004). To

support this hypothesis, three qualitative resistance genes, whose protein products are

RLKs, have been identified in rice (Song et al., 1995; Sun et al., 2005; Chen et al., 2006).

Therefore, rice appears to be an excellent model system to study RLK-mediated defense

signaling.

As describe din Chapter 1, a putative proteolytic cleavage motif (P/GX5-7P/G) was

identified in the juxtamembrane (JM) domain of XA21 (Xu et al., 2006). This motif was

originally described in the extracellular JM region of animal epidermal growth factor

receptors (EGFRs) (Yuan et al., 2003) and has recently been implicated in the stability of

XA21 (Xu et al., 2006). In this study, rice RLKs are searched for this motif.

Chromosome maps depicting locations of the RLKs are then created to investigate the

distributions of rice RLKs carrying this motif.









Materials and Methods

Identifying Putative Proteolytic Cleavage Motifs in the JM Domain of Rice RL~Ks

Raw protein sequences files were downloaded from the University of California

at Davis' Rice Kinase Database, which included a total of 1429 receptor kinases (January

1, 2006, http://rkd.ucdavis. edu). The Rice Kinase Database incorporates and shares

genomic and proteomic data resulting from a collaborative rice proteomics study, and

enables the user to easily view this data. The database creators have additionally made a

phylogenetic tree of these kinases using the nearest neighbor method. To quickly

determine if each kinase qualified for this study, its ID was entered into the PlantsP

web site (http://plantsp. genomics.purdue. edu/) to obtain detailed motif and domain

structure information. Because the JM domain is loosely defined as being located

between the transmembrane domain and the P-loop of the kinase, proteins which did not

clearly carry both of these were simply eliminated and the resulting number of rice RLKs

totaled 796. Each protein's JM domain was then searched by eye (using Microsoft Word

and Excel) for the putative cleavage motif, as well as additional proline and glycine

residues which may also contribute to the exposure of the cleavage motif to proteases.

All proteins were subsequently placed into one of three categories: motif-positive,

motif-like, and motif-negative. Motif-positive proteins are those containing at least one

perfect motif, while motif-negative proteins contain none. The motif-like category

contains proteins having a cleavage motif(s) with +/- one amino acid outside of the

normal range in the variable region of the motif. That is, these cleavage motifs may be

P/GX4P/G or P/GXsP/G. This allows for the investigation of a group of proteins which

may be recognized by less specific interactions and can also allow for some sequencing

errors.









Additionally, 27 previously described plant RLKs are investigated in the same

manner as described above. These included RLKs functioning in both growth and

development and defense responses originating from various plant species. These

published data provide an opportunity to quickly examine the putative cleavage motif in

species other than rice without examining whole kinomes--many of which are

unavailable.

Average Length of JM Domains of Rice RLKs

Upon the collection of the rice RLKs sequences and identification of the JM

domains, a marked difference in the length of the JM domain was noticed between the

motif-positive RLKs and the motif-negative RLKs. Each motif category was then

analyzed separately for an average JM domain length. All JM domains of proteins

making up a group were compiled, with careful elimination any character except those

representing an amino acid, and counted using Microsoft Word's character count. The

total was then divided by the number of proteins belonging to the category.

Creating Rice Chromosome Maps Depicting the Locations of Rice RLK Genes

To view the distribution of the rice RLK genes in this study with respect to their

motif categories across the rice genome, rice chromosome maps are created. To further

analyze this relationship, seven different subfamilies of rice RLK genes are also mapped

with respect to their motif category. All chromosome maps of rice RLK genes were

created using the freely available Genome Pixelizer software

(http://www. atgc. org/GenomePixelizer/GenomePixelizerWel come. html ). Genome

Pixelizer was created to help visualize relationships between genes and following

"footprints" of evolution. Using data provided by the program user, Genome Pixelizer

rapidly creates a chromosomal map, indicating the location of each gene with a colored










square. Each square is interactive, providing all input information when clicked--though

this feature is only available when using the program. The maps shown in this chapter

are snapshots only and are not interactive. For the RLKs studied here, protein IDs, their

subfamilies, 5' end positions of genes, and other necessary information were obtained

from http://rkd.ucdavis. edu. Technical assistance was generously provided by Chris

Dardick of the Ronald lab at University of California at Davis.

Results and Discussion

A Significant Number of Rice RL~K's Contain the Putative Cleavage Motif

Of 796 rice RLKs analyzed, 47. 1% were categorized as motif-positive. This

represents a significant portion of the total proteins analyzed. Motif-like proteins

comprised a group of only 5.9% of the total proteins analyzed, while motif-negative

proteins represent 46.9%. Table 2-2 shows all rice RLKs involved in this study and their

JM domains. Residues involved in putative cleavage motifs have been highlighted in red,

while proline and glycine residues flanking the putative cleavage motif are highlighted in

blue. As discussed in Chapter 1, these flanking proline and glycine residues may

contribute to exposure of the site by disrupting the local secondary structure.

A Significant Number of Select Plant Proteins Contain the Putative Cleavage Motif

Table 2-1 shows the previously described plant proteins implicated in development

and disease defense chosen for examination in this study. Putative cleavage motifs have

been highlighted in red, while flanking proline or glycine which may contribute to

exposure of the site are highlighted in blue; ellipses indicate amino acids which have

been left out for convenience of space in the figure and have no bearing on the immediate

study. The maj ority of defense-related RLKs contain the P/GX5.7P/G motif although

some RLKs that function in developmental processes also carry the motif.













Table 2-1. Distribution of the P/GX5-7P/G motif in the JM domains of plant RLKs.
Perfect motifs are highlighted in red, while motif-like sequences are in blue.
Proline and glycine residues which may contribute to the disruption of
secondary structures are also highlighted in blue.


Protein
BAK1

EXS

HAESA

NORK

PRK2

SERK3

SYMRK

CLAVATA1

HAR1

ERECTA

BRI1

XA21

XA21/A1

Pi-2(t)

RKL1

RKS1

SFR1


SIRK

WAK1

WAK2

WAK4

LRK10

PBS1

RKC1

SFR2

XA26


Function
development

development

development

development



development

development

development

development

Devel.,defense

Devel.,defense

defense

defense

defense

defense

defense

defense


defense

defense

defense

defense

defense

defense

defense

defense

defense


Reference
Li et al. (2002)
Canales et al.
(2002)
Jinn et al.
(2000)
Endre et al.
(2002)


Hecht et al.
(2001)
Stracke et
al.(2002)
Clark et al.
(1997)
Krusell et al.
(2002)
Torii et al.
(1996)
Li and Chory
(1997)
Song et al.
(1995)
Song et al.
(1997)
Jiang and Wang
(2002)
Ohtake et al.
(2000)
Ohtake et al.
(2000)
Pastuglia et al.
(1997)
Robatzek and
Somssich
(2002)

He et al. (1997)

He et al. (1997)

He et al. (1997)
Feuillet et al.
(1997)
Swiderski and
Innes (2001)
Ohtake et al.
(2000)
Pastuglia et al.
(1997)
Sun et al.
(2004)


Juxtamembrane Domain
RRKKPQDHFFDVPAEEDPEVHLGQLKRFSLRELQVASDN
RRWAMTK... EESRLKGFVDQNLYFLSGSRSREPLSINIAMFEQPLLKVRL
G...

RKITRSKNIGYVWILLTIFLLAGLVFVVGIVMFIAK. .. LAASKWRSFHKLH
RKTKRADKGDSTETKKKGLVAYSAVRGGHLLDEGVAYFISLVEA
DN
RRNKN... LGGSSLTSSSPTSQDQKLIP ... GQSSAASTPDRACNDGGKRA
EVAGQKL

RRKKPQDHFFDVPAEEDPEVHLGQLKRFSLRELQVASDN

RYRQKLIPWEGFAGKKYPMETNIIFSLPSKDDFFIKSVSIQ

RQMNKKKNQKSLAWKLTAFQKLD

RKRRLHRAQAWKLTA

RPHNPPPFLDGS LDKPVTYSTPKLVILHMNMALHVYEDIMRMTEN

REMRK... YAEGHGNSGDRTANN.. .NLAAFEKPLRKLTFADLLQATNG

HKRTKKGAPSRTSMKGHPLVSYSQLVKATDG

EVPATTSMQGHPMITYKQLVKATDG

KRKRHPPPSQDDAGSSEDDGFLQTISGAPVRFTYRELQDATS
CRKKSNKRS.. .EIPGDKEAVDNGNVYSVSAAAAAAMTGNGKASEGNGP
ATKKLV

RERRSIEVFGKLRPVPFDFDESFRFEQDKARNRELPLFDLNTVAN
RFWKR... LSGDMKTEDLELPLMDFEAIATATHNF SS TNKLGQGGFGIVY



RRFKKKQQRGTLGERNGPLKTAKRYFKYSEVVNITNN
RMKHLKDTKLREQFFEQNGGGMLTQRLSGAGPSNVDVKIFTEGK
ATNG
KIKHRKNTELRQKFFEQNGGGMLIQRVSGAGPSNVDVKIFTKME
TNG
EHKMKNTKDTELRQQFFEQNGGGMLMQRL SGAGP SNVDVKIFTEEGM
KEATDG

RTRYNEEIHLKVEMFLKTYGTS KPTRYTF SEVKKIARR

HTFAFRELAAATMN

KRRKQKQEIELPTESVQFDLKTIEAATGN

WKRKQKRSILIETPIVD...ISSRRHISRENNTDDLELPLMFEATN

RKKANHQNTSAGKADLISHQLLSYHELLRATDD









Average Length of JM Domains of Rice RLKs

The average length of the motif-positive RLKs JM domains was 75 amino acids,

while motif-like and motif-negative categories' were 41 and 40, respectively. This is

interesting to note and suggests some correlation between length of the JM domain and

containing cleavage sites.

Rice Chromosome Maps Depicting the Locations of Rice RLKs

Analysis of the chromosome maps shows that the rice RLK genes are well-

distributed within the genome (Figure 2-3,) even when separated into their respective

motif categories (Figures 2-4, 2-5, and 2-7.) Many of the genes exist within clusters,

which may have originated from duplication events. To address what types of motif

categories of RLKs any one subfamily contains, chromosome maps were created showing

seven large subfamilies of rice RLKs (Figures 2-7, 2-8, 2-9, 2-10, 2-11i, 2-12, 2-13).

Generally, many proteins within a subfamily are clustered. However, for subfamily L-

LEC and LRR-la the clusters contain proteins of the same motif category. This further

supports similarity and evolution within a subfamily. Subfamily CrRLKIL-1 and WAK,

though not having significant clusters, is composed of proteins of a particular motif

category. Subfamily DUF261c contains one maj or cluster which is interspersed with both

motif-positive and motif-negative proteins. Subfamily SD-2b though, appears to have no

clear significant clustering or motif category preference. These findings are crude and

the significance of the phenomena noted within separate subfamilies is better determined

through further examination of each subfamily.










Table 2-2. The P/GX5.7P/G motif in the JM domains of predicted rice RLKs. Proline and
glycine residues within a motif are shown in red, while proline and glycine
residues possibly contributing to the disruption of local secondary structure
are sl own in blue.

Protein ID Category Juxtamembrane Domain

RRSKRRRRSREGS ST SATALVPPLWKVFT SEELRS ITKNF S
EGNRLPGNAKTGGTYSGILPDGSRVAIKRLKRSSLQRKKD
FYSEIGRVAKLYHPNLVAVKGCCYDHGDRFIVYEFVANGP
LDVWLHHVPRGGRCLDWPMRMRVATTLAQGIAFLHDKV
9629.m00042 motif-positive KPQVVHRDIRASNVLLDEE

RRSKRRRRSREGS ST SATALVPPLWKVFT SEELRS ITKNF S
EGNRLP GNAKTGGTYS GLPD GSRVAIKRLKRS SLQRKKD
FYSEIGRVAKLYHPNLVAVKGCCYDHGDRFIVYEFVANGP
LDVWLHHVPRGGRCLDWPMRMRVATTLAQGIAFLHDKV
9629.m00043 motif-positive KPQVVHRDIRASNVLLDEE
WYKKKRRRATGYHAGFVMPSPASSPQVLGYSGKTNYSA
9629.m00111 motif-positive GSPDYKETMSEFSMGNCRFFTYEELHQITNG
WYKKKRRRATGYHAGFVMPSPASSPQVLGYSGKTNYSA
9629.m00112 motif-positive GSPDYKETMSEFSMGNCRFFTYEELHQITNG

P SVEDIEP SCGYLAMTPIGGGWNSVNLPEYTSYAEVVKSM
RGGFAVKFPTPWLNWFGLIKKCLNKSVSHRTMSTNGGLE
HLD SLFAANSLLPIPPTSPPFAGTRRTS SAVQLPRDLLCLGQ
SYMAGLVVRRGQGPSAAWRRGEGPPLVAGGGGDVDRAG
9629.mO001 53 motif-positive DD GGGEGD GQATK STMD GVRRLRMAKVCVRTVGRDD
KWIADDPYCYILVLD CLAFYPQVS CTLAAFTNEIC SGPTKS
NSRYRYFTL GMTGIVRSNDRKQFGAVKYRYLEVGGGGGA
9629.m00167 motif-positive PLVVMTFLAHKCWKKRITIDAV
KDKDFF SACPAKQCGKVEIRYPFRFEP SNTS S SCGLPCMKL
TC SDRQETILDIKNYLGRPYKVTAIDYKRATLTIVPLADD S
SLPPTPGCPLPNLISEGALDHRCEPYAMWLLKSINIEFPFR
AVHVSE SLQTRYNEEIHLKVEMFLKTYGT SKPTRYSF SEIK
9629.m00191 motif-positive KIARR
PRLSGRRSARRLREGEKIVYSSSPYGATGVVTAAGGTFER
9629.m00341 motif-positive GKMVFLEDVSSGGGKRFELDDLL
RRKRKRSASFEGLIHGGTPLP SLTKEF SLAGLAYTHIFTYEE
9629.m00363 motif-positive LDEATDG
9629.m005 13 motif-positive RRRKPVHRKKGQSLTPVVEEQFERVSYQEL SNGTKG
RRKEEKEELRTPTTSQP STAWMPLLGRI SFRS APP SAVGSR
SPSFTIDTNANTPGGGATPGMAAAASSSPSYRFPFAALQDA
9629.m00554 motif-positive TGN
RRKEEKEELRTPTTSQP STAWMPLLGRI SFRS APP SAVGSR
SPSFTIDTNANTPGGGATPGMAAAASSSPSYRFPFAALQDA
9629.m00555 motif-positive TGN


RRRQPLFGRGST SPAD GD GKDADMLPPWDVTLYQKLEI S
VGDVARSL


9629.m00688


motif-positive












Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain
RFRG SD GLAQRFPH SALPKF SRS SGTGQTLLAGRYS SP SGP
9629.m00937 motif-positive SGSLGS SIATYAGQAKTFKFAEIEKATNS
9629.m01024 motif-positive QDKD GEDEFAELPGMPTRF SFQMLKLATKD
9629.m01041 motif-positive MARMGISKGGS GKEAKKPPLLLGR
9629.m01044 motif-positive RKRGRAAIGPWKTGLSGPLQKAFVTGVPKLNRPELEAACE
9629.m01045 motif-positive RKRGRAAIGPWKTGLSGPLQKAFVTGVPKLNRPELEAA
9629.m01193 motif-positive KLFATRPLDARS GLMVF SYAQVKNAT
CNRS GGGGDEEVSRVVSGKS GEKKGRE SPE SKAVIGKAG
9629.m01202 motif-positive D GNRIVFFEGPALAFDLEDLL
9629.m01205 motif-positive RYRRDLFGSSKFVVEGSLVVYSYAQIKKAT
FKKKKRHHHHHPPPPPPPHLLHYYGHPPPPPPPPPPFKGDH
YGGVYQNWQQNGPPPPPDHVLKKVP SHP SPPPPPAPLNVH
SGGSGSNYSGGDNSQPLVSPGAALGFSRCTFTYEDLSAAT
9629.m01240 motif-positive DG
KKWARKKRIIENSLTGGKMVMFRSAAMQ SL SPKSFLTMI
9629.m01429 motif-positive MG
KLRKGKRKVPPVETPKQRTPDAVSAVDSLPRPTSTRFLAY
9629.m01474 a motif-positive DELKEATNN
SNKRSKKL GGGGAD SHT SAWLPLYHSHTS GK S SGHITANI
9629.m01474 b motif-positive AGMCRHF SFAEIKAATKN

RSRKRVRRASANIPITQIPAI SKEIKEVRVEQVPTSDFAAHD
GVLMTIQDKSSEKESDKVMVHLGVSKSKRGDESHSGSFR
YMDKDL GFQ SADEGG SGTFRHN SAHAITAP SPLVGLPEF S
9629.m02103 motif-positive YLGWGHWFTLRDLEVATSR
WRAVS SKEKGNGGAAGSKGSRCGKD CGCF SRDESATP SE
9629.m03098 motif-positive HTEQYDLVPLDQQVRFDLDELLKASA

DSPGGSLASLPEWQPMPCKSVSVNPLCSSYLYVTPEGRNL
SKVASDFSGNASLFQRITRLSGSEDLLVNVPCVCEAINATM
TGLFHDTNYRVKD GDMGDIINSKTF SGLALNVGD GQILHK
EEKLIIHLPCGC SSTAPEGVL SYAVQD GDTLGNIASLFRS S
WKDILDLNPRVANPDFIKPGWILFIPMGVAGP SNKKIDPFQ
9629.m03450 motif-positive TERPVIFSLRAIEDATSN
RWRKQRVVAGSPAAVGGRCSTDAAGKD SFRKSAS STLVS
LEYSNGWDPLAD GRGGIGF SQEVAQ SFRFNMED VESATQ
9629.m03772 motif-positive Y

RKRGNRFGDTAEPEIPDITKEIAVDEARNRVAAENVQRQE
SYTL SLKERQTNKGSRKMLAHFL SCKS SGSHNLVGC SSMY
QNDKAQC SYS SDEGT SGHNEREYSQYATMSTSPQIGLPEF
9629.m03992 motif-positive SHLGWGYWFTLRDLEDATNG


RRKTKRTMDNLRQTQIPIFSKEIPVDRVGGRSLAQTMHER
EQP SFPPQDKHTNREPGKTLGHMAL SKS SEPDNMSQ GS SV
CNVDRAGSVHSGEDGSTGHGRKPYSPAAFVSASPLVGLPE
F SHL GWGHWFTLRDLELATNR


9629.m04252


motif-positive











Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain

MRRKKKTAVGFDGRSHMEIPIVSKDINVDRVDAQ SLHD S
GTPIMPVQDKYEQMKGVSHLAE SRSVDVDAF SQC SSVYNI
EKAGS SYSEDYS SSGPGRKGS SSYGYASASPLVGLPEL SHL
9629.m04552 motif-positive GWGHWFTLRDLEYATNR
9629.m04594 motif-positive FLFKMHNIPKSMEKGYKMITSQFRRFTYRELVEATGK
9629.m04595 motif-positive KKHNIPKSMED GYKMITNQFRRFTYRELKEATGK
9629.m04597 motif-positive SKHNIPMSMEAGYRMVTSQFRMFTYRELREAT
9629.m04603 motif-positive SSKQSIPSSLQAGYKMVMTSQFRRFTYRELKGAT
9629.m04613 motif-positive SSKQSIPSSLEAGYRRVMLTSQFRRFTYRELKDVT
9629.m046 15 motif-positive LRSKQNIPKSVMD GYELMTEHFRKF SYRELKEAT
CRNK SVATIGPWKTGL SGQLQKAFVTGVPKLQRSELEGA
9629.m04657 motif-positive C
RKRKHKKVNS SSKLLKYS GSGGTPRSMGGDME SGSVKDL
9629.m04784 motif-positive QTHLFSYEELEEATDS

PENIDPITC SFIGLIGP SYVLPKD QVPPGNW SQFCKTFEVPV
VKYQQMDPKGDAWRKGGYGQVLRQGFLLSVND SRRPPN
CTQCEE SKGRVFKLKGENETFLKKYRHRRI SKGTPRIESFL
9629.m04795 motif-positive QRNGTLHPKRYTYTEVKRMTKS
CHKKHHGQQPPVQELTTPPKAEP SQKKQRAQHLKRY SYS
9629.m04796 motif-positive EVERMT
9629.m04824 motif-positive KFCTGKKLKGLPITMSLE SNNNHRAI SYYELVRATNN
FKIRNRNHLTEE SPMPPKPAGPGSAVVGSRLGSRPI SASP SF
9629.m04969 motif-positive S SSIVTYKGTAKTF SLIEMERATQR
KKKRRRIEPPASLPTQQPAPPPPPNYFP S SGGS SLTSDAFFIS
PGYHPVRLFSAGSHGYPYSPADSAIGYSRMLFTPENLAEFT
9629.m05220 motif-positive NG

KLSQYINPQAPNNQGKSPDQSISHKFQLLKSGSFCYGSGR
LCCQFGNVKQSRTDGSDHHMNTPKGVVVDVFDREKPIVF
9629.m05240 motif-positive TYQEILASTDS
9629.m05252 motif-positive RD IRRRRKRVAQREDWKITPFQTDLGF SEAAILRG
9629.m05253 motif-positive RD IRRRRKRVAQREDWKITPFQTDLGF SEAAILRG
SKRRRRRQHPPPPHHPGYPPFPAEFYDPHRPPSQQQSHALS
P SP SSTPPLLLQPH SFVS SGGASEAASAVPGIAMMGGAFGY
9629.m05335 motif-positive DELAAAADG
KRRSRVGKD TGMSD GHS GWLPL SLYGNSH S SGSAKSHTT
9629.m05510 motif-positive GSYAS SLP SNL CRHF SFAEIKAATNN
FVSQWRKAGELAD GDIDEEMGYDELADEEFFVE SGPRRF
9629.m05592 motif-positive RYSDLAAATKN
FWRNRVRTRRNETAAAAAGGGDDVLPFRVRNQQHPASS
9629.m05634 motif-positive VKRD QRLDVKRECDEKDLDLPLLDLKAIVAATDD


RNKARRKQHTEMEK SSDADDLPFRVRKSPAL SPARD QWF
DENRGAEDDLDLPLFDLEMIFNATDR


motif-positive


9629.m05637












Table 2-2. Con inued

Protein ID Cateor Juxtamembrane Domain
TRKKKRARKTG SSKWS GG SRSTGRRYEG SSHHDDDLELPI
9629.m05640 motif-ostive FDLGTIAAATDG
RQKKIAKEAVERTTNrPFASWGQGGKDNGDVPQLKGARYF
9629.m05897 motif-poitive AFEELKRCTNN
RRARATTSRSAPATAL SDDYL S SPEND AS SGKLVMFGKG
9629.m05964 motif-poitive SPEFSAGGHAL
9629.m06449 motif-positive RRRMVKGTTQVEGSLISFTYRDLKSMTKN
WRRRQD SIRSKSRRL SGERRL SRPRPNVGSVLF SL GELAKA
9629.m06547 motif-poitive TCG
9629.m066 10 motif-ostive GRRRTIGINRDD GKLITFKYNELQFLTRN
RRKLVRSRPLAFESASKAKATVEPTSTDELLGKKSREPLSI
9629.m06844 motif-ostive NLATFEHALLRVTADDILKATEN
GAGEVQLAAPQPEKIAAVQKDES GWPLWL SSAAGDALAG
9629.m06926 motif-poitive WAPRSADAFHKLEKR
9629.m06939 motif-postiv RVAILHAKYAPAIVRGPVSFTPQI SPKNLH SA
9629.m06940 motif-ostive RVAILHAKYAPAIVRGPVSFTPQI SPKNLH SA
9629.m06957 motif-ostive RHRRRASDSSESSSSSPAQPELQGARCMTLEELSSATRN
RVARRSRRNSDTGSSETPPTLVEWGRCGRTLSAPEYQGAR
9629.m07015 motif-ostive QFSLEELAHATKN
RRRAGD GGTTTPEKELESIVS SSTKS SKLATGKMVTFGPG
9629.m07193 motif-poitive NSLRSEDFVGGADAL
RKQLQGASAGGNSKSKSIVVSAEQKKKATPVAGGGGGEI
9629.m07395 motif-ostive DNMMAAMAARGPLEFEVRELEEATS
RHCL SGLRRRHRAAAAADVDAEAAEGGAGARQLMPPCS
9630.m00072 motif-ostive PPPQQELPLLRPAAKLVPSTKEEGEPWKLTWREVEALTGG
RRKDAL SPH SHAYSFDKYTSW SSRSNLVSHRS SPLPQPKP
KPRIS VLKEFLC SCNPICGNEGGPLPGVIVRF SYSELEQATG
9630.m00103 motif-ostive K
RRKDAL SPH SHAYSFDKYTSW SSRSNLVSHRS SPLPQPKP
KPRIS VLKEFLC SCNPICGNEGGPLPGVIVRF SYSELEQATG
9630.m00104 motif-ostive K
RRKDAL SPH SHAYSFDKYTSW SSRSNLVSHRS SPLPQPKP
KPRIS VLKEFLC SCNPICGNEGGPLPGVIVRF SYSELEQATG
9630.m00105 motif-ostive K
YAYRRWYVDGAGCCDDENLGGESGAWPWRLTAFQRLGF
9630.m00115 motif-ostive TCAEVLAC
9630.m00168 motif-postiv DD GVYPEGQILEAPNLRTFTFIELRTATKN
KRRRET SLH GGFLRGVEMSTPDWS GKP SGQ SAVVKVDKE
Q STVAEEKDTKGS IS SYQKNVQE SLQNHPLQFKFTIFTVAS
9630.m00356 motif-poitive LQQYTNSF SEQNLMRQTL
LTLLRSTSFLSKNRRYSNDGTEAPSSNLNSEQPLVMVPQG
9630.m00518 motif-ostive KGEQTKLTFTDLLKATKN


SLRDAIPKIENKSNTSGNLEAGSFTSDPEHLLVMIPRGSGE
NKLTFTDLMEATDN


9630.m00525


motif-positive











Table 2-2. Con inued

Protein ID Cateor Juxtamembrane Domain

9630.m00733 motif-postiv KRGGRRDEHGRLVFVQESRKRFEIEDLL
LSKYQERQSRRDYTTSQVGRVrHQRVEEPKVKQASVQSRN
DAKKGSTEVPERRQMYKITIQLLAAALEKPPEKRKEHQYT
9630.m00752 motif-poitive NS

RNAQKRKDD TS SNSKDFVGPL SVNIERASNREIPEQ SPENT
SVATMKISPAEKMTPERIYGKTGSMRKTKVPITATPYTVA
9630.m00890 motif-poitive SLQVATNS
RRRRGVGEESVRPGKVVGDVSSSAEYGALASGKQTTTAT
9630.m00912 motif-ostive SMSSLSAARSLMASEVREALESLTVYK
RRRKRGKKAAAAWEDDEVAVGAVKVAATAPVVLVERPL
9630.m01346 motif-poitive MELTLADLAAATSGFG
9630.m01660 motif-postiv RRKLAKKGKEIIPGDFVSIWNFD AKVAFQDALYATEN
9630.m02590 motif-poitive RNRS SLKRRQS S CSEEVEDIK SVLLDP SVIRSATGN
9630.m02923 motif-ostive KGKFKTRKELPAPSSVIGGINNHILVSYHEIVRATHN

RRRRRRRGPNT SPVQQLPVSAPPKNPQKVKAPKDIQEVPA
QATAAAAAKTPLAQVLQMPAPPPPPPPMAAAAPPPETVQI
ATGKEHRITYPEPPHRSGSSSHGSGEAPSVPEVSHLGWGH
9630.m03277 motif-poitive WYTLKELEAATEM
9630.m03316 motif-postiv KRKKYGEQSENGVGDAKVFSVWNFEGGEACRQIFETTKY
KS SFRRQDYIVKAVADTTEALELAPA SLVLLFQNKDD GKA
9630.m04006 motif-ostive MTIGDILKSTNN
9630.m04684 motif-ostive RWRKRNAVRRAQIE SLRPL SNSDLPLMDL SSMYD ATNQ

KQYEHLEPGNQLH SLP SP SEGNE SIEKSADEFWDKQNFEID
HGQDNTLDQEKD SAEVRQDAERT SDKS SGTE SAKSEITLD
9630.m04997 motif-poitive DVAEFEIQWEE

KQYEHLEPGNQLH SLP SP SEGNE SIEKSADEFWDKQNFEID
HGQDNTLDQEKD SAEVRQDAERT SDKS SGTE SAKSEITLD
9630.m04998 motif-ostive DVAEFEIQWEEI
RKQERRAGRRASRSRDVQLRRHRSPASDSAHLVYGNDDD
9630.m05216 motif-ostive GNDIVIPGLPTRFTHEEIEDMTN S
PHWAQVPKDVSLCKPDIHALP SSNVPPKLVILHMNMAFLV
9630.m05315 motif-positie YEDIMRMTEN
KRKLIRTKQRFFEQNGGVLLQQQMSSYGGTSGGAGGFKIF
9630.m05589 motif-poitive SKEELEKATNS
QKRKLIRTRQKFFEQNGGIFLQQQMRSYGGAGGGVGGFKI
9630.m05597 motif-ostive FSTEELKNATNN
KWYKLRQLQEAVSPATTPAVNRRYGGRSTLSVSRVSSAS
9630.m05673 motif-ostive ASMLSTVATCTTSVKTFSL SQLEKATDG
RRGRNKRVAGDVDKGAMPEEEEEQQQQQPQAQPREEINA
SASASASVASERRGGREFSWEREGIGKLVFCGGVAEMYSL
9630.m05830 motif-ostive EELL


KSCTYSPKSTANNAKSPPANVEKVPKANEVLYSWNSL1VNDCE
SSSDVIKPERA1VKTKVWAKTSKNFLTAKQFQAVDILAATRN


9630.m05880


motif-positive











Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain
RRRRQAPTPAPEEKESTQLPWSQHTQDGSSWVDMSNASG
9631.m00239 motif-positive AGMTGGLHRMSMQLNI SLADITAATEN

SRRRRRRDDLASNLYPADTKILKQHLQQPTPPKDIQEIVRR
QQRQQQTPTPTPPQPPPPAAQHGVQLAKAETPPPPQRTQPP
VLPAGSTRSTAAS GMSATTS GGSERD GATPRSTAS GSAGP
963 1.m0025 1 motif-positive EVSHL GWGHWFTLRELEEATD G
AMNIKAYMRRSKEEQEGKEEDEVLE SE STPMLASPGRQG S
963 1.m00268 motif-positive NAIIGKLVLF SKSLP SRYEDWEAGTKA
RRRKRRAALEEHFEQHQPFT SFP SNEVKDMKPIEE STTIDV
ESLPSPASFSLKPPPKIERHKSFDDDDLSNKPVLKKTNVAP
9631.m00800 motif-positive KATVYSVADLQMATES
9631.m01136 motif-positive RMRRRRGKVTAVQGSLLLLDYHAVKTATRD
RKKSSSSTPATAVEKGRDLQMAPMDMEPKGQNGSAAGN
GAHVGAAAAAPAAATSAAVAAAAAAAKTGGATGGSKK
9631.m01146 motif-positive LIFFGPMAAAPPFDLEDLL
CCSKKKKRPPHMHMPYYTDENGKVYYANSMPRWQNSV
DQGGGWHAQYSPGQAPP SSEMS GSHGAGPLPPP SPGMAL
9631.m01182 motif-positive GFSKSSFSYDELALATGG
CARRRRGAKHLSMSRVEHAPSSGSLRQASSSSAPKEKDHA
EAGAGTGTGTS SSDVAS SSAAASYLESPVRRKPERIS CAAA
9631.m01193 motif-positive MDMGWGRWYDLEELEAATGG
RRGRKRGAGAGGGAAARMGVGVGVGARTQPAPALRRLS
9631.m01412 motif-positive CQQLRRATGG
963 1.m01555 motif-positive KNFGKKDIHGFRVEL CGGS SIVMFHGDLPYSTKEILKKLET
RRCRRQRRRRRQAQPFPLPPPIYN\PNPYYKGDLPPQPFVA
QPP SDHYFIQHQHPTPPQT SGTF SD AGSERPH SIDILTELPT
9631.m01585 motif-positive GGSLSYDQLAAATDG
RMLRRKKKPVKQPSNTWVPFSASALGARSRTSFGRS SIVN
963 1.m0 1698 motif-positive VVTL GQNGAGAGAGYRFPFAALQEATGG
RRRAQKAREELGGPFASWKRSEERGGAPRLKGARWF SYE
9631.m02117 motif-positive ELKRSTNN
RRRAQKAREELGGPFASWKRSEERGGAPRLKGARWF SYE
9631.m02118 motif-positive ELKRSTNN
SKRKEKKDD GLDNNGKGTDNARIEKRKEQVS SGVQMAE
963 1.m02 148 motif-positive KNKLVFLD GC SYNFDLEDLL
CKRRQRAGKD SGMSD GH SGWLPL SLYGNSHTS SSAKSHT
963 1.m02 15 1 motif-positive TGSHA SSLP SNLCRHF SFVEIKAATNN
RVRTPGSH SAAELEL SDGYL SQ SPTTDVNS GKLVMFGGGN
9631.m02173 motif-positive PEFSASTHAL


KRKSTRHQHGGDPEKNEPLTLRPIA SGKFNQLRTI SII SPTA
KEGLQKTVSMNLKPP SKIDLHK SFDENDLTNKPVLAKNV
DLSSIRATAYTVADLQMATES


9631.m02769


motif-positive











Table 2-2. Con inued

Protein ID Cateor Juxtamembrane Domain
KAKKNRKAIPSALNNGQVTPFGQRNHTASALNNWEITPF
WQRNHVAASND AQDNNSMRPAGQGNHQDLDLP SFVIETI
9631.m03402 motif-poitive LYATNN
KNRWKRRRRPAQVMNLARRRTLVVPERVASPEVYQPSN
963 1.m03 557 motif-ostive GPTASP SGTS SYEF SGTT SWFTYDELAAVTGG
RRWRRRRRRQQAQPLPLPPPMLYNPNPYYKGDQPPLPFVF
MQQHHHHPTAPQT SGGTF SDAGSERPH SI SID GGSL SYDQL
9631.m03714 motif-ostive AAATGG
KRKQKPFGRVQ SPHAMVVHPRH SGSDPDMVKITVAGGN
VNGGAAASETYS QAS SGPRDIHVVETGNMVI SIQVLRNVT
9631.m04965 motif-positive NN
RRRHRPGWQKTNSFQ SWFLPLN STQ SSFMSTC SRL SSRNR
963 1.m05382 motif-ostive FGSTRTKSGFSSIFASSAYGLGRYFTFVEIQKATKN
SRKTAPRRKRKKKPHNPVTHFDADTSGSKGGGGRDTSGP
9631.m05526 motif-poitive KPPPPPPWLAEPRAAPSTSDAAGMSKGTFTYEQLAAATGG
RRYRGRGPAPAAGPTNAAARAAAFLRRNGLHQHRP SFTY
9631.m05779 motif-poitive EQLRAATAG
9632.m00032 motif-ostive WRNRFKWCGVPLHRSQGGSGIIAFRYSDLDHATKN
9632.m00033 motif-ostive RNRFEWCGAPLHD GED S SGIKAFRYNDLVHATKN
9632.m00260 motif-ostive KWRGTGRGFDKGTRGVRRFKYHQLVSATNQ
RARGMGIVGGKGGHGGGS SD SES GGDLAWPWQFTPFQK
9632.m00342 motif-poitive LSFSVEQVVRNLV
RK SGKFQLRIIGKNSNPKENIEELLDNYGSLAPKRYKYSQL
9632.m00847 motif-poitive KDMTGS
9632.m01083 motif-ostive RRKTTKTMIPPDNTGNEEDNDYVDPPTLNLTVLRAATRN
9632.m01131 motif-ostive RKKTQL SQLP SNS GLP SKIFTYRELEKATGG
9632.m01135 motif-ostive RKDVQPLQP SRDPGLPLKAF SYAELEKATD G
KRRMI SF SWFNYGHRQCTDVLRQF SGMLNML C SSNPKRR
9632.m01392 motif-poitive EVPTTPINNETLKKVSYGDILKATNW
RRARKRRRRDD GVSYDD SIDDDDEEDMES GTGPRRIPYA
9632.m02040 motif-ostive HLAAATGG
9632.m02719 motif-ostive RGNKFKCCGAPFHDNEGRGGIIAFRYTDLAHATKN
RHKQKIKRQALLRQTDEFFQQHGGQILLEMMKADGNDGF
9632.m02858 motif-ostive TLYKRGEIETATNN
KYKQRIKRQDIMRKRGEYFHLH GGQLLTDMMNIENNI SFK
9632.m02868 motif-poitive LYDRDDIELATKG

KYKQRIKRHDLMRKRGEYFNLHGGQLLTDMMNIENNI SF
9632.m02875 motif-poitive KLYDRDEIELATKG

RHRQNTKKQALLRQTDEFFQQHGGQLLLEMMKVEGNVG
9632.m02879 motif-ostive FTLYERGQIETATNN


9632.m03307


motif-positive


WRIKGKWIIAHPLEKSEDSIGIIAFRHIDLRRATKN











Table 2-2. Con inued


Protein ID Category Juxtamembrane Domain

SRRRRRRRGVAVATPVLHLATAVAPPKHPGKPPKDIQEVP
SRAAAAAAAPKAQPAQVIQAPPPQPPP SE SIQIETGKEHRIT
FREQQHQpPPQPPPYHQRSGGPSSRGGSGESRGGGGGGGG
9632.m03373 motif-positive GAEPGVPEVSHLGWGHWYTLKELEDATAM
RRHRGS SSPVS GRIHAEPTGTAPRVERRL SMALL SKGPNTTV
9632.m03459 motif-positive EQFPLVALRAATDC
WWRRRKPEEHFFDVPAEEDPEVHLGQLKRFSLRELQVAT
9632.m03631 motif-positive DN
QRKPRPHH SRDVAAELALH SKEAMSP SVYTPRVSDARPPP
9632.m03678 motif-positive PPAAVVPAIQPAVAANVAGKKKLFFF GRVPRPYDLEDLL
S CHLRRRAHNLKRSKKDIEVTAVSVEYEEVTCKQMCTKEI
9632.m03707 motif-positive YDATEN
9632.m03789 motif-positive KKSKKIDPPKQSYSTGGLPLKSFTYEELHEATGG
9632.m03791 motif-positive KKTDLPKQ S SSTGGLPLKSFTYEELHEATGG

GSGKVQQTTDVDVKVKLDNGSYHGVAYMHNSFDLYSQL
YD GGSW SMQ GPDPATD SAGKIVG SQVKSNLKSKRKTIIEK
SRVS SDEVNVAKGLLPDNAGQKNIMKHDVIRETVP SLHV
VAEETEND SKTVS TSNRENTS GTPERSF SSVHQLED SDL SD
EDWNDED SGS GS GFSNTP SFDMFDDASRNKKKDLILVHLL
RLACASKD SL SASLPAIS SEL CNIGIL SEWAKDLI SKSPAVF
9632.m03956 motif-positive GETFGHFFGPQMTS SEC SLFWRADNS SSRPN SRYLND
HMRKPLETVAPLQDKQIF SAG SNMQVSTKDAYTFQELVS
9632.m04082 motif-positive ATNN
WKYKGFEKSRGTGRVSNS SATRKTGMRS SFS SMTS STASF
9632.m04 117 motif-positive VSTIATCPPTVKTF SI SELEKATENF S
RWRRRFIEKRD GVKKRRRSP GRGSG S SGT STD SVSQPKLI
9632.m04727 motif-positive MFNSRITYADTVEATRQ

SRGRENQWPPLEGNSQLTTTKNLSCDEVPYPFGLKGKSAP
GFRVTCRKND SAAMLRIGHQKFRIDQVS SQEGFVVIFAGPI
YRL CYDRNGRPVVGS TGIGPTNLTD TPFFF SKRNTLVATG
CY SNFTATFTS SLHHHGW STNGS CTTNGRVNSD GLCP GTA
CCDAYGMPLDD AQEVTFEFNKTSA SVAGTCSAAFILYQKE
QIFKVSGNSKPMHLHQEEHIFRAGGGD SKPVHLEDVLVPL
GERRMVLDWVIGRATCEQARNNSFKTQYRCNNES SCMD
RFMGEGYVCRCKAGYDQHNGNPYEAGGCQAYSVITLPED
ILALAPIIRLVMATGQ GQGALTPIGHLAPLPTTNRKKRKVE
RNRAELFRKNGGLLLQQRF SMMT SQGED SSAKIF SAEELK
9632.m04923 motif-positive DAT
9632.m05098 motif-positive WRQNRRKRKL SLEQQELYSIVGRPNVI SYGELRSATEN
9632.m05 100 motif-positive WMQKRRKL SLEQQELYCIVGRPNVF SYGQLRSATENFN
9632.m05 101 motif-positive WRKKRRKL SLEQQELYSIVGRPNIF SYGELRSATEN
RRYRRKRRAADIAGD SPEAAVVVPELRRF SYGQLAAATN


9632.m05116


motif-positive












Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain
KCRVL SGKRHQNKVVQKRGIL GYL SASNELGDENLELPFV
9632.m05247 motif-positive SFGEIAAATNN
RKCRGKRQNKVVQKRML GYL SALNEL GDENLELPFVSFG
9632.m05252 motif-positive DIAAATNN
RRRQRPRVSDDDAGVPAATAAVHARPNPALAAPSINLSSV
9632.m05258 motif-positive KEATGN

NYTGVVERLVWVAS SRAWQRFFQGPRDPCD SYARCGPFG
LCDADAAATSFCGCVDGFTAASPSAWALRNTSGGCRRGV
ALD CAGGGGG SRTTDKFKVVRGVKLPDTRNAS VDMGAT
AAECERRCLGNCSCVAYAAADINGGGCVIWTDDIVDLRY
9632.m05263 motif-positive VDRGQDLYLRLAKSEFDVIPDNP SMGVASVNLATIKS ITEN
RRIQAPRLITKEAGPPADEAIFRSDSVKSAVLSSPLVEFSTI
9632.m05264 motif-positive SATNN
MKRGKKRRRP S SAAYP SPKK SAAMSEVSRDNTDL GYVEC
VPDEETAAMMMPEEKARRLERS GCLTFCAGEGASYSLEQ
9632.m05414 motif-positive LM
9632.m05461 motif-positive RAD SVAAGRVGDEGYSLVFSHFRRFTYDEL SDATCG
KCAKIAIKMWYS SSRDHHTPIANGGGS S SSRGGIGGADAD
9632.m05466 motif-positive VVEMG SMSHFIEGLQNERPVRF SARQLRA
MRCVEAKHAERARRREEEAVPVSPPASGTYSSVDVRVEM
9632.m05467 motif-positive GSVDRFLDDILREKPARFTPENLREFTG
WKFLRPDIMRRLMRPKRAP SEVPEYF SGNMS GNLRTITYF
9632.m05492 motif-positive DYATLKKATRD
RKKRGSVPPNAASVVVHPRENSDPDNLVKIVMVNNDGNS
9632.m05758 motif-positive S STQ GNTL SGS SSRA SDVHMIDTGNFVIAVQVLRGATKN
TTKKKKKQRRRDNGYRAGFMSPTSPLS SHHPS SGSGASAN
VGSSLDPSFKTNYSAGSPKLKACMSDISMGNSRFFTYQEL
9633.m00004 motif-positive YQITDA
FGFCSHKKVHNSVQPNIASNNNGGGGGGAAAAVGSGAPS
PYGSPNGSLGRLRRQLSRVMTRQRSGPSSFKDPAEEFTFA
9633.m00288 motif-positive QLAAATKD
RRKKKMKKPQTPLT SRP S SSWTPL SLNAL SFL STGTRTTSR
9633.m00622 motif-positive TTYTSGTNSDTSYRIPFVVLQEATNH
9633.m00690 motif-positive HRRGGGARGGEDKD GEMSPPWDVTLYQKLEIGVSDVARS
APAAPPDVLSSPAAAAGEGEAEALLAVKAALHDTANVLA
DWNAGSGGVVVAGGGGGGGPCNWSMVTCSKTGHVSVL
DLAHRNL SGTL SPAIGKLRRLRLLFLQHNAI SGPIPDTIGRL
KVLQTLDLAYNHFTGTIP SIL GH SKGIFLMDL SFNNL SGPA
PVFSANSVLFSALTSVQKVILRGSETFVSRYSGHIFPYQRP
9633.m00702 motif-positive PIYLGHLKQFMIKEIKEATNN
TRKVSRRLHSLDLPKHHRRSSSSSPPPMPPPLPPPPPSANA
TLGKESPSSNSASDGAAAAVVVGGERGAVQVFSYRQLHA
9633.m01041 motif-positive ATGG


TRKVSRRLHSLDLPKHHRRSSSSSPPPMPPPLPPPPPSANA
TLGKESPSSNSASDGAAAAVVVGGERGAVQVFSYRQLHA
ATGG


9633.m01042


motif-positive











Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain
TRKVSRRLHSLDLPKHHRRSSSSSPPPMPPPLPPPPPSANA
TLGKESPSSNSASDGAAAAVVVGGERGAVQVFSYRQLHA
9633.m01043 motif-positive ATGG
QSKKRKVLNNSASHSSGWLPVYGGNSHTSTSKSSGGRSA
9633.m01831 motif-positive ALINPNITAMCRHF SFGEIKSATKN
RQKKRAQKLVSINDPFASWGSMGQDIGEAPKIKSARCFTL
9633.m02137 motif-positive EDLKLSTND
RRQQRIRLAKEKLAKEREEILNANNS SGRTAKNF SGRELR
9633.m02285 motif-positive RATAN
RRKKKVAKD TGKSDEGRWTPLTDFTKSQ SAT SGKTTNTG
9633.m02291 motif-positive SH SMLPANLCRHF SFAEIQAATNN

PFYFFLP SLFCLQGPWWLTGHDLEGS GCVWEWGLHPWLP
KSKLEGGKAFLAVGTSSVAAEFVINGTSLGLGEEREVQQL
SRLEYLVDHG SVP SGMVAPNTRSF SLDKSKSQGGLD SRKD
9633.m02806 motif-positive AFIPRDANGQPIAAHTFTFRELAAATKN
TRRWKPGTVD GAGGAS CNGDKPGGAPAS SCGS SVRGYN
NSRYYAAAAAGCIYGGRL GF SVQPRNRGAQVFTYRELES
9633.m03039 motif-positive ATDG

RRRKPAAKRAS SAAAAAAAP GGAAVPL SPATIPPVSKEIQ
EVAVHVGSLRHYLEAGATFLKEGGGVGGAVVDGD SLGG
STVYGSQRVHIEAGKGRRMVAYAD GEVGPVASDLAASA
9633.m03361 motif-positive QAAVGVGVGPEVSHL GWGHWYTLRELEEATAA
RRVRSAASH SAVPTAL SDDYD S QSPENEANPGKLVMFGR
9633.m03735 motif-positive GSPDFSAGGHALLN
RQKRRAKELKERADPFASWAAGQKD SGGAPQLKGARFF S
9633.m03811 motif-positive FDELKICTNN
RQKRRAKELKERADPFASWAAGQKD SGGAPQLKGARFF S
9633.m03812 motif-positive FDELKICTNN
KRNRKPHKHMMIGSVDL GDEDEMRGSE SLLYDL STLRAA
9633.m03875 motif-positive TAN
RRRRRRRRRQPT SPGDFLGPLPVT SRHHQQ SQFIKPTVTYP
PQLNAH SPLQ S SSNSDPP SPLLQP SPPPPAAS GGTVSYGDL
9633.m04105 motif-positive VAATNG
9633.m04180 motif-positive KRRKRLARTEPAWKMTPFQPLDFSEASLVRG
KKKQGSMNT SIKPQNEANYVPTND SD GHG SSMQLENRRF
9633.m04201 motif-positive TYKDLEKITNN
RKKKQGSMNTSVKPQNETASYVPTNGSHGHGSSMQLENR
9633.m04202 motif-positive RFTYNDLEKITNN
RKKKNKSKGAVKPQILGNGVQ SH SQNGS GGSLLELHNRQ
9633.m04204 motif-positive FTYKDLAVITNN
KKFWHGLLSSMGKSKEAPNIESFLQKHEAQHPKRYSYSEV
9633.m04448 motif-positive KTMTKS
RKRRQYKMT S SSRLLKYTTS GRTPRSKGS SDKFVES GSFH
9633.m04450 motif-positive YLQTHHFAYEELEEATDG












Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain
KWYKRPQDWERRNSF SSWLLPIHTGQ SFTTSKGGS SKS GY
9634.m00267 motif-positive TF S STL GLGRFF SFAEIQAATKN
9634.m00625 motif-positive RKWETRPEITDEGYAIIS SQFRRF SYKELQKATNC
9634.m00626 motif-positive KWGRRPEIRDEGCTIIS SQFRRFSYKELEKATGF
9634.m00628 motif-positive KWGRRPEIQDEGYTIIS SQFRRFNYKELEKATD C
KKKYFQQHGGMLLLQEIGLKQGQSTAFTIFTEAELIEATN
9634.m00665 motif-positive K
9634.m00812 motif-positive KKLTTTSVSPHL SSGGVMDERNSEHPRISHRELVDATGG
9634.m00813 motif-positive RRDARRSMLLAGGAGDEPGERDHPRISHRELAEATGG
RPH SPPVFKD VSVSKPVSNVPPKLVILHMNL SLLVYEDIMT
9634.m00996 motif-positive MTEN
NKQYEYLEPGCQLL SLPS S SGANELIPKGRHDFWDNQLEI
DHGQTSVPEKEKDLVEVPQEAERVSDKSVGTESSRSDIAL
9634.m01240 motif-positive DGVAE
RCVKKNGLPAVNINTNPTAAAAMYAVVPDSQIRDATVER
9634.m01323 motif-positive FLKEIAGEKPIRFTAQQLAGFTNN
RRRRASASVAAPARSPES STETLRANG SLNS SVSL SVASD
WDHHPPPAKRAAAFWAWRGGANNGSHSPPPVSVSGIPKY
9634.m01742 motif-positive HYKDLQKATNN
9634.m01764 motif-positive SIRKLIELTSRKNAGFLPELVKGPRKF SYKEL SAATRG
RKKKTLEKQH SKTWMPF SINGLTSL STGSRT SYGTTLT SGL
9634.m02243 motif-positive NGSYGYRFAFSVLQEATNN
9634.m02283 motif-positive PRKWLRNRKPKKLGSFIKKSHPLVSYEELNQVTSS
RRKKKMD GLVYHYD GNNYFVP SSQFGGS SRNHHPPP SAI
MLNS GGASAD GGGYYNS GTF SGGEGTGPAGSK SRF SYEE
9634.m02784 motif-positive LTGITSN

RRRRRQAERYYPGFAVP SYTPQHMS GEAPFLRPP SAS GSM
NFSAGQSQGVSPMMSSGQAYGQSTSYGQQQRLTSANYST
9634.m02811 motif-positive GSQGGGAARSVAASGELSVGNTKAFTFDELYDITAG
KRKRHPPP SQDDAGS SEDD GFLQTI SGAPVRFTYRELQDA
9634.m02861 motif-positive TSN
9634.m03396 motif-positive RMRTRRERVD GENIEHLP GMPRKF SFEELKVVTGD
RSRKRVPQNSRKSMQQEPHLRLFNGDMEKITYQDIVKAT
9634.m03685 motif-positive NG
9634.m03690 motif-positive RIYGMKEMQANPHCQQINDHVKNITYQDIVKATDR
RSRTLAGKKAMSMSVAGGDDF SHPWTFTPFQKLNFCVDN
9634.m03723 motif-positive ILECL
9634.m03838 motif-positive RMEGRQLTGVWPAESGYEMITSHFRRYTYKELQRATRK
RK SKD GGI SE SKDVASTFAVNIDRASNREIWDHTQQDAPV
SSSVLPPMGKMTPERVYSTNSSMSKKMKVSVTANPYTVA
9634.m04131 motif-positive SLQVATNS
RRT SSVHQNK SSNNGDIEAA SL SSVSEHLHDMIKGTILVM
9634.m04643 motif-positive VPQGKGGSNNLKFKD ILKATNN
RKVMSNGAVRD GGKGVEVSLFD SMSELYGD CSKDTILFM
9634.m04648 motif-positive SEAAGEAAKRLTFVDILKATNN











Table 2-2. Con inued

Protein ID Cateor Juxtamembrane Domain
RRVMSNGAVHD GGRGVGASLFD SMS SELYNDND SSKDTI
9634.m04650 motif-ostive FFMSEVAGEAAKAVTFVDVLKATNN
RKLMSNAAVRD GGKGVDVSLFD SMSELYGD CSKDTILFM
9634.m04652 motif-poitive SEAAGETAKSLTFLDILKATNN
RRVVSNGAVRD GGKCVESTLFD SMSEMYGD SSKDTILFM
9634.m04653 motif-postiv SEAAGEAASGVTFVDILKATNN
RRRSATQRWQNHHAAAF GYQGNTTAYYYHHTGGARPQ
WAATKTGAPSTPPNMMMHPTNMTGPHVVVRPPLVPPPPP
9635.m00383 motif-ostive PVPAGLDENAFGYDELAAATGG
RCVKKNGLPAVNINTNPTAAAAMYAVVPDSQIRDATVER
9635.m00413 motif-poitive FLKEIAGEKPIRFTAQQLAGFTNN
RWKKKTS ANKTKDNPP GWRPLVLHGATTPAANSRSPTLR
9635.m00473 motif-poitive AAGTFGSNRMGRQFTVAEIREATMN
9635.m00841 motif-positive NKSKWCGVPLYGSQ GND GGIIAFRYTGLVRATKC
RRRRRS TTTAGDVSDDESVA SLPPLPREGLYIFTK SELKQA
9635.m01226 motif-poitive TNG
9635.m02031 motif-ostive RKARRCKKVRVLDETGDFITGCPTYPFEIIRAATNG

KYCRQFVIRFLKPFMRDEKLMDPRGKSEGTSKRRKARKK
D GLINSTQIF SASDKEGNGTGGSTEAQ SNKAHD STNVELP
9635.m02803 motif-poitive NGLNGRQIGKL
RRRRS GEVNCTAF S SSLGGGGNNTNGRQLVVKGSARREF
9635.m03088 motif-poitive RWEAIMEATAN
KQLPFCSNANTITHMPEGTYKTNLLQLAKNLITNVNQTQL
HSANGTAGAAGPDTVYGAVLCRGDSSAESCATRLQRVLD
TASINGT SGDD SGYFQNQKNVTLYDHDFQALL SF SDKDFI
SSFSNAPECTVSAYLNPPPDADRAQFSQLFSELMEKIAAAV
VSRRPVNYLTGRGWFDLKSQTVYALAQCTDGMPPENCRS
CLD GIIDEGKKMVGGGLTGGAVLGMRCSLWYQTDVKFF
AGDPEVSLHMPTQQARFELRLL SMAVQNVINLWRIEEGNS
9635.m03453 motif-poitive GFSLYDFSQIKEATQN
9635.m03481 motif-postiv KRKTERARKPSIADPTDPADIESIDSLILSISTLRVATNN


KAWHFCGSSGDVFAPRSTYQSNLALLSAGLAKNASASPA
LFAAGGVGDPPDTVYGLALCRGDTTNATACGACVAAAF
QDGQQLCAYAREATVFYDPCYLRFSGRNFLAADGDNFAA
YF SKVRNVTAPAEVFDAAVVALLNATADHAAAS SPRRFA
TGVEAFRGWGVRDIYALVQCTPDMSPAGCRS CLAGII SWV
NDPDYF SGSPTGRVL GVRCNYWYDVHPFFPG SPLLRLDAP
AFDVSPPAP SPAPVAADTTPPADRAVDRDLKFF SYTF SVS Q
CSVFICLKRRKASKNQNTPIIPAPNKIKRGNCAIFDLPTLQ
ATDN


9635.m03483


motif-positive











Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain
YPWGLCNDTAGEFPARRS SYLASINLIAATLPGNASASPDL
FATAEGVGAPPDQVSALALCRGDANASTCLACLTQAFLD
LPNACAYHKVAAIFYD SCLLAY SNATIAAGDF SSEKIPIYG
FYSNANATTEQARFNRLVAALVNATADYAARNSTRRRYA
S GEADFNAEFPKVYSWAQ CTPDLTPAS CRS CLAQIIGTYIG
FFENRVGGFVRAVWC SFQYSTTPFLD GPMLVRLQGT SGAS
PAP SPAAVVPAVNQTPPAATPTLEGDANYS TEAEDIENLD S
9635.m03495 motif-positive MLIDISILRSATGD
QPWEICGENGNYTANSTYQANLKQLAAALHKNVSSGTGG
GRLFAS GAVGAVPDAVYALAL CRGDINAS ACAD CVGTIF
QDAQQL CPYRKEVSIVYD SCYLRF SNLDFL SSADNS GVVD
LYNTGTVSGDVGRYDRAVTGLLNATARYAAGNTNASSRL
FATGVMVGFDAQFPKIYAMAQCSPDLSPAQCGLCLGAMV
ARWWQTFEPNTQGARSVGARCNMRVELYSFYNVP SMLQ
LQAEAVAP SP SPAPAPAGKPPAVPGTTGAASRSEDFES IES
9635.m03516 motif-positive LFLDLSTLRIATDN
9635.m03524 motif-positive RRRTPARKASPVPYSTNPDDIQSIDSLLLDLSTLRAATDN
9635.m03526 motif-positive RRKRTPANKAS SLPF STNPDDIQ SID SLLLDL STLRAATDN
DAQPMPWHRCNTSSGNYTANSTYQSNIQYLATSLPAYASS
SP SLFAS GS SGAPPDAIYALAL CRGDTTNAS SCATCVAAAI
QAAQKHCALVKTVAIYDDP CIVRF SNLVFPVSPPYNKGMF
VAWDDNNVSAAAAAAFD AAFARLANATAEHAAAD SVR
RFATGEEAALAVAGEVYPKIYSLAQCTPDMSADACRSCLE
DILVRMVPTYLAGRKGGRVLGVRCNFRFETYPFFFGQPLL
QLPGSPASSSAPVNGVPTESSTDDMQSIGSLILDLSTLRVA
9635.m03530 motif-positive DD
9635.m03531 motif-positive RKKRRRGKAEHFTGPDAAEDFESVK STILL SLASLQVATDN

CGTSGGNYTAGSTYESNLLRLASTLRANASASPTLFASGV
RGVGPDAVYGLLLCRGDMNPSDCFDCGTNVWRDAGPTC
NRTKDAILVYNQCYAQF SDRGDFLAATNNS GEVSLLIS GT
NITSTDVAGYDRAVTELLNATVRYAVENSTKLFATGQRV
GNDTGF SNPIYSMAQC SPDL SPAQCRS CLD GLVGQWWKTF
PLNGKGARVAGPRCYLRSELGPFYTGNPMVRLPVKADEL
9635.m03539 motif-positive TQRRLAKAERHPGTDTNEDFES VKSTLL SLASLQVATDN
DKLFRNKVANPVRFQ SPQRFT SFD SSIPLNQVQDRKMEDE
9635.m03613 motif-positive TRH SNELNVTLFDFNTIAF STDN
KCRRRIKEKL GIGRKKAQLPLLRPARDAKQDF SGPAQ SEH
9635.m03616 motif-positive EKSEEGKNCELPLFAFETLATATDN
KRGRNIKDVMHKSWRSMHT STRSQQNS GMLD ISQ SIPFED
9635.m03618 motif-positive DTED GKSHELKVY SFDRIKAATCN
RARFPVDQDPDPESL SCENPKCGGGGGGGGKCGAFHMSA
TSSPPSGCSSSCVTGCSSSSEGVKVFRLDKTAFTYRDIVAA
9635.m04060 motif-positive TSG


KKKS GGKKVRPRGGGGGGLRRL SFTDLTGAADQDL SVSL
VGSNLHVFTVAELRDATRG


9635.m04226


motif-positive











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain

KKKYWSLPRGGDPEQKEPL SPIVS GFKD SLKQMKSIKII STI
GKEELQKTVSMNLKPPTRIDLHKSIDEND VTSKSFTRKISF S
9635.m04270 motif-ostive SIRTPAYTVADLQVATGS
RRRHQRKKMREAEEANDDDDDTEGDPIMEIENGMGPRRF
9636.m00204 motif-poitive AYHVLVNATKS
RRRHQRKKMREAEEANDDDDDTEGDPIMEIENGTGPRRF
9636.m00205 motif-positive AYHVLVNATKS
RRRQRKKMREEEEDD SEGDPIVEIEMGTGPRRFPYHILVN
9636.m00208 motif-poitive ATKS
RRRRIKNRKEAEDEQDIS SD SEDND GEPIVEIEMGTAPRRL
9636.m00210 motif-ostive PYYELVEATKN
RRRRSKKRREVEEAEEARHVGLARDDDDDDD GEPIVEIE
9636.m00214 motif-poitive MGMGPRQIPYQDLIEATN S
RRRRIKNRKEAEDEQDIS SD SLDDD GEPIVEIEMGTGPRRF
9636.m00216 motif-poitive PYYELVEATKS
RRRQ SKKRREAQD GSWH GSDDDDD GELIMEIEMGTGPRR
9636.m00218 motif-ostive FPYHKLVDATKS
RRRQ SKKRREAED GGWHGSDDDDD GEPIVEIEMGMGPRR
9636.m00223 motif-ostive FPYHELVDATKS
RRRQ SKKRREAED GGWHGSDDDDD GEPIVEIEMGMGPRR
9636.m00224 motif-poitive FPYHELVDATKS
RRRQRKKMREEEEDD SEGDPIVEIEMGTGPRRFPYHILVN
9636.m00225 motif-poitive ATKS
RRRRIKKRREAEDEENASTDSDNGEPITEIEVGTGPRRLPY
9636.m00229 motif-poitive YELVEATKN
RRRRSKKRREAEEAEEARHVGLAGDDDDDDD GEPIVEIE
9636.m00233 motif-ostive MGMGPRQIPYHELVEATKS
RYRRSQAQIRS S SSRRASTIPIRANGVNACTIL SNSTTGQES
PREVEDRGASMWLEGPGRKSVI SAS GIPKYAYKELQKATS
9636.m00365 motif-ostive N
RWLQWRGGDDTAAADALGPGGARHPDLVVGPWRMTAF
9636.m00457 motif-ostive QRLSFTADDVARCV
RGKQRGDEIYGGLMLGD ISTSREL SDRKVDFPIF SFREIASA
9636.m00764 motif-positie TNN
9636.m00975 motif-postiv TQKRKRLEVEMEELL SIVGTPNVF SYGEIKSATDN


9636.m00986


motif-positive


RQKRKKMEAEMGELLSVVGRPDVF SYGEIKSATNN











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
RNCKISDNLRTVNF SKLGRLTLLDL SFNNITGEVPQ SILNLN
NLGYLFL GNN SLTGSLPDAKS SSLTNLDF SYNQLTGSFP SW
VTN\NNLQLNLVANKFNIRENNNSILP SGLNCLQQDTPCLL
GSPEYYSFAVD CGSNKSMKGSDNTIYEVDAANLGVASYY
VTRNTRWGVSNVGIFNDAS SRNYVINS SQQFQNTLD SELF
QTARMSPSSLRYYGLGLENGNYSVKLQFAEFAYPDSKTW
ESTGRRIFDIYVQGVLKEKNFDIRKAVGGKSFTAVNKIYNT
IVSKNFLEIHLFWAGKGTCCIPTQGYYGPMISALSVTPNFT
9636.m00990 motif-ostive PTVRNGEPKKKSKAELYNLAGRPNVF SNAELKLATEN
9636.m00993a motif-ostive KKRRRLAQQQGELYNLVGRPDVF SNAELKLATNN
9636.m01372 motif-ostive KRRPFLRYTRAPQHHETEFDEESIGIRPYSFHDLELSTDG
9636.m01496 motif-postiv KTRMKPNIVDNENPFLYETNERI SYAELQAATE S
9636.m01743 motif-postiv RSRRARRRSPTLPF SPPPAPARPLRRY SRRALRRATGG
KKTRQALD SKDKKL SSTTKGHMLLPMF GKLNSMKTSKKE
9636.m01856 motif-ostive VVAMMDFSVLDSATGK
9636.m02427 motif-ostive KKHKCLLPWQRSTTAPRLH SLLRSQLKSYTYSEVRKMTKS
KFHKNKTKEIQAGCSE SLPGS SKS SWKL SGIGEPL SINMAIF
9636.m02489 motif-poitive ENPLRKLTFSDLHQATNG
RRRRRHRCLL SGPS SVLGILEKGRDVED GGGGEVMARL G
9636.m03449 motif-poitive NVRQFGLRELHAATDG
9636.m03477 motif-positive KPKINSDL GILFQGS SSKLNEAVEVTEN
9636.m03886 motif-ostive RSRRSD S SGAIHGAGEAWEVTLYQKLDF SVDEVV
KRRRKVS GGDD GS GITGTMIRSLAGGPREFEYRELRKATN
9636.m04075 motif-ostive N
TEAAGD GCS AGCDLALASFYVTPNQNVTNMADLFGIGAA
NYRSLAPYNPNIPNLDFINVGGRVNVYFTCGCRSLPGSPGA
TYLAGAFPFQMSRGQIYTSVAANYNNLTTAEWLQATNSY
PANNIPDTAVINATVNC SCGDASI SPDYGLFLTYPLRAEDT
LASVAATYGL SSQLDVVRRYNPGME SATGS GIVYIPVKDP
NGSYLPLKSPGRRKAKQATLLQ SSED STQLGTI SMDKVTP
9636.m04332 motif-ostive STIVGP SPVAGITVDK SVEF SYEEL SNATQG
RKKRQREEAAKQRMGVVFKKPEPDESPD GIGRSL SCCLRK
9637.m00068 motif-poitive KAGDESD STEEVTDTSASKEGVVAAKAKT
9637.m00536 motif-postiv KKWGSGFKKGLGAKAAVGKPRQYTYQHLFSATKG
RRKTKGSANNTINPHNEPTSHSHGSGSYGHGSMQFENRRF
9637.m00673 motif-ostive TYKDLQMITNN
KLRMNQKTEEVRTGYVESLPTSGTSSWKLSGVREPLSINV
9637.m00942 motif-ostive ATFEKPLRKLTFAHLLEATNG
RRWRRRRRRQQAQPLPLPPPMLYNPNPYYKGDQPPLPFVF
MQQQHHHPTAPQT SGGTF SDAGSERPH SI SID GGSL SYDQL
9637.m01166 motif-ostive AAATGG


KCRRKRMAL STKHQHIKAAATHE SEPNELRDAEAGALEP
VAS SAGPNHGKEYGGDPAAAAGPRQYEYGERVVSD GPR
HGAAYNELVAAGPRLYEYGELAAATRD


9637.m01453


motif-positive











Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain
PFVS SVNLRTL GS GVYHPVLAANQ SMCLFDRRNMG SNVSI
LRYPDDPYDRYWWKMRSDPTWKNLSTASTIEQNDNFVVP
LPVMQTAIEASNNDTIIKVTRKDKTAHKCMIFAYLADFQN
SQLRQFNITL SDTKPLLYSPPYL SAGIVDI SDWDMPNNGM
YTITLEPTSASKLPPMLNAFEIYTLIP SDNPMTFPRD SWD GV
KC SNP SDNTSRIISLDL SNSNLH GPI SNNFTLFTALEHLNLA
GNQLNGPIPD SLCRKNNTGTFLL SFD SDRDTCNKS IP GINP S
PPKSKLVFVGIVSADVPH SEPELEIAPASRKYHED GLQRVE
9637.m01534 motif-positive NRRFTYKELEKITNK
RQKRKPKIL SYNLYFLEPVS TDDPPREPELDIAPASRNNHG
9637.m01540 motif-positive GTLLKVENRQFTYKELEKFTNN
RAKRKLNT S STDLAMVPELMGAPGHITNHWDHLQKPENR
9637.m01545 motif-positive RFTYQELEKFTEN
RIVHDNPTTFSQDFDAIMAIKYEYGIKKNWMGDPCFPHEY
VWD GVKCSD AVIFPTANCMGRYPIL SHCLLP SST S SSTDLA
9637.m01547 motif-positive MVPELRGAPGHITNHWDHLQEPENRRFTYQELEKFTDN
GTPFVNTVELRQLD SMLHFRKIMGNSSPIYLYERRNMGPS S
RDNPIIRYPND TYDRFWYPWGSEDDPTY SNL SAP STLIIPP S
P SYAVP SPVLETAVVPADNNK SVL SIIQTNDKEIHEYLVLV
HYADFQSTLQRQFQAYSNGDPIQGTGGPYVADYTGQTVG
TIDWI SAETS GKYNITLAATD SSQLPPIVNAFEVYGRIPLDN
PSTFPTDYTCKII SLAYNKLNRWIKELRLIKVPHKVDAIMTI
KFEYGIKKNWMNDP CFP SNLVWNGVRCSTGSDNTMRII SL
DL SNSNLHGSI SNNFTLLTALEYLNL SGNQL SGTIP SSL CEN
NAGSFVFRFSYLFNVDIGDNFVHLDSTYGPEFLNAPGSTK
9637.m01568 motif-positive NHWDHMQKTENRRFTYEELEKYTDN
RAKRKHNNDPPTVLELTGAPGHKTNHWDRLQKPENRRFT
9637.m01571 motif-positive FEELQKFTDN
RAKGKHNVSTFDPPRVPDPKKAPGSTTDHW SHLPINGSRQ
9637.m01581 motif-positive FTYEELKNFTLN
9637.m01604 motif-positive KKQAIVKSRGQEQYGDHIHIPENREFTYEELVKITNN
RVKRK SNIFAYNPPRVPEPTNASRNEKYHWDHLQENENR
9637.m01629 motif-positive QFTYKELEKITDN
9637.m01633 motif-positive RAKGKSNISIPGSEKYHWDRLQKNENRHFTYDELKKLTDN
KAKRKPNTSAYNPPRVPEPMNAPVSEKYHWDHLEKNENR
9637.m01645 motif-positive QFTYEELEKFTNN
RKIRERLAAVREEFRRGRRRGGGGS SPVMKYKFPRITYRE
9637.m02208 motif-positive LVEATEE


RNKMSKNGRKKKGKS STMKVYL GRQKSP SRD TGYNADA
DDD GGGDDDDIVIPGMPARF SYQEITTMLT SN


9637.m02488


motif-positive











Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain
CQTRCGDVDIPFPFGI GDHCAIHEGFRLECDNATKGTSNQK
PFWGDFEVIKI SMED GKVWVKAYMSRQCYDQ STGGMSY
SDASANL SGS SFWL SD TDNKITVIGCKTLAYMTTD SYVIG
CS SACDNKVNKLTPKNGS C SGAGCCQANVPKSIQYYQGY
FNEGYNTTKIWMSSPCSYMAVMETAAFNFSTSYLTSSVFY
DTYKGGVPVVYDWAITSKTCTESRRNKTSYACI SNNSQCI
DNLTNAQGYRCKC SNGYEGNPYIKD GCKDIDECLNNATY
PCKGICTNTL GNFTCS CSP GSYMMNGD CMPKKKLRFD SVP
9637.m02629 motif-positive VVVELQEATNR
RMRRGGGAGGGGRVVHGEGAWEVTLYQKLDISMDDVL
9637.m02693 motif-positive RGLTSAN
AIPYPLPLPRGGGDTYDAVAANYADLTTAAWLEATNAYP
PGRIPGGD GRVNVTINCS CGDERVSPRYGLFLTYPLWD GE
TLESVAAQYGF SSPAEMELIRRYNPGMGGVS GKGIVFIPV
KDPNGSYHPLKSGVGIVLLFCELLCIYAKVAKVQEGHIASI
9637.m02882 motif-positive SRRNQPPCCYYLCDDASQAEGIKVERSIEF SYEEIFNATQG
FVSLVHQVAVDTEHDDSIGVTDESGWPCIELDRRMRRMN
GRNPEKNIKMEHEND SSLAWVFHAPYREHENEFPDIPEEK
9637.m02917 motif-positive EGNGFAPKSDDPTKAPPPIEVPEL SFDELKEKTDN
EKIMTGKLTDFTSAYAYKCRLFFIRILSAWPAGSIDCCEEM
INRCFCCVTGGD SDPEPAATS SRRRTNPARASKNRTSVDY
9637.m03043 motif-positive PWETYTLKELLQATGNF SESN
PKFKEEMIVKHD GKNNKKRALRVL SVSDEFGKEIPAQDLD
9637.m03212 motif-positive FPFVEYNEIATATENF SDAAMN
KIKGKKRNREKHRKLIFD GANT SEEIGQGNPVQDLELPFV
9637.m03219a motif-positive RFEDIALATHN
KIKGRYIQ CSFPIYLDAYGKKRKREKHRKLFLD GACTSEEIE
9637.m03220 motif-positive DGSPIQDLELPYVRFEEIALATHN
KACKKRNREKHRKQILFGMSAAEEVGEGNPVQDLEFPFV
9637.m03224 motif-positive TFEDIALATNN
RRNH SSPVS SHYYTDES GRRNS SAVNMKSLEH SP SMGCKT
PPAVPRKSMSDNEFENKLNH SRRSTDPI SLMNH S SSDLQA
9637.m03295 motif-positive ATGN
9638.m00012 motif-positive RKYS QYREMARTL GLEYLPAGGPRRF SYAELKAATKE
KITKKKKRPPPPNMPFFTDEKGNVYYATGGLPPMWQQHG
S SNYSIPPPPPPGWHMS SSAGGFS GEMGMGYS SGPYGPAL
9638.m00059 motif-positive PPPSPNVALGF SKS SFSYEELAAATSG
9638.m00828 motif-positive KQHLKLKKFYEQNGGPVLKGVRNIKIYTKKELKQIT SN
RKLQAKQCSPLAATAQQLIVKQAASKQIKQQSTYKSKQA
MLAVAD GDGAALGEDVGEVGVLDECAVAVVEGEAVVA
ALSVARGPGDDVGTRSALVLRHLMVAPRKSSATASWSST
ARAMRPPPRPTAA SP SHRLGPAAASPDRRHLTPADTADQD
9638.m01519 motif-positive VEAGSLLFDLATLRKATAN
9638.m01657 motif-positive RKQKWF SRGVENAQEGIGIRAFRYTDLQCATKN
9638.m01661 motif-positive RRKGKLFARGAENDQGSIGITAFRYIDLQRATKN


RKKLRP SSMRTTVAPLPD GMYPRVSYYELFQ STNGFNVN
N


motif-positive


9638.m01937











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain

KRKRKGTRQEHVEQRQPFNSYP SNEVKDVKPIPE STKIEVE
PLP SPVAVSLKPPPKIERNQ SFDDDDDDF SNKPVAKKSNSA
9638.m02114 motif-ostive SVKATVYSVADLQMATDS
HHSKKSRVVRLGGGDIKDKAAEQAGKKVSSGSGNGSRST
9638.m03092 motif-poitive TES GKGAADQLQFFRPEKATF SLDELF

RRKPKKSFDKIPVSQIPD VSKEIAVDEVREHAVVENFRVQE
SHAISVQEKHYEKDSGKMLAHLVRSKSSDADNLSQCSSV
YQCDRAGSSYSGDEGSSGNARRHFSQYATVSASPLVGLPE
9638.m03136 motif-ostive FSHLGWGHWFTLRDLEHATNR
KAYKNKRRREQQQHDDEEEILVSDSAAIVSPGSTAITGKL
9638.m03492 motif-poitive VLFRKNSSASRYEDWEAGTKAVLDRN
RRCRRRRC SRLAPAPPHHGRSNRSLKQQQ SMVSDKD IEEA
ARWPPPP SFQPPIEVIKAEQTAPLIMVEAARTS GETAT S SGG
9638.m03505 motif-poitive STRGW STES GGSDAAEPEA SRRGWGRRYTRRELEEATNR
RRRQRRATLPVPEEEEKES VGTPW SPFTPD GEG SFGSAVV
9638.m03516 motif-ostive TPRRMNMKLHIPLAEIMVATGD
KRSRRTFKEIPITQIP SASKDIKEVRAVDEFLPNDFVVHD GL
LLAIQNEPVEPVDKDVrNQFAQEDKTIQGEENSPVPLHYVD
NYDVIQ SVS TCEQ S SSHAPVD SVLLPGLPEF SYLGWGHWF
9639.m00075 motif-ostive TLRDLELATNC
RHRHRHRVVVKPRCRCLQPL SSAATLPVTAP SSRS CECVS
9639.m00613 motif-poitive SW SFYGGGGDAGDRSLKML SLDDLAGATGG
9639.m00642 motif-postiv REKQKRKSVSLP SFD SSFPKVSYHDLARATD G
9639.m00647 motif-postiv RGKQKKKCTSLTPFD SKFPKVSYNDLAKATEG
9639.m00648 motif-ostive RGKRKRE SL SLP SFGTNFPNF SYNNLFKATEG
9639.m00651 motif-ostive RAKLKRESVSLPFFGSNFPRI SYNALFKATEG
9639.m00653 motif-ostive GRGKRKKK SI SFP SL GRKFPKVSFNDL SNATDR
9639.m00659 motif-postiv RGKQKGHSISLPLSDTDFPKVSYNDLARATER
ANNLRGEVP SNLGNSLPNLQYLIL SDNFFHGHFP SSLINS SK
LNLIDMAENNFTGVIP S SIGKLAKLNVL SLQLNQFQAGTK
KEWEFMD SLANCTELEVF SVARNHLQGQ SFPPI SYFGDIPN
TL SNCESLEDIRLDRNAFTGIIPTSLGNIRSLKVLNL SHNKL
TGSIPVSLGNLQLLEQLDL SFNHLKGKVPTNGVFMNETAI
QID GKSWALWRRKHEGNST SLP SFGRKFPKVPYNELAEAT
9639.m00662 motif-ostive EG
9639.m00663 motif-ostive KGKQRTNSISLP SFGREFPKVSYKDLARATNG
9639.m00664 motif-postiv NRKQNRQ SISSPSFGRKFPKVSYSDLVRATEG
9639.m00665 motif-postiv KRKHKRQ SISSPSFGRKFPKVSYHDLVRATEG


GGAVGLEERSDAAEDSLVSVLAVYDGFCNLKQINLELKV
CGGS SIRKTLVKEAASYGAAHLIL GVAKNSL SF SRS SSI SV
AKYCAKRVPTGCSVLAVNNGKILFHKDAVQQEPYHSAST
MTETPRRSYRKLLT SVIGEKLRDECEQDNRSIFRAVTMPP S
SPAPTREVSLALVPMKVHRRE SPEVATGW SFLRKKFLPDR
KPASHDRSKMSVVQWAMRLP SRYS SASPVC SEYRTTTPD
GITSASRILRDRVAVPSRSNSGKSSVVIEELDNSSDKEIPEE
IALREKFPSVYSTFSHSELAKITSD


9639.m00844


motif-positive










Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
9639.m00968 motif-ostive RIKFNCCGVPLHHNQGNSGIIAFKYTDL SHATKN
9639.m00970 motif-ostive QNKLKCCGMPLHHTQ GNS GIVAFRYTDL SHATKI
9639.m00973 motif-postiv EKKHKKAKAYGDMADVIGPQLLTYHDLVLATEN
RRRRRRGRGGQAD QAAAMSLMLPRHGS SKGPGSVVEHF
9639.m01092 motif-poitive ALEALQAATDG
LYSDMDIDLASYFKITGSPFDTFSSSKIKMHVTTILKSGEF
RQNNKSEIKSQLT SKPTLQ SSQGITDNFP SEQDFEHGAFKG
PYVGF SDF GQ SEMKGES STQNKAGNKSQYPITIPKNPTLD S
9639.m01134 motif-positie LKVITDN
RARS CRGGGGPD GGGAWRFTAFHKVDF GIAEVIE SMKD G
9639.m01209 motif-positie IN
RRNKRPKLQPQPRSP SYASWDIKS TSI STPHLQGARVFTFD
9639.m01301 motif-poitive ELKKITNS
D GS SSVANGVTIS SEMTP SGESFNS SYNS STTPVD ISLAAIE
9639.m01600 motif-ostive ACTDG
KLRDRRGD GCGGD GDDDEERKRGLFPFP CMRADD SSDD G
SDAGDDVKRNNTTTTTTASGGGGGGEEGQLVAIDKGFKM
9639.m01817 motif-ostive ELDELLR
HHRKKKNVEKFRPVSTKT SPAESEMMKIQVVGANGI SNGS
9639.m02341 motif-ostive SAFPTELYSHVSAANS SNI SELFESH GMQL SVEVLLKATNN
RYMSKKSKADETID STRS SQDNKVHGEVINRWS GLYKF SK
9639.m02342 motif-poitive GEIEKAIN
9639.m02532 motif-postiv TRQ SKKPLKPRRGDILGATELQGPTSFYYKDLKVATNN
9639.m03250 motif-ostive RKNIKTNIPSTTSMEGHPLISHSQLVRATDN
9639.m03256 motif-ostive HKKIQTEIP STT SMRGHPLVSYSQLVKATDE
9639.m03257 motif-ostive HKRRKKEVPAMT SIQGHPMITYKQLVKATD G
9639.m03259 motif-postiv HKRTKKGAP SRT SMKGHPLVSYSQLVKATDG
9639.m03260 motif-postiv KLLARYKKIKSKIP STTCMEGHPLISYSQLARATD S
9639.m03261 motif-postiv RKPMSKLP SATSMQ GYPLI SYQQIVRATDD
RRYKKGEVSLQGDMNMQTDEEALAWGREAC SSEFTSFKL
9639.m03532 motif-ostive SQVLDATNN
9639.m03591 motif-ostive QRMRHRPEIYVDVPGQHDHNLEFGQIKRF SLRELQIATNN
9639.m03592 motif-postiv QRMRHRPEIYVDVPGQHDHNLEFGQIKRF SLRELQIATNN
DVITLVKVIKSRVETVRQNKEDCELLAERADMILDLLRRV
QASKVIEDPDMWKPTEGLKSTL CRAGAIVKS CQEEW SYA
YRF CKGGRIARELRKVLKDLKFYILHLIGMITIINHDQNTR
YYYIPETDVVKPQDATASNAGKPVALEETGLKKFTL SELE
963 9.m04143 motif-ostive VATDN


DVASLVKEIKERVQTVSQNKED CELLAERAELILDLL GRL
QKSKVIEDPDMWKPTERLRSTLRRACEVIEFCRERSCTYRF
CKSDHTAKELRKQTPDVVQLQD GVQVPALGLPAQHFKYN
DRNDRGETLGI SGKAQLVTEP SSVNEPGLKRFAF SQLEVA
TDN


9639.m04147


motif-positive











Table 2-2. Con inued

Protein ID Category Juxtamembrane Domain
ICGSSKYTANSIYQSNLDSLLSSSFLVVSGDSSSGALFAKGS
RGAAPDTVYAVALCRGDANASACSGCVDAAYAAATARL
CPL SKDAAVFYDECALRF SDEDILNMDAF GRVNTSAAVG
VAPLVLMNITSEPML SGWNTNIQ GTKNFTQFFIKTMNYIV
AQALSTTKHYAAIRVDMDDADASNTVTLPRRLFCLAQCA
PDLVEDICYNCLQNFSDLATANFAGRQGGRILALRCNLRY
DTDKFFAGKTNADEDEALIWGLQGRSSEFTIYDFSQVLEA
9639.m04236 motif-positive TDN
9639.m04368 motif-positive KWRRRWFFDNNGGRLLEGMGITIFTEKELD S
GNESFRLP SLWFF SIDANNFTGPIPQGFAACQQLQVF SLIQN
LFEGALP SWLGKLTNLVKLNLGENHFD GGSIPDAL SNITM
LASLEL STCNLTGTIPADIGKL GKL SDLLIARNQLRGPIPAS
LGNLSALSRLDLSTNLLDGSVPSTVGSMNSLTYFVIFENSL
QGDLKFL SAL SNCRKL SVLEID SNYFTGNLPDYVGNL SSTL
QAFIARRNNI SGVLP STVWNLT SLKYLDL SDNQLH STI SESI
MDLEILQWLDL SENSLFGPIP SNIGVLKNVQRLFLGTNQF S
S SI SMGI SNMLTKLVKLDL SHNFL SGALPADIGYLKQMNIM
DLS SNHFTGILPD SIAQLQMIAYLNL SVNSFQNSIPD SFRVL
TSLETLDL SHNNI SGTIPEYLANFTVL SSLNL SFNNLHGQIP
ETVGAVACCLHVILKKKVKHQKMSVGMVDMASHQLL SY
9639.m04369 motif-positive HELARATND
9639.m04384 motif-positive RMEYEKRKLRDHFNKNGGQLLKNIGIKIFTKEEVGKITNN
9639.m04387 motif-positive RKKWKLKGCYDRNGGQMLEKTSVKIFTKQELDKITNN
9639.m04402 motif-positive KKMKNPDITASFGIADAICHRLVSYQEIVRATEN

KRSRRTFKEIPITQIP SASKDIKEVRAVEEFLPNDFVVHD GL
LLAIQNEPVEPVDKDVrNQFAQEDKTIQGEDNSSSVPLHYV
DNYD GIQ SVSTCEQ S SSHAPAD SVPLPGLPEF SYLGWGHW
9640.m00074 motif-positive FTLRDLELATNC
DDLYIYVrHLVFFRLAEQMPTADAETVTSESTAVIQQDSTM
VNVDAAGLHEQDELPHWMSQLFEKLDLEVDEDVVDEDIC
SIGNMTHEEEEADLERGIH S LKNHPIMKKLNKGIHPVVLA
LHVLLEEEEEEEVVQEEDMAKGLAELDEYL SRHTYHTIEE
9640.m00282 motif-positive ATAS
GCWLF SSKGLFRH SRVYAIDQEGYKLITSHFQRYTYADIK
9640.m00283 motif-positive KATAN
KVKDRKKQGRS STVAAGDENESRHGL CRCIWGHRGVD S
DTDTDDSSASENGGGGGKYGEGELVAIDRGFRVELDELLR
9640.m01285 motif-positive SS
9640.m01512 motif-positive KRKPREGGIRRSVSPGITSIDRVTLQNATEN
NQ SIGKTVVWTADRDVPVNGRGSRIELRD GNMVLLDFNS
RLVWS TGTTS GQVRSAKLLDTGNLVLLGHD GSRIWQ SFD
SPTD TLLPTQPIAANLKLVS GKYML SVDNNG SLALTYDTP
EGH SKYWPRNINATPF SGDQPQGLDML GCI SAGNHIRKFT
9640.m03313 motif-positive LKELVAATAK
9640.m03756 motif-positive HRYKNRDVVEPWELDYGPHRYSYAELRRATRG


RCRRSGGGGGRSGFDRLAAKRLLSEAASSSGVPVYSYHEV
ARATNS


9640.m03953


motif-positive











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
HRRRRD SKIRPVKLP S SSRDE SVEPDLEHGGGPRRF SYGEL
9640.m04040 motif-ostive AAATND
RRRRKRAGEARELEMDEGDFFDDEADDFEKGTGPKRFHY
9640.m04064 motif-poitive GELAIATDD
RFVLRRRRKHAGLTKEQEMEEGGIFDDETAMEDDFEKGT
9640.m04066 motif-ostive GPKRFRFGELAIATDD
HRRRKHAGLTMEQEMDEGDFFDDEAGDFEKGTGPKRFR
9640.m04068 motif-ostive YGELAIATDD
RRRQWRRNNAKLTVKMARKHLPKDARFFRGKPIEDELEL
9640.m04069 motif-poitive EAAGPRRFHYGELAAATAN

EQRSAL CLFHATSQ SCNQLKSIPVGHLLVD SHIFRD SGQRA
IHTLS SSFSSSPRVLCWVLAVLGYSYRLQASSLSAGELTDL
9640.m04089 motif-ostive QGKFFMETFFRKDTPIIRNVKIYSSKELRKATKN
KKRRRAEAEAEEAAGGKVFGKKG SWDLKSFRVLAFDEHE
9640.m04300 motif-ostive VIDGVRDEN
RRWVLRARQD GEHDGLPT SPAS S SSYDVT SFHKL SFD QHE
9640.m04302 motif-poitive IVEALIDKN
KSRQASNKKKKKKQGGSRSWFKLPMLSSQQASYASEEQQ
9640.m04368 motif-poitive GEEDD GD GDEVLIPGLPARFTYAELEEATEG
FKRKKSTEPTTAS SSKGKTVAGGRGENPKEEYS SGVQEAE
9629.m05927 motif-ostive RNKLVFFEGC SYNFDLEDLL
9634.m03594 motif-ostive KQLRGVWPAEAGYEMIANHFRRYTYRELVLAT
9629.m00 145 motif-like KRRIRRHQEMQEEEQEFEELPLQ GMPRRFTFQQLQEATDQ
HRRKQTLGFIIHHKYTGNE SNTEEELKRYQ SL SPKRYRYSD
9629.m00370 motif-like LKKITKC
9629.m00524 motif-like HRKLKGRQNSQEI SPVIEEQYQRISYYALSRGSNE
9629.m00695 motif-like RHRRNQQIFFDVrNDQYDPEVCLGHLKRYAFKELRAATNN
RYENYKLEQFH SKGDIES GDD SD SKWVLE SFHPPELDPEEI
9629.m06510 motif-like CN
9629.m07224 motif-like RRRSRYS SKRRSAKRIPMKID GVKDF SFQEL SHGTND
9630.m01244 motif-like RHQKRQQDLAGWKMTPFRTLHF SECDVL GN
KGRRKSHLREVFVDVAGEDDRRIAFGQLKRFAWRELQIA
9630.m01729 motif-like TDN
9630.m01864 motif-like RKRYNHGELREDWEVEFGPHRIPYKDLRRATER
QKRKLIRTKQRFFEQNGGVILQQQMH SGGGTGGFKIF STE
9630.m05595 motif-like ELKKATNN
KRKLIRTKQRFFEQNGGVILQQQMH SGGGAGGFKIF STEE
9630.m05599 motif-like LEKATNN
QKRKLIRTKQKFFEHNGGVILRQQMH SGGGTHGFRIF STE
9630.m05621 motif-like ELKRATHN
KQLYHRWYVHGGCCDDAAVEEEGSGSWPWRLTAFQRLS
9631.m00434 motif-like FTSAEVLAC


9631.m04841


motif-like


NGRRKSHLREVFVDVS GEDDRRIAF GQLKRFAWRELQLA
TDS











Table 2-2. Con inued

Protein ID Cateor Juxtamembrane Domain
9632.m00264 motif-like QKRVRNTFGKGTGGARRFEYDDLAIATGN
9632.m00511 motif-like KRIRHTFGKGTGGTRRFEYDDLAIATDN
RK SRGNQP SKKVQ SKYPFQHMND SNEVGSENVEL SSVDL
9632.m01387 motif-like DSVLTATNN
RHKRSIKRQALLRQNDEFFQQHGGQLLLEMMKVEGNAGF
9632.m02841 motif-like TLYGRQEIETATNN
9632.m03289 motif-like RRHRKKLHCQALNSIYAGTGVIPFRYSDLQRATKN
9632.m03297 motif-like KWRNKTKL SGGTRKDYQFCNGIIPFGYIDLQRATNN
9632.m03298 motif-like KIPRNKSWLL GHRRKNFH SGS GVIAFRYADLQHATKN
9632.m03302 motif-like WRRKGKWFTRTLQKPEGGIGVVAFRYINLQRATKA
9632.m03306 motif-like WRRKGKWFTLTLEKPEVGVGIIAFRYIDLQRATKN
9632.m04086 motif-like RRNKRNCS SVGRIICGTVAFRYKDLQHATKN
RRKRKADEKEAPPGWHPLVLHEAMKS TTDARAAGK SPLT
9632.m05124 motif-like RNSSSIGHRMGRRFSISEIRAATKN
9632.m05259 motif-like WCRRKHKI SEGIPHNPATTVP SVDLQKVKAATGN
RRKNRELKNADLHAQNPENAFCQSQSWRCPEGQSPMFQR
9632.m05801 motif-like YSYKETMKATNN
9633.m01394 motif-like RRRKTNPDVLPEADPYKSRRFKYKELQVITN
TLWRRSRRSTGGKVTRS SDAAKGIKLVPILSRFNSVKMSR
9633.m02300 motif-like KRLVGMFEYPSLEAATEK
RRKKQGSMNNSVKRQNETMRYGPTNNGSGHNSSLRLEN
9633.m04198 motif-like RWFTYNELEKITNK
9634.m01190 motif-like RRKPHDQFFDLLEEETPEVrHLGQLRRFTLRELQVATDN
9634.m03596 motif-like RREGKLARGI SEVGYEMVTNHFRRYTYRELMIATRK
9634.m03679 motif-like RSRKGMKLKPQLLPFNQHLEQITYEDIVKATKS
9636.m00717 motif-like RRRKPQEHFFDVPAEEDPEVrHL GQLKRF SLRELQVATDT
9636.m04 116 motif-like RKKRPAD VTGATNPFENRRFKYKELKLIAD S
RQRKRRRKNSPPPANND SDQYS SD GQRQHGTADLERAVT
9637.m01434 motif-like GGGPRRYQFHELAAATRD
HKKRKQQMTL GLVHQY SVQPTGI SNSVSHVD IKGHVLMS
9637.m01597 motif-like DDHEFTYEELVKITNN
ERNKLHSIKQKYFRQHGGRLLFEEMKGTAFKIFTEEELQK
9637.m02621 motif-like ATNN
9638.m00203 motif-like HNKRKPQESTTAKGRDMFSVWNFDGRLAFEDIVRATED
RW SRRFRKDRVRLREKRSRRFRGDELICEMEGEI SEF SVFE
9638.m00382 motif-like FREVIKATDN
KRKKKS SDETVVIAAPAKKLGSFF SEVATESAHRFAL SEIE
9639.m00021 motif-like DATDK
9639.m00293 motif-like NKGVFRP SQVSVLEEGYRIVT SHFRAYRY SKLERGTKK
GCWLF SSKGLFRH SRVYAIDQEGYKLITTHFQRFTYVDIK
9639.m00297 motif-like KATAN
9639.m02693 motif-like RKKPKAQ SKKTIGFQLIDDKYPRVSYAELVQGTNG


9639.m03758


motif-like


KKRS GPERIGINH SFRRLDKI SYSDLYKATYG











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
KRKKKS SDETVVIAAPAKKLGSFF SEVATESAHRFAL SEIE
9640.m00020 motif-like DATDK
9640.m00279 motif-like KGVFRP SQVSVLEEGYRIVT SHFRAYRY SELERGTKK
KITN\KIKQRRAKKLRRKFFKKNHGLLLQQLI SSNKDIAER
9632.m05428 motif-negtive MKIFSLEELDQATNK
9629.m00 140 motif-egtve KTKNDDEIQLKVEMFLKTYGTSKPTRYTF SEVKRITRR
9629.mO00141 moti-natv KYRKTRISIDAVEKFLRMQQAHGPKRYAYTEITAITGH
9629.m00142 moti-natv KRRTRRRREIREEEQELEEITLQGMPRRFTFQQLQEATDQ
9629.m00 143 motif-negtive WKTRITIDAVEKFLRMQLML GPTRYTYTDIIAMTSH
9629.m00 144 motif-neatv KVEMFLRTYGTSKPTRYTF SEVKKIARC
9629.m00 147 motif-negtive KYWKTRIKID AVEKFLQMQLMLGPTRYAYTDIIAMTSH
9629.m00148 motif-egtve KQRYNEEVHLKVEMFLRTYGTSKPTRYTFSQVKKITRR
9629.m00151 motif-egtve RNKITIDAVEKFLQMQLTLGPTRYAYTDLTAITGH
9629.m00 152 motif-egtve KSRYNEEIHLKVEMFLKTYGTSKPTRYTF SEVKKIARR
9629.m00 154 motif-neaie SLKSRYDEEVHLKVEMFLRTYGTSKPTRY SF SDVKKITRR
9629.mO001 55 motif-negtive KYWKARITIDAVEKFLRMQEML SPMRYGYTDIIAIT SH
9629.m00 156 motif-negtive KTRYNEEIHLKVEMFLKTYGT SKPTRY SF SEVKKITRR
9629.m00 168 motif-egtve SLKSRYNKEIHLKVEMFLKTYGTSKPMRYTF SDVKKITRR
AHKYWKTRLAIDAVEKFLQMQQVL GPTRYAYTDLTAVT S
9629.m00169 motif-negative H
9629.m00170 motif-egtve KIRITTDAVEKFLRMQLMNGPTRYAYTDLIAITGH
9629.m00171 motif-neaie WKTRVTIDAVEKFLRMQQMLGPTRYAYTDITAITGH
9629.m00 172 motif-negtive KSRYNEEIHLKVEMFLKTYGTSKPTRYTF SEVKKIARR
9629.m00173 motif-negtive EKFLRMQQMLGPTRYAYTDIIAITGH
9629.m00180 motif-neaie KTRIAIDAVEKFLRMQDMLGPKRYAYTDIIAITSH
YI SLKSRYDEEVHLKVEMFLRTYGTSKPTRYNF SDVKKIA
9629.m00181 motif-negtive RR
DTYGYHSICHRYHQALFWLMLAPLVVFIFLAHKYWKTRIT
9629.m00185 motif-egtve IDAVEKFLQMQQMIGPMRYAYTDIIAITSH
9629.m00 187 motif-egtve KTRYNEEIHMKVEMFLKTYGT SKPTRYTF SEVKKIARR
9629.m00188 moti-natv KYWKTRITIDAVEKFLRMQQMIGPMRFAYTDIIAIT SH
9629.m00 189 motif-negtive L SLRTRYNEEIHLKVEMFLKTYGT SKPTRYTF SEVKKIASR
9629.m00 190 motif-negtive KYWKTRIAID AVEKFLRMQEMLGPKRYAYTDIIAIT SH
9629.m00192 motif-negtive KYWKTRITIDAVEKFLRMQQMIGPTRFAYTDIIAITSH
9629.m00 193 motif-neaie L SLKTRYNEEIHLKVEMFLKTYGT SKPTRYTF SEVKKIARR
9629.m00195 motif-egtve KTRITIDAVEKFLRMQQMIGPTRFAYTDIIAITSH
9629.m00 196 motif-neaie L SLKTKYNEEIHLKVEMFLKTYGTSKPTRYTF SEVKKI SRR
RRKKQ GPMNNSLEQQNEMSTST SHVLIN SGYGDNVSLRL
9629.m00252 motif-negtive ENRRFTYKELEKITNK
HKRKKRKQTRDLKDLMHS S SSMQ SYSKDLELGGSPHIFTY
9629.m00358 motif-egtve EELEEATAG


RQRQKFRSAICGVYSGNTKNEEEMLKKCE SLALKRYKYS
ELKKITKS


9629.m00364


motif-negative











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
TCRQKEKGSLLTLQKYVANESKIEEALKEYD SLAPKRYNY
9629.m00366 motif-egtve SELKKITRS
HRKGQILCYLLCNKTRSRNESNIQKLIVSYGSLAPKRYKYS
9629.m00367 motif-negtive EVAKITS
HGKGKQLRYFLYTKTS ST SERNIEALII SYG SIAPTRYKYSE
9629.m00375 motif-egtve VTKITS
KCRRRMQNRF SFLNAMD GASRTDTAKVEKLLQ SYG SLAP
9629.m00376 motif-egtve RRFRYSELKKITKS
9629.m00490 moti-ngtv HQRKFKQRQNRQ ATSLVIEEQYQRVSYYALSRGSNE
9629.m00520 motif-negtive HNRKLKRRQNRQAT SLVIEEQYQRVSYYAL SRGSND
9629.m00522 motif-negtive RKLKRRQNSRATIPGTDEHYHRVSYYALARG SNE
9629.m00550 motif-negative ILMFIIRRNKDKNRSENYGSLVAFRYKDLRSATKN
9629.m01025 motif-egtve RKRCNRQRADE SDFADLPGTITRFTFKMLKAATND
9629.m01192 motif-egtve WRGKRKLFTEKPVNSD SRLMIF SNSQLKNATKG
9629.m01203 motif-egtve LIYKKRS SCVASQAKMEGFLAVY SYAQVKKAT
9629.m01204 motif-neaie MYKRSS CVARQTKMEGFLAVYSYAQVKKAT
9629.m01206 motif-neatv CRYRRDLFASSKFEVEGSLIVYTYAQIRKAT
9629.m01247 motif-negative MIRRK SKNKKED SSHTPGDDMNHLIVTYHELARATDK
9629.m01357 motif-egtve WFYWRYRSFNNSKL SADRSKW SLT SFHKL SF SEYELD C
9629.m01868 motif-egtve RRKKYKIHDAGDLQQENMF SVLNFD GGNAYEQIIEATEN
EGVAIGALIQLHTKGFLWAGNHLLL SNS SAGLRMD CQEN
9629.m01991 motif-negtive NLKSFLQTNGHVVLQRVDNNY SLRYFTKNEVWHITNG
RRKLTDVKKKYIQEHGGLLLFEKMK SDQGLAFKVFTQAE
9629.m02541 motif-negtive LEQATNK
RLMFERRKLTDVKKKYFQQHGGLILFDKMKSDQGLAFKV
9629.m02553 motif-egtve FTQAELEHATNK
RRWKRGIQKKIRRAYFRKNKGLVLERLI SSDESVAH STKIF
9629.m02555 motif-egtve SLEELERAPDH
RRGRKGEVNMQNNIAAVNRLEEDALVWRLEERS SEF SLF
9629.m02564 motif-negtive EFSELLEATDN
RRRRRRAAAAALAKAAAD SRSKRSQAME SI SASTTLVQFT
9629.m02756 motif-egtve YDEIKAATGG
9629.m03 184 motif-egtve KGRKQTDRKNAT SDLMLNEKYPRVSYHELFEATD G
WKKKIFRNKKRSKSFIDIYGD GVPVRIAQ SSLNFKYEELRK
9629.m03474 motif-negtive ATNY
9629.m03865 motif-negtive RSRRAKALRRLED S SSFLTVFTYRDLQLVTNN
9629.m04349 motif-negtive RKRNL SEANKVEG SLVVFRYRFLQHVTKN
KPYQALL SCIERRLRRKAAAAATAAEPLPPEKKEDAGRPL
9629.m04404 motif-egtve VQAERLRAAFRID GLLREYSH GEIQAMT
9629.m04617 motif-neaie SIRNDIQNSAEGGYMMIRNQFRGFTYQELKEAT
9629.m04791 motif-egtve KKSRYRRISKGTPRIESFLQRNGTLHPKRYTYTEVKRMT


9629.m04980


motif-negative


KNKKPERPRASSRTSSMAREETVRFDGCCVEFDVCTLM











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
KRRRLKNEEASTSRDIYID SRSH SATMNSDWRQNL SGTNL
9629.m05047 motif-egtve LSINLAAFEKPLQNLTLADLVEATNG
WRQKSKAKGVDTD SAIKLWESEETGSHFTSFCF SEIADAT
9629.m05632 motif-egtve CK
9629.m05849 motif-negtive CKGKPKEHDDYDMYEEENPLH SDTRRFTYTELRTIT
9629.m05851 motif-negtive KGKSRKSEEEDYDMYEEETPLHIDIRRFTYAELKIT
MRRRSKRRTT SRRSLL SRYS VKVD GVRCFTFDEMAAATN
9629.m05922 motif-negtive D
9629.m06444 motif-neatv FRRRMVKETTRVEGSLIAFTYRDLKSVTKN
9629.m06446 moti-natv RRRMVKATTRVEGSLISFTYRDLKSVTKN
9629.m06570 motif-neatv TARSRPAT SEQWAAEEGYRVVTDHFRRFTYGELRKATKN
9629.m06607 motif-neaie RRSRRLKALRRVEGSLTAFTYRDLQVATKS
9629.m06614 motif-negtive GRRRIS SMNHTD GSLITFKYSDLQILTKN
9629.m07414 motif-negtive KIKMRVDEVEKFLQLQQMLTPTRYSYTDIIAIT
9630.m00079 motif-negtive RRKQKPCLQQ SSVNMRKI SYEDIAKATD G
KRKLMKEKERFFQQNGGMLLYEQIRSKQVDTVRIFTKEEL
9630.m00113 motif-egtve ENATDN
TWRAW SRWQEDNARVAADDE SGSLE SAARSTLVLLFAN
9630.m00153 motif-egtve DDDNGNGDD GERTMTLDDVLKATGN
9630.m00498 motif-neaie RRMLKAKDKRRAAGPTYE SALLENREF SYRELKHITNN
RGKNFVTENRRCRND GTEETL SNIKSEQTLVML SQGKGEQ
9630.m00519 motif-negtive TKLTFTDLKATKN
ATVKGTDCITNNRSSENADVDATSHKSDSEQSLVIVSQNK
9630.m00520 motif-egtve GGKNKLTFADIVKATNN
SI SGMSFRTKNRC SNDYTEAL SSNIS SEHLLVMLQQGKEAE
9630.m00522 motif-egtve DKITFTGIMEATNN
ERSKRFITKNS SDND GDLEAASFN SD SEH SLIMITRGKGEEI
9630.m00523 motif-negtive NLTFADIVKATNN
RATKLMRKGELANNRNEETASFNPNSDH SLMVMPQ GKG
9630.m00524 motif-egtve DNNKLTFADIMKTTNN
9630.m00544 motif-egtve RRWKKDFDQLAKSMQSLPGVPVKISFADIRKATNN
RH SHRAHD SKNIRS SSDTARVALVPMLNKFNSMKTNKKG
9630.m00812 motif-negtive LVAMMEYNTLETATGK
RRRRKMLRRRQFVLVPAGDNAMADHETTL SNNLL GRRR
9630.m00926 motif-negtive MKKREPPSINLATFEHAPVRVTVDEIMRATGN
9630.m01077 motif-negative KRREEKPILTDISMDTKIISYKDIVQATKG
9630.m01086 motif-egtve MKRRKEEPNLQH SSVNLRKI SYED IAKATD G
9630.m01088 motif-egtve MKRRKEEPNQQH SSVNLRKI SYEDIAKATD G
9630.m01126 motif-egtve HKRAKKTNANRQT SLIKEQHMRVSYTELAEATKG
9630.m01128 motif-negtive RNKKAKPNPQISLI SEQYTRVSYAELVNATNG
9630.m01130 motif-negtive RLRTKLRRANPKIPL SDKQHMRVSYAQL SKATNS
9630.m01131 motif-negative RNKTQAKSDLALND SHLRVSYVELVNATNV
9630.m01182 motif-negatve RRKKHLQDHLSWKLTPFHVLHFTANDILSG


9630.m01185


motif-negative


RRKKLQDHLSWKLTPFHILHFTTTNESGGL










Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
9630.m01236 motif-negatve RRKKGPQDVTSWKMTQFRTIDFTEHDIVSN
9630.m01273 motif-egtve KRKGKSNEHIDHSYMELKKLTYSDVSKATNN
9630.m01310 motif-negtive KYCRWRLPFA SADQDLEIEL GHLKHF SFHELQ SATDN
9630.m01866 motif-negtive KRKFQRYVELREDWELEFGAHRL SYKDLLQATER
9630.m01874 motif-negtive KRKFQRYVELREDWELEFGAHRL SYKDLLQATER
9630.m02460 motif-egtve RSVRRKNQEHAVASEDMGEATLSMEVARAATKG
RRKMKSVEQ SENGAGNTKVF SVWNFD GGDVCKQ SFEAT
9630.m03311 motif-negative EN
9630.m03878 motif-egtve KKTPRRTYL SLL SFGKQFPRVSYKDIAQATGN
9630.m03880 moti-natv KRTSRRTDLLLLSFGKQFPRVSYKDLAQATGK
9630.m03884 motif-egtve KMKPREKYIS SQ SFGENFLKVSYNDLAQATRN
9630.m03885 motif-negtive KMKPREKYISSQSFGENFLKVSYNDLAQATRN
9630.m03886 motif-negtive KMKPREKYISSQSFGENFLKVSYNDLAQATRN
KRRLAKIKREHFRQHGGLLLFEEMKSRQGL SFALFTQEEL
9630.m03961 motif-egtve EQATNR
KRRLATVKRRYFNQHGGLLLFEEMKSNQGL SFTVFTKDE
9630.m03963 motif-egtve LEEATNK
KWKRGIQRRIRRAYFKKNQGLLLEQLIIDENTKDKTRIF SL
9630.m04029 motif-negtive EELEKATYN
KWRKGIQKRIRRAYFKKNQGLLLEQLI SNE SATNKTKIF SL
9630.m04033 motif-negative EELEEATNN
KWKKSIQKRIRRAYFKKNQGLLLEQLI SDE SATNKTRIF SL
9630.m04037 motif-egtve EELEEATNN
9630.m04056 moti-natv WYKNRLTKSP STMSMRAHQLVSYQQLVHATD G
9630.m04102 motif-negtive RRRRRYAELKEEWEVAFGPHRF SYKDL
LRPRLRGLRLDRLTSRLPACLRRSRTANTMLPYFAPIADRL
9630.m04231 motif-negtive GALQPYLAPIADRL
RKRRRRS GGDP SSAFNAAIDFRKIP GLPKEFDYMELRRGT
9630.m04698 motif-negtive NN
RKRRKRIGDDP S SVFNTTIDFRSIPGVPREFDYRELRRGTN
9630.m04699 motif-negtive N
9630.m04857 motif-negative RHRRNHQILFDVDEQHTENVNLGNVKRFQFRELQVATEN
RKIKNRRANMLRQMFFKQNRGHLLQQLVSQNTDIAERMII
9630.m05594 motif-egtve PLAELEKATNK
9630.m05879 moti-natv KRRKQKP SLQQ SSVNMRKI SYEDIANATD G
9631l.m01 172 motif-neaie RRRSAALRSQKSTKRLL SEAS CTVPFYTYREIDRATNG
9631.m01199 motif-neaie KARSLKRSAEARAWRLTAFQRLDFAVDDVLDC
9631.m01470 motif-neatv KRWFRHAELREDWKVEFGPQRF SYKDL
RYKLRHCQCSKNELRLAKNTTYSFRKDNMKIQPDVEDLKI
9631.m04208 motif-egtve RRAQEFSYEELEQATGG
RRKHIQEKQQYFKQNGGLRLFDEMVSRQVD TVRVLTEDE
9631.m04253 motif-negtive LKKATNN


RRKHTIEKQEYFRRNGGLRLYDEMVSRQVDTVRVLTVDE
LKKATDN


9631.m04258


motif-negative










Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
9631.m05490 motif-egtve RRRLKYAELREDWEVEFGPHRFTYKDLFRATEG
9631.m05492 motif-neaie FRRQRRAIYVELVEDWEVEFGPHRFAYKDLHKATKG
963 1.m05502 motif-negtive KARSLKKASEARVWKLTAFQRLDFTCDDVLD CL
963 1.m0599 1 motif-negtive RSRRYAEEEEEWEIEYGPHRI SYKDLHGATKG
WVFCRH SPKYGAA SAQYALLEYAS GAPVQF SYRELQRST
9631.m06141 motif-negtive KG
FKRWKRSTRKKIRRAYFRKNKGLLLEQLI SS SNNVTPNTRI
9631.m06166 motif-negtive FSLEDLEKATNN
9631.m06406 moti-natv SRWDLDALEIQAVEQGYKV MASNFRRYNYKELAKATRK
9632.m00228 moti-natv KSKYLRIDATKVGTAVDDSILKRKLYPLISYEELYHATEN
RRWRRHMQRKIRREYFQKNKGLLLEQLMS SDENVAHDP
9632.m00291 motif-negtive KIFSLEELEKATDN
9632.m00377 motif-neaie KWKRVLNFFHKGTAGARRFEYRDLATATKN
9632.m00403 motif-negtive KVFHKGTASARRFEYHELATATEN
9632.m00458 motif-egtve QKRIRNVFDKGTGGARRFEYRNLAAATDH
9632.m00467 motif-egtve WKWRKTNREFDKGTRGACRFNYHRLAAATNH
9632.m01129 motif-egtve KKNIPLSQAS SKSQLPLKTFTYKELEKATAG
9632.m01308 motif-negtive RKKRKLRAYFNRNGGQLLKSIKID IYTKEKLDQITKNY ST
9632.m01335 motif-negtive MRKKAKQQDRII SPDMEDVLNNRLI SYHDIVRATDN
KYKRRLVRQDLMNKRDAYFRQHGGQLLLDMMKLENQV
9632.m01893 motif-egtve SFKLYDREEIELATNN
RRWKRD IQRQLRRNYFRKNQGLLLEQLI SSDENASDKTKI
9632.m02006 motif-egtve FSLEELEKATNN
RRWKRD IQRQLRRNYFRKNQGLLLEQLI SSDENASDKTKI
9632.m02009 motif-negtive FSLEELEKATNN
9632.m02077 motif-negtive KKRRALAYQKEELYYLVGQPDVFNYAELKLATDN
9632.m02192 motif-negtive QKRRKYRERDEELDFDIMPGMPTRF SFQKLRKSTED
9632.m02 196 motif-egtve RRRRKYQETDEELDFDILPGMPLRL SLEKLRECTED
9632.m02204 motif-egtve RRRRKYQKLDEELDFDILPGMPMRF SFEKLRERTED
9632.m02208 motif-negatve QRRKYQEIDEEIDFEPLPGMPVRF SYEKLRECTKD
HRWKKDIQKQLRRKHFQKNQGLLLEQLI SSDENASENTKI
9632.m02260 motif-negtive FSLDELEKATNN
RRWKNDIQKQLRRKHFRKNQGLLLEQLISSDENASDKTKI
9632.m02267 motif-egtve FSLDELEKATNN
RRWKRDIQKQLRRKHFQKNQGLLLEQLILSDQNATDKTKI
9632.m02268 motif-egtve FSLEELEKATNN
RKRKNDIQKQLRKKYFRKNQGLLLEQLI SSDECATD STKIF
9632.m02809 motif-negtive TLEELKEATNN
HRRS IKRQRLIRQRDEYFQQHGGQLL SDMMKID CNLEFTL
9632.m02818 motif-negtive YRQEDIEVATND
KYKQRIKKQALLRQADEFFQQHGGQLLLEMMKVEGNAG
9632.m02835 motif-egtve FTLYERERIKIATNN


RHKRS IKKQALLRQTHEFFLQHGGQLLLEMMKVEGNVGF
TLYERGEIETATSN


motif-negative


9632.m02838











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
QHKRSIKRQALQRQTDMYFQQHGGQILLELMKVESSAEFT
9632.m02843 motif-egtve LYDREKIEVATNN
RHKRS IRKQALLRQTDEFFQQHGGQLLLEMMKAEGNIGFT
9632.m02866 motif-negtive LYKRVEIETATKN
RGRGNATVKKELSALKDANGNVISAQTFTFRQLAAATRN
9632.m03079 motif-negtive FREEC
9632.m03291 motif-negative RKGKRYNLTMDNVQ GGMGIIAFRYVDLQHATKN
9632.m03294 motif-egtve RRSKTKFS GDRLKD SQFCNGII SFEYIDLQRATTN
9632.m03301 motif-negatve WKSKGKWFACTQEKPED GIGITAFRYTDLQRATKN
9632.m03304 motif-neatv WWNKSKRYNCTSNNVEGESGIVAFRYIDLQHATKN
9632.m03761 motif-negtive CRRLHAAAKLVLFYPIKITADELLAAL
QRRKLTKIKKEYFRQHGGMILFESMKSKKGLAFTVFTEAE
9632.m04193 motif-negtive LIHATNN
9632.m043 16 motif-egtve MRRRRMF SELKEEWEVTFGPHRF SYKDLFHATD G
9632.m043 17 motif-egtve RRRVRYAEVREDWEVEFGPHRF SYKELYQATKG
9632.m04800 motif-egtve WRRSTRKRRLAYRNLEKMIDAHGPVKFKLKELRRATAN
WTMKKRKVARKRAELFRKNGGLLLQQRFLMITSQGEESS
9632.m04925 motif-negtive AKIFSAEELKNAT
KKRRLAKQKQRYFLQNGGLLLQQQIFTHQAPARIFTTSEL
9632.m04926 motif-negtive EDATNN
KKRKLAKIRQRYFMQNGGMLLKQKMF SQGAPLRIFT SSEL
9632.m04927 motif-egtve EKATNS
9632.m05095 motif-egtve WRQKKRKILLELEELYNIVGRPNVF SYNELRSATEN
9632.m05097 motif-negtive WRQKRRKLTLEQQELYSIVGRPNVF SYSELRSATEN
9632.m05099 motif-negtive MWRQKRRKL SLEQQELY SIVGRPNVF SYSELRSATEN
RKWQ SKASVLLGKRRNNKNQNRMLL GNLRSQELIEQNLE
9632.m05246 motif-egtve FSHVNFEYVVAATNN
KWI SKGEKRNNENQNRAMLGNFRASHEVYEQNQEFPCIN
9632.m05253 motif-egtve FEDVVTATNN
RKWQTKGKQRNDENKKRTVLGNFTT SHELFEQKVEFPNI
9632.m05257 motif-negtive NFEEVATATNN
9632.m05462 motif-negtive RVRDD GY SLVF SHFRRFTYDEL SD ATCG
RIIHSRMQEHNPKAVANADDCSESPNSSLVLLFQNNKDLGI
9632.m05627 motif-egtve EDILKSTNN
9632.m05838 motif-egtve RRRRKYQELDEELEFDILPGMPTRF SFEKLRECTED
RRARARRRGTTALAAVADKRD SLASAAALARSPREFTYK
9633.m00270 motif-negtive ELSAATRG
KMRNRRNDHHDDMD GS SEIIRTIAASQL SFKYEEL CKATD
9633.m00320 motif-egtve D
KWKRGVQKRIRRAHFKKNQGLLLEQLILDEKAQDKTKIF S
9633.m00373 motif-negtive LEELEKATNY
9633.m00657 motif-neaie RKKLRFCGAQLHD SQCS GGIVAFRYNDLCHATKN


9633.m01194


motif-negative


RKKAGPQEDWEMKCRPPSFIYKDLYNATSG











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
MRKRARFNHHRLNRKTEILIEMPMEEEVHNTRRF SYAHLL
9633.m01241 motif-egtve AATEN
9633.m01466 motif-negtive KKRRTMAQQKEELYNLIGRPDVFSNTELRLATDN
9633.m01467 motif-negtive KKRRTMAQQKEELYNLIGRPDVFSNTELRLATDN
9633.m01492 motif-negative KKRRTMAKQKEELYNLVGRPDVFSNSELKLATDN
9633.m01499 motif-egtve KKRRAMAQQKEELYNLVGRPDVF SNVELKLATDN
9633.m01517 motif-negatve KKRRTIAEQQEELYNLAGQPDVF SNTELKLATDN
9633.m01570 moti-ngtv KKRRALAQQKEELYINLVGRPDVF SYAELKLATDN
9633.m01571 motif-negtive KKRRALAQQKEELYNLVGRPDVF SYAELKLATDN
9633.m01572 motif-negtive KKRRALAQQKEELYNLVGRPDVF SYAELKLATDN
9633.m01594 motif-negative KKRRALAQQKEELYNLVGRPDVF SYAELKLATDN
RRKKVAKH SGKTDKKCLTYQTELYKSP SNLCRNFTFHEM
9633.m02281 motif-egtve QIATSS
KKVAKH SFMTDKKCMTYRTEFYH SPSNL CRNFTFDEIQV
9633.m02283 motif-egtve ATRN
RKKRVDHGNTN~KELLLATLL SKKSNLCHQFTFLQIQEATS
9633.m02289 motif-egtve N
RRKKQAMSNSVKPQNETVSNVS SNGGYGH S SSLQLKNRR
9633.m04197 motif-egtve FTYNELEKITNN
RRQKQKIGS SLEVSD SRL STDHYQQKEVCRRS ASPLI SVEY
9634.m00350 motif-egtve SN
RMKLEKEKQRFYDQNGGHILYQKII SGQVNTVEIFTEEVL
9634.m00424 motif-negtive KNATNN
AGERRRAARMARRLPSMEDERIRVEYSYFRKVAGLPRKL
9634.m00426 motif-egtve TLESLAAATDG
RSMAAARAKRQ SVRLVDVEDYQAAAEREHPRI SYRELAE
9634.m00815 motif-negative ATGG
9634.m01052 motif-egtve RRRAALADTLEEWELEHPQRIPYKELYKATKG
9634.m01423 motif-egtve WRRRRRRHAEVREDWEVEFGPHRFAYKDLVRATRG
9634.m01613 moti-ngtv RKKVKHQNIS SGMLDMI SHQLL SYHELVRATDN
9634.m01642 motif-neaie RHRRNRQILFDVDEQQIENVrNLGNVKRF SFRELQAATEG
9634.m01788 motif-negtive RMALNCVRGGYRSKSD TVIFIPKLIKSKEHLAFLEKDQD G
KKRRRRVGDDPE SL SSTAAFKFNKS SINLRSLAGTPKEFEY
9634.m02185 motif-egtve TELRKGTED
9634.m02894 motif-negatve RWRKRNAVRRAQMERLRPMS SSDLPLMDLASIHAATD S
9634.m03394 motif-egtve RRKRDEPLEDEYFIDQLPGLPTRF SFVDLKSATGD
9634.m03445 motif-negtive KVLPRRQNENTTTPRWKLTAFHNINFNYQDIICGLADNN
9634.m03602 motif-neaie QREDKQLRELAEVGY EMITNHFRRYTYRELVTATRR
9634.m03654 motif-negtive KRKKAKNPTDP SYKKLEKLTYADLVKVTNN
9634.m03671 motif-egtve KKHKS S SGPT STRAVRNQLPRVSYTEL SMGTNG
9634.m03687 motif-neatv RRKEMQANPHCQLI SEHMKNITYQD IVKATDR
9634.m03694 motif-egtve WRKRMQVTPKLPQ CNEHVFKNITYENIAKATNK
9634.m03697 motif-negtive RKRIQVKPNLPQ CNEHKLKNITYEDIAKATNM


9634.m03700


motif-negative


TKRMQAEPHVQQLNEHRNITYEDVLKATNR










Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
9634.m03716 motif-egtve KRMQAEPHVQQLNEHRNITYED VLKATNR
9634.m03999 motif-negatve RRINNNDHDIVRHFTKKELHRATNG
9634.m04004 motif-negtive RRIRRRSSSDHDMVMRLFTRKELYDATNG
9634.m04433 motif-negtive RWRRKIRSSRTGNAAARSVLNGNYYPRVTYAELAKATDD
RQRRALADVKRKYFERHGGLLLYDELSTRPGNTFTIYMEE
9634.m04788 motif-negative QLEQATNG
QRRALADIKRSYFKRHGGLLLYEELNARKSNAFTIYTEEQ
9634.m04798 motif-egtve LEQATNG
9634.m04920 motif-egtve RKGC SAWRSAARRRS GAWKMTAFQKLEF SAEDVVECVK
SKREVSIIDDEEINGS CHD SYDYWKPVLFFQD SAKELTVSD
9635.m00073 motif-negtive LIKSTNN
9635.m00209 motif-negtive RKKMFRKQLPLLP SSDQFAIVSFKDLAQATEN
9635.m00303 motif-neaie RRRLKYTEIQEDWEVEF GPHRF SYKVLYDATEG
9635.m00304 motif-egtve RRRMRYTELRED WEIDF GPHRFAYKDLFHATEG
9635.m00306 motif-egtve RRRSRYAELREDWEVEFGPHRF SYKELFRATD G
9635.m00307 motif-negtive RRRKRYTELREDWEVEFGPHRFPYKDLHHATQG
9635.m00308 motif-negtive RRRQRYAELREDWEDEFGPHRFAYKDLLHATDG
9635.m00309 motif-negtive RRRMRYTELRED WEVEFGPHRF SYKDLFRATD G
9635.m003 10 motif-egtve RRKLTYTELREDWETEFGPNRF SYKDLFLATEG
9635.m00311 motif-neaie RRNLRYAELREDWEVEYGPRRFCYKDLFDATEG
9635.m003 12 motif-egtve RRQQRYAELREDWEVEFGPHRF SYKDLFNATEG
963 5.m003 13 motif-negtive RRRLRYVELKEDWEIEFGPHRF SYKDLFHATH G
9635.m00314 motif-negtive RRRLRYAEIREDWEVEFGPHRF SYKDLFCATEG
9635.m00315 motif-neatv RRHLRYKEVREDWEVEYGPHRFAYKDLFDATKG
9635.m003 17 motif-egtve RRRLRYAELRED WEIEFGPHRF SFKDLYLATEG
9635.m00318 motif-egtve RRKRYAELYEDWEVEFGPYRF SYKYLFDATEG
9635.m00320 motif-negatve RRQLVYKEVREDWEVEYGPRRFAYQDLFRATRG
9635.m00322 motif-negtive RRRLRYAELRED WEIQF GPHRF SFKDLYFATEG
9635.m00324 motif-negtive RKKNAKQREVIMD SAMMVDAVSHKII SYYDIVRATDN
9635.m00325 motif-negtive RRWFKYAELREDWEIDFGPHRF SFKNLYFATEG
9635.m00326 motif-egtve RRRQMRYAELREDWEVEF GPHRF SYKDLFHATEG
9635.m00329 motif-neaie RRCRRYQELHEDWEVEFGPHRF SFKELFKATNGFV
9635.m00339 motif-neaie RWCWKKNARSRENWEAELGPRRFAYRDLRRATD G
9635.m00347 motif-negtive KARSLKKASEARAWKLTAFQRLEFTCDDVLDS
963 5.m005 12 motif-negtive KSKIPDLVSNEERKTGF SGPHYFLKERITFQELMKVTD S
KQERKREARFKLRLI SMAIQNVINLWRIEEGNS GF SLYNF S
9635.m01265 motif-egtve QIKEATQD
9635.m01838 motif-egtve RRRLRYMELQEDWEVDFGPHRF SFKDMYHATEG
9635.m01839 motif-egtve RRHMRYTELREDWEVEFGPHRF SYKDLYHATEG
RRLRNHKSMLRKKDTMAREEVLKLWRLEESD SEFMLFDF
9635.m02971 motif-negtive SIEDATSN


RRLRNHKSMLRKKDTMAREEVLKLWRLEESD SEFMLFDF
SQIEDATSN


9635.m02984


motif-negative











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
KKLRKGD GRKSNRQLEAH SRNS SKTEEALKLWRTEE SST
9635.m03469 motif-egtve DFTLYDFGDLAAATDN
RRRKRSRSKQQH SYSMQMGEDIE SVD SLFIDL STLRAATG
9635.m03484 motif-egtve N
9635.m03485 motif-negtive RWRKRSRSKQQS SYSIQMAEDIESTD SLFIDLSTLRAATGN
9635.m03488 motif-negtive KRKKAAKKDNLLKKMARGKCTIFDLATLKEATEN
9635.m03489 motif-negtive RRRSKVTETDHQLRKITRAQCLIFDLPALQEATDNF SDNN
9635.m03490 motif-egtve RRRSKVTETDHQLRKITRAQCLIFDLPALQEATDNF SDNN
RRKRPVITKAKQTNANYYAEADDVDSVDSMLMDISTLRA
9635.m03493 motif-egtve ATGD
9635.m03494 motif-neaie RFRRRTKVTDAVHPLKKITRAQCMIFDLSALQEATEN
9635.m03497 motif-egtve RFRRRTKAAETDHPLKKITRAQCMIFDLPTLQEATEN
9635.m03511 motif-neaie RFKRRTKAVEADHPLKKITRAQCMIFDLPTLQEATEN
9635.m03523 motif-negtive RRRRPEEQTFLPYDIQ SID SLLLDL STLRAATDD
9635.m03527 motif-negtive KRIKKRRPEEQTFL SYSVS SDDIQ SID SLILDLPTIRVATDD
9635.m03528 motif-neaie RRRRLARKTLRPKS SEDEMQ SFASLVLDLQTLRTATDN
9635.m03532 motif-egtve RRKRRSRKAEHF SELDASEDLESVKS TLITLASLQVATDN
9635.m03533 motif-egtve RRKRRSRKAEHF SELDASEDLESVKS TLITLASLQVATDN
9635.m03537 motif-negtive RKKSRATKAEHL SELDASEDLES VKSTLLTLGSLQVATDN
9635.m03639 motif-negtive KCRRDRTLRI SKTTGGALIAFRY SDLQHVTSN
FNKREKNPQKKDCSSTRNPVFEECSTHKATNSAVQQLSLK
9635.m03785 motif-egtve SIQNATCN
FRRIRRTTRSREKEKEKLD CDESID SEFEKGKGPRRFQYNE
9635.m03803 moti-egtve LVVATDN
RRRRI SRRRTREEYEMGGSDDFDMNDEFEQGTGPRRFLYS
9635.m03805 motif-negtive QLATATND
9635.m03867 motif-negtive RRWHRQFAEVREDWEVEFGPHRFTYKDLFHATQG
9635.m04367 motif-neaie RRRTKATKLSLSYS SRSEDIQNIESLIMDLPTLRIATDN
9635.m04368 motif-egtve RKKRLPTKTPLIENTEDLEDFESIFIDLSTLQSATSN
KRRRRRARVRSELRRL SMAVQNVITLWRLEEGNS GFKLY
9635.m04760 motif-egtve DFSDIKDATNN
9635.m05024 motif-negtive RRRRRFAEVRED WEDEFGPHRFAYKDLFRATD G
9636.m00991 motif-negtive KKRRKAARQQEELYNLVGRPNIF SSAELKLATDN
9636.m00993b motif-negtive KKRRRT SQRKEELYNMVGRRNVF SNAELKLATEN
9636.m01497 motif-egtve KRKMKLNVVDNENLFLNETNERI SYAELQAATNS
9636.m01501 motif-egtve KTRMKPNIIDNENLFLYETNERI SYAELQAATE S
9636.m02876 motif-egtve SRNTYFAERMIINLEELEKATNN
RRRATAPRSRSTAAAAAAHDVAEPITVTVARTDMDAAVK
9636.m03313 motif-negtive QSHSPPPP
9636.m03475 motif-negtive KFYHPKTKNLESVSTLFEGS SSKLNEVIEATEN


KQRREKKLKEKFFKQNHGLLLQQLI SRNTDFGERMIITLEE
LQKATNN


9636.m03956


motif-negative











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
KMKLRKMKRMKETFFRQNHGLLL GRLVSQNAD IGQRMI
9636.m03957 motif-egtve MTLQELEKATDN
RKIKLRKMKKTKERFFKQNHGLLLQQLISQKVDIGERMIIT
9636.m03960 motif-negtive LSDLEKATNN
RRKFKSRRAKKLKEFFFKQNRGLLLHQLVDKDIAERMIF S
9636.m03961 motif-egtve LEELEKATNN
RKVKLQRVKKMRDKFFMQNHGLLLQQLI SRNTDFAERMI
9636.m03963 motif-egtve ITLQELEIATNN
RMARARRSMERRRQERLEHTLTNLPGMPKEFAFEKLRKA
9636.m04074 motif-egtve TKN
9637.m00704 motif-neaie RRKLLYAELREDWEIDFGPQRF SYKDLFHATQG
9637.m00705 motif-negtive RRHLRYSEVREDWEVEFGPHRF SFRDLFHATEGFK
9637.m01303 motif-negtive NKRAIEVD SENSEWVLT SFHKVEFNERDIVNSLTENN
9637.m01504 motif-negative KWRRKNSMHKDLRAFDLQTGSFTLRQIKVATRN
RQKRNSNYSTEDPTRDRSNQLENSLEKSQNHGDVLQIVEN
9637.m01544 motif-egtve RQFTYIELEKVTNK
RRKLQHIKNNYFQQH GGLILFEEMKSQQGHAFKIF SEEEL
9637.m01775 motif-negtive QQATKK
REKRKLQYVKRRYFRQHGGMLLFEEIKSQQGI SFKIF SEEE
9637.m02620 motif-negtive LQQATNK
RERRKLQHIKQKYFKLHGGLLLFQEMNSNERKSFTIF SEAE
9637.m02623 motif-egtve LQHATNK
HERRKLQHIKQKYFKLHGGLLLFQEMNSNERKSFTIFSEAE
9637.m02625 motif-egtve LQHATNK
HDRRKLQHIKNQYFRRHGGLLLYEEMKSKQGLAFKIF SEE
9637.m02627 motif-negtive ELQQATNK
TN~MIKARRAKKLRAVFFKQNRGLLLLQLVDKVIAERMV
9637.m02724 motif-egtve TLEELEKATNR
KVAKVQEGHIASISRRNQPPCCYYLCDDASQAEGIKVERSI
9637.m02896 motif-egtve EFSYEEIFNATQG
PKIKEIIKKKYGENNKRRALRVL SISDDLGQEIPAKDLEFPF
9637.m03211 motif-negtive VEYDKILVATDN
RGKKRSVKEHKKSQVQGVLTATALELEEASTTHDHEFPFV
9637.m03213 motif-negtive KFDDIVAATNNFSKS
KSRENRRKRDSQKTLVPGSRNTSSELLEENPTQDLEFPSIR
9637.m03219b motif-egtve SDIVAATDN
KIKGKRRNRQKHRELILDVMSTSDDVGKRNLVQDFEFLFV
9637.m03225 motif-egtve KFEDIALATHN
RRIKQRRARTLRQKFFKQNRGHLLQQLVSQKAD IAERMIIP
9637.m03306 motif-negtive LAELEKATNN
RKIKQRRARTLRQKFFKQNRGHLLQQLVSQKADIAERMII
9637.m03310 motif-egtve PLAELEKATNN


9637.m03311


motif-negative


KRKVKKQRARMLRQKFFKQNRGHLLQQLVSQKADIAER
MIIPLSELEKATNN











Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
RKQKRRRAKKIRQKYFKQNRGQLLQQLVAQRAD IAERMII
9637.m03317 motif-egtve PLGELKKATNN
963 8.m00128 motif-negtive KRKMNKYFKKNGGSVLQKVDNIMIF SKDEVKKILKNN
9638.m00133 motif-neatv RRKMNEYFKKNGGSILQNVDNIVIFSKDEMKKLL
RARRREVRSAMML SSLQD GTRTATTWKL GKAEKEAL SIN
9638.m00154 motif-egtve VATFQRQLRKLTFTQLIEATNG
963 8.m00177 motif-egtve RKEKQKMREFFIRNGGPILENAKSIKIFRKEELKRITK
RRGHRKGIMGLQARRTDNLQGEEELVWDLEGKNPEFSVF
9638.m00383 motif-negtive EFDQVLEATSN
S CQQFKLRP SSIYLKQERSYCIIMDL SE SKHKEYIERAQWIE
9638.m00436 motif-negtive ENI SNIKPFTEEDIKRITSDYN
9638.m00516 motif-egtve HKKRKMNEYFKKNGGSVLQKVDNIKIFTKDELKKITKNN
9638.m00522 motif-negatve QKRKINEYFKKNGGSILQKVDNIMIF SKDDLKKITKNN
9638.m00529 motif-negatve HQKRKMNEYFKKNGGSVLQKVDNVKIF SKDELKKITKNN
9638.m00590 motif-negtive KRS QRL SANRVSMRNLD STEELPEDLTYED ILRATDN
9638.m00592 motif-negtive KRSQRLSTNRVSVRNMDSTEELPEELTYEDILRGTDN
9638.m00835 motif-negtive HREKRKMREFFEKNGGPILEKVNNIKIFKKEELKPILKASN
KLLFDERRKTKEFFIKNGGPVLEKVDNIKIFKKEELKPIIQS
9638.m00843 motif-negtive CN
RRWKRHAQKRLQTKYFRKNQGLLLEQLI SSDENASEKTKI
9638.m00886 motif-egtve FSLEELKKATNN
9638.m00929 moti-natv RHRAIKKVALAGPRSYSYEELYTATNG
9638.m01234 motif-egtve RKKVKHQKI STGMVDTVSHQLL SYHELVRATDN
9638.m01485 motif-negtive RQKRRMNEYFRKNGGSVLQKVENIKIFTKDELKKITKNN
9638.m01489 motif-negative KKLQAKQYAADKNVD SG SLLFDLAIIRKATAN
9638.m01490 motif-negtive RKLQAKQYTDENDIYSGSLLFDLATLRKATAS
KDQRTIPKDIDHEEHIT SLLIKKYPRI SYVELYAATD SL SSE
9638.m01591 motif-negtive N
9638.m01608 motif-neaie KRLREKS SKVNQDQGSKFIDEMYQRI SYNELNVATG S
9638.m01814 motif-negatve KRKWKSNEHMDHTYMELKTLTYSDVSKATNN
KKRLRPL SSKVEIVAS SFMNQMYPRVSYSDLAKATNGFTS
9638.m01940 motif-egtve NN
9638.m02622 motif-negatve RRLRYAELREDWEVEFGPHRFAYKDLFVATAG
9638.m02623 motif-negtive RQRLRYAELREDWEVEFGPHRF SFKDLYDATGG
SRKARRAAVVVEKAETSASGGGGSSTAAAVQASIWSKDT
9638.m02876 motif-negtive TFSFGDILAATEH
9638.m02881 motif-negative RRRPREKKEVE SNTNYSYESTIWEKEGKFTFFD IVNATDN
9638.m03509 motif-egtve RRRAALADTLEEWELDHPHRLPYRELYMATKG
9639.m00291 motif-negatve RKEMRSSQVWIAEEGYRVMTSHFRMYSHRELVKATER
9639.m00654 moti-ngtv NRKQKRKSVDLP SFGRKFVRVSYNDLAKATEG
RSFKLEELKKRDMEQGGGCGAEWKLESFHPPELDADEICA
9639.m01200 motif-egtve VG


RRIKHRRKIKLRQKFFILNRGQLLKQLVSQRADIAERMIIT
DELEKATNN


9639.m03230


motif-negative










Table 2-2. Con inued

Protein ID Categor Juxtamembrane Domain
9639.m03255 motif-egtve KYLNRRKKNNTKNSSETSMQAHRSISFSQLAKATEG
9639.m03742 motif-negatve KKRNNLGKQIDQS CKEWKFTYAEIAKATNE
9639.m04370 motif-neaie RMKVKKHQKISSSMVDMISNRLLSYQELVRATDN
9639.m04371 motif-negtive RKKVKHQENPADMVDTINHQLL SYNELAHATND
9639.m04373 motif-negtive RATDD
9639.m04376 motif-negatve QMTRKKIKRKLDITTPTSYRLVSYQEIVRATES
9639.m04388 motif-egtve KRRKRRMFANNNGGRLLKDMNIVLITEKDLNKMTKN
9639.m04389 motif-negatve KYKVKHQKMSVGMVDMARHQLL SYHELARATND
9639.m04398 motif-negtive RKKIKRKLDTTTPTSYRLVSYQEIVRATES
9639.m04401 motif-negtive RKKANHQNT SAGKPDLI SHQLL SYHELRATDD
9639.m04403 motif-negtive RMTRKKIERKPDIAGATHYRLVSYHEIVRATEN
9640.m00278 motif-neaie RKEMWS SEVWAAEEGYRVMTSHFRMY SYRELVKATER
9640.m00281 motif-egtve KGVFRRCQVSALDEGYRMVTNHFRAYSYVELRNGTRN
9640.m00711 motif-neaie KTHSARSAIWAAEEGYRVVTDHFRRFTYKELRRATRN
RRKRSPKQTEDRSQ SYVSWDIKS TST STAPQVRGARMF SF
9640.m01015 motif-negtive DELKKVTNN
REIKHRRAKRVKQKFFKQNRGHLLEQLISQRADIAERMILP
9640.m01616 motif-negtive LVELEKATNN
9640.m02587 moti-natv RRCNDHRRRVQQKELELLGIMGP SRFQLQDLVAATGN
9640.m03046 motif-neaie RQTRNSRKGEHALLLI SDQHVRVSYTELVTSTNG
HRRRNKQDTWITNNARLISPHERSNVFENRQFTYRELKLM
9640.m03691 motif-egtve TSN
KLQRRKYRKEKEEYFKQNGGLRLFDEMRSRQVD TILILTE
9640.m04130 motif-negtive KEIKKATENYSDD
RRRHKKEKIEYFKQNGGLRLYDEMI SRQVDTIRILTEREIK
9640.m04131 motif-negtive RATENYN
RKRHKKDKDEYFKQNGGLKLYDEMRSRKVDTIRILTEKDI
9640.m04132 motif-egtve KKATDNYSED
9640.m04 177 motif-neaie KPMKQVMQ SNETSPRPLLMEQHWKL SYAELHRATD G
SRKKSIGG SYVKMDKQTIPD GAKLVTYQWNLPYS SGEIIR
9640.m04345 motif-negtive RLELLDEED
9639.m04391 motif-neaie RMKVKKHQMISSGMVDMISNRLLSYHELVRATDN
9639.m04405 motif-negtive KKMKNPDITASFDTADAICHRLVSYQEIVRATEN






































a .dp


b


m6 pas Am off a m







D g

amaP D ma~ n 8


Secric ~s4 ind ce~go&C1 teor[SnlabUI


aP n ~ a~ can ad a W










.. d. .. ]Mtfpsiv


.b a. Y s Motif-like

., Motif-negative


DeatedwinGenoneroetr


Figure 2-3. Rice RLKs selected for study mapped on the twelve rice chromosomes.
Numbers along the top represent length ofchromosome in megabases.


-chromosome
















00 B.B 1X~ r7.6 PO 26~~ 30~8 35~7 16 Up
~P~e ~%C~8~~~ ~ ~m ~W ~~

Cb~ora a P ~P O~~

Og _B s sss s _eos

P ~P ~ PPPP~


a DR@a a A oP ca a a


~ .B a


Ftau;iceUo~ariJDavmprpaetrstiv pLenghhUF)


Cleased*Mr~enomaPheabe


Figure 2-4. Motif-positive rice RLKs mapped on the twelve rice chromosomes.
Numbers along the top represent length of chromosome in megabases.


[]I Motif-positive

-chromosome








63


m mI





-

. .


Rm aRLK s JMD Caegayow MoPoe (Song ab UF)


Deated Wh GenmePbotr


Figure 2-5. Motif-like rice RLKs mapped on the twelve rice chromosomes. Numbers
along the top represent length of chromosome in megabases.


SMotif-like

-chromosome








64


C ~r~r~Y r IC r Ir

_r _r _r -5-r

~I I rl


.3 .


a ..... ... .


Ir,


I -~ rl


Ftc SUC a ndMD Category Mainlepanv (Sorg ieb uIF


Cleeledd wh enmae~uero


Figure 2-6. Motif-negative rice RLKs mapped on the twelve rice chromosomes.
Numbers along the top represent length of chromosome in megabases.


M Motif-negative
- chromosome




























3_

Ei


I


.


aD ~ C8 117 rZ6 pp aAl 18 15~2 31~ uO
I





_~o -9


I


[]I Motif-positive

E Motif-like

M Mtif-negative

-- chromosome


Reenua AF s61a4 cna CR;L Cked aadnordn toMD Cadega 5rqp (Sngb UFI


Created wh Senae~instr


Figure 2-7. Rice RLKs in subfamily CrRLKIL-1 selected for study mapped on the
twelve rice chromosomes. Numbers along the top represent length of
chromosome in megabases.





R.*e MLKI Slbian DUF26b: oh~e~d~eornngtoD tsategr (SngbbUF)


go


112 lf.6 fLO aA~


18 HZ


GentetdhbraamPbhir


Figure 2-8. Rice RLKs in subfamily DUF261c selected for study mapped on the twelve
rice chromosomes. Numbers along the top represent length of chromosome in
megabases.


[]I Motif-positive
i Motif-like

SMotif-negative
-chromosome








67





go _B$ _Its r2o _*4 _me _.396 _uo
-r

I Ir __t -r_

r r

p~a ..r.


I~ I

II *

, ,


[]I Motif-positive

M Motif-like

SMotif-negative

-chromosome


o B


Stra AL~r hrarb LAI Carta~nealac~mbs IMd lgyp (EmO lb LIP)


taeshi wmatah Gndka


Figure 2-9. Rice RLKs in subfamily L-LEC selected for study mapped on the twelve rice
chromosomes. Numbers along the top represent length of chromosome in
megabases.







68


m

a H


O I


HiseRLK' s~agerrsD2b edAccagsledMD Co~eley ISerghb UF


Figure 2-10. Rice RLKs in subfamily SD-2a selected for study mapped on the twelve
rice chromosomes. Numbers along the top represent length of chromosome in
megabases.


[]I Motif-positive

SMotif-like

SMotif-negative
-chro moso me



C~rosseah~nonp uber







69


D



r _r..





AiceSLK S~kledyW ColoadAccordngtoJMD Cele [Songlob UF]


CmatdwhGenowPuere


Figure 2-11. Rice RLKs in subfamily WAK selected for study mapped on the twelve rice
chromosomes. Numbers along the top represent length of chromosome in
megabases.


[]I Motif-positive

M Motif-like

SMotif-negative
-chromosome







70


* *


_P


fREO RtL~ 5ub~emtLRRI tdgmdured u $lMD CL~gy Sag Idb FI


Crocomhiarthewnc


Figure 2-12. Rice RLKs in subfamily LRR-la selected for study mapped on the twelve
rice chromosomes. Numbers along the top represent length of chromosome in
megabases.


[]I Motif-positive
E Motif-like
SMotif-negative
-chromosome







71


[]I Motif-positive

E Motif-like

SMotif-negative

-chromosome


I I


I


I


Ree FLEN S~gItadP~b~pledaJM D toJM aley (Ser ab lJF


Created wth Gernarda hir


Figure 2-13. Rice RLKs in subfamily SD-2b selected for study mapped on the twelve
rice chromosomes. Numbers along the top represent length of chromosome in
megabases.
















CHAPTER 3
MOLECULAR CHARACTERIZATION OF THE XA21T2AS5/96 MUTANT

Introduction

The rice disease resistance protein XA21, a receptor-like kinase (RLK), contains a

putative proteolytic cleavage motif (P685SRTSMKG) in the juxtamembrane (JM) domain

(Xu et al., 2006). Previous studies have suggested that autophosphorylation of three

serine and threonine residues within this motif play a role in the stabilization XA21.

Sequence analysis led to the identification of a second putative cleavage motif

(P922TDSTFRP) located in the intracellular kinase domain of XA21. Similar to the first,

the second motif carries three serine and threonine residues (Thr923, Ser925, and

Thr926) that can be potentially phosphorylated. Therefore, it is hypothesized that

autophosphorylation of these three residues also contribute to the stability of XA21.

Here, transgenic rice plants with these three phosphorylable residues mutated to alanine

are analyzed for the steady-state stability of XA21. Consistent with previous studies, the

XA21T923A/S925A/T926A mutant accumulated to lower levels than wild-type. This work

provides additional evidence supporting that autophosphorylation of XA21 plays a role in

the stabilization of XA21.

Materials and Methods

Mutant Rice Plants

Transgenic lines expressing a c-Myc-tagged XA21 T2AS5/96 were provided

by Dr. Xuihua Chen of the Song lab at the University of Florida.









XA21 Detection

Protein isolation. Rice plants approximately three months old were used for this

study. Total protein isolation was performed by first grinding leaf tissue in liquid

nitrogen. Ground tissue was transferred into a 1.5 ml Eppendorf microcentrifuge tube to

a volume of approximately 700 uL. An equal volume of extraction buffer (50 mM Tris-

HC1, pH 7.5; 150 mM NaCl; 1 mM EDTA; 0.1% TritonX-100; 1 mM 4-(2-aminoethyl)-

benzenesulfonyl fluoride (Sigma); 2 ug/ml leupeptin; 2 ug/ml antipain; 2 ug/ml aprotinin;

5% v/v P-mercaptoethanol) was added and the tube was vortexed. The tubes were

incubated on ice for five minutes. To pellet cell debris, tubes were centrifuged at 4oC,

12,000 x g, for ten minutes. The supernatant was saved for application in western blot

analysis. Protein concentrations in the extracts were measured using the Bio-Rad protein

assay (Hercules, CA).

Western blot analyses. Each protein sample prepared above were subjected to

SDS-PAGE and subsequently electrotransferred to a PVDF membrane (Millipore

Corporation, Bedford, MA) for one hour at a constant 265 milliamps. The membrane

was incubated at room temperature for three hours in Blotto [5% non-fat dried milk in

TTBS (100 mM Tris-HC1, pH 7.9; 150 mM NaCl; 0.1% Tween 20)]. The membrane was

washed in approximately 150 ml of TTBS three times for 8 minutes each. Blots were

incubated with primary antibody (anti-c-Myc, 1:700) in 3% bovine serum albumin in

TTBS or in Blotto at 4oC. The membrane was then washed as previously described.

Secondary antibody (anti-mouse IgG, Amersham Biosciences) incubation was performed

in Blotto at room temperature for one hour. The membrane was then once again washed

as previously described. The blot was developed using Amersham Bioscience's

ECL+Plus kit.









XA21 Transcript Levels

To address the issues that lower protein levels may be due to lower transcripts, real-

time PCR was carried out as in Wang et al. (in press). Primers used in this experiment

recognize the LRR region ofXa21 and have been scrutinized for specificity. Template

RNA was isolated from each mutant plant using the Qiagen RNeasy mini kit. Analysis of

raw data, with the assistance of Dr. Li-Ya Pi of the University of Florida, was carried out

to obtain values representing relative amounts of transcripts.

Results and Discussion

All XA21 T923A/S925A/T926A mutant rice plants surveyed exhibit lower levels of XA21

compared to wild-type levels (Figure 3-1). In contrast, Xa21 relative transcript levels in

the mutants are comparable to those of wild-type XA21 or higher, confirming that lower

steady states of XA2 1T923A/S925A/T926A iS not due to lower transcripts. Ponceau staining of

the membrane used in the Western blot ensures approximately equal amounts of proteins

were loaded into the gel. Together, these indicate that Thr923, Ser925, and Thr926 are

involved in the stability of XA21. It must be noted however, that this Western blot

analysis only indicates decreased level of protein and that degradation product cannot be

seen. For future studies, it would be interesting to purify the membrane fraction of

XA2 1T923A/S925A/T926A mutant rice extract as in Xu et al. (2006) to allow detection of a

cleavage product.

If the putative cleavage motif examined here and the one described by Xu et al.

(2006) are true cleavage sites acting independently, three cleavage products of XA21

would result: 100 kD, 25 kD, and 15 kD (Figure 3-2). However, because of the N-

terminus location c-Myc-tag, only full-length XA21 and the 100 kD cleavage product









could be detected on a Western blot. It is possible however that both putative cleavage

motifs may act in conjunction when the protein is folded.

Instability of XA21 T923A/S925A/T926A mutant examined here is possibly due to a lack

of autophosphorylation of XA21 on these residues. Alternatively, autophosphorylation of

these three residues may function as docking sites for downstream proteins. Yeast-two-

hybrid analysis has led to the identification of 27 proteins that associate with XA21. A

complex of proteins could protect XA21 from a protease(s) by limiting access to the

recognition site. Residues mutated in D1 plants are contained within a motif of perfect

identity to the proteolytic cleavage motif P/GX5.7P/G found in the animal EGFRs (Figure

3-3). Glycine flanking the putative cleavage motif may contribute to the exposure of this

motifby disrupting its secondary structure. These findings are consistent with previous

studies by Xu et al. (2006) and support that autophosphorylation of XA21 plays a role in

the stabilization of XA21.




















I III


WT 9 11 2 19 24 35 36
D1 Mutants















Figure 3-1. Analyses of XA21T923N/S925N/T926A mutant. Relative transcripts are illustrated
by the bar graph. The Western blot film at center shows amount of full length
XA21 in wild-type (WT) plants and in seven XA21 T923A/S925A/T926A
mutant plants (numbers represent the plant line). Ponceau staining of the
membrane was performed to ensure equal loading.


I I


I I


I I


Ponceau


n,













15 kD
40 kD
140 kD


c-Myc7 A


N-terminus C-terminus


Transmembrane
Juxta mem bra ne


A: putative cleavage motif (ZSy et al. 2006)

B: putative cleavage motif studied here


Figure 3-2. Schematic representation of XA21 indicating putative cleavage motifs (A
and B). Triangles mark the approximate location of putative cleavage motifs
and the N-terminal c-Myc tag. Bars above indicate approximate weights in
kD of various portions of the protein before and after cleavage at the indicated
sites. The transmembrane and JM domains are indicated.


XA21 Intracellular Domain
677HKRTKKGAPSRTSMKGHPLVSYSQLVKATDGFAPTN LL GSG
VYKGKLNIQDHVAVKVLKLE N PALS FTAEC EALRNMRHRNLVKIV
TICSSIDNRGNDFKAIVYDFMPNGSLEDWIHPETNDQADQRHNH
RVTILLDV~ACALDYLHRHGPE PVVHCDIKS SNVLLDSDMVAHVGDFG
LARILVDGT SLIQQCST SSMGFIGTIGYAAPEYGVGLIASTHGDIYSY
GILVLE IVTGKRP922TDSTFRPDLGLRQYVELGLHGRVTDVVDT
KLILDSENWLNSTNNSPCRRITECIVWLLRLGLSCSQELPSSTT
DIIDELNAIKQNLSGLFPVCEGGSLEF






Figure 3-3. Amino acid sequence of the intracellular domain of XA21. The putative
cleavage motif characterized in the study is underlined and the mutated
residues of Dl mutants are shown in red.
















CHAPTER 4
DIRECT MUTATION OF THE PUTATIVE CLEAVAGE MOTIF INT THE
JUXTAMEMBRANE DOMAINT OF XA21

Introduction

Previous findings suggest that auto phosphorylation of XA21 in the

juxtamembrane (JM) domain plays a role in the protection of XA21 against proteolytic

degradation (Xu et al., 2006). The phosphorylated residues Ser686, Thr688, and Ser689

exist within a putative cleavage motif flanked by conserved residues of proline (Pro685)

and glycine (Gly692). Herman and Chernajoysky (1998) demonstrated that Proline211

of the human p75 TNF receptor is required for its shedding. Pro211 is located in the

juxtamembrane region of this protein. It would be interesting to investigate whether the

conserved Pro685 and Gly692 of XA21 are important for the instability of this RLK. In

this chapter, Pro685 and Gly692 are mutated to alanine. The constructs will eventually

be used to confirm the role of the predicted cleavage motif in XA21 stability. Residues

within the putative cleavage motif in the juxtamembrane domain of XA21K, a non-

functional kinase mutant, are mutated for future research.

Materials and Methods

The codons encoding proline and glycine residues flanking the putative cleavage

motif located within the juxtamembrane domain of XA21K were mutated to encode

alanine via site-directed mutagenesis (Figure 4-1). The resulting mutants were named

P9A and Gl6A, respectively. The construct used as the template, GST-XA21K,

containing the putative intracellular domain of XA21 (XA21K), was used as template for









site-directed mutatgenesis. The primers used are listed in Table 4-1. Each reaction

contains 25 ng template DNA, Pfu buffer (Invitrogen), 200 uM of each dNTP, forward

primer, reverse primer, and water to total 50 ul. Reactions were placed in MJ Research

PTC-2000 Thermocycler and allowed to complete one denaturation cycle for ten minutes

at 990C. The cycler was then paused to allow the addition of 2.5U of the DNA

polymerase Pfu (Invitrogen) to each reaction tube. Next, PCR conditions were run

continuously: 5 cycles of 94oC 1 min, 50oC 1 min, 680C 16 min; 16 cycles of 94oC 1 min,

56oC 1 min, 680C 16 min; 1 cycle of 72oC 10 min. The reactions were then subj ected to

restriction enzyme digestion by DpnI, at 37oC overnight to remove template. Each

reaction was checked by agarose gel electrophoresis. Next, 1 ul of each reaction was

mixed with 19 ul of ER2566 competent cells (New England Biolabs,) electroporated at

400 mV, and allowed to recover in 1 ml of Luria Broth (LB). Cells were then plated on

solid LB supplemented with 50 ug/mL ampicillin and incuated at 37oC overnight. Single

colonies were picked, cultured, and subj ected to DNA isolation by Qiagen miniprep kit.

DNA was sequenced by the University of Florida DNA Sequencing Core at Fifield Hall.


Table 4-1. Primers used in site-directed mutagenesis. "F" and "R" indicate forward and
reverse, respectively
Mutation Primers (5' to 3')
P9A F-AGAACTAAAAAGGGAGCCGCTTCAAGAACTTCCATG
R-CATGGAAGTTCTTGAAGCGGCTCCCTTTTTAGTTCT
G16A F-TCAAGAACTTCCATGAAAGCCCACCCATTGGTCTCT
R-AGAGACCCATGGGTGGGCTTTCATCATGGAAGTTCTTGA










XA21 Intracellular Domain

HKRTKKGA PSRT SMRHP LVSY SQLVKAT DGFAPTNLLGS G SFGSV
YKGKLN IaD HVAVKVLKLEN PKALK SFTAE CEALRNMRHRNLVK IVT
ICSSIDNRGNDFKAIVYDFMPNGSLEDWIHPETNDQADQRHLLR
VT ILLDVACALDYLHRHGPE PVVHC D IKS SNVLLD SDMVAHVGDFGL
ARI LVD GT S LI QQS TSSMGF IGT IGYAAPEYGVGL IASTHGD IYSYG
I LVLE IVT GKRPTD STFRPD LGLRZYVE LGL HGRVTDVVD TKL ILD S
ENWLNSTNNSPCRRITECIVWLLRLGLSCSQELPSSRTPTGDIE
NAI KQNLS GLFPVCEGGSLEF


Figure 4-1. The intracellular domain of XA21. Mutated residues Pro685 and Gly692 are
larger and highlighted in red. The putative cleavage motif is underlined.

Results and Discussion

To confirm that the sequence PSRTSMKG in the JM domain is a true cleavage

motif, the conserved Pro685 and Gly692 were individually mutated to alanine. The

P685A and G692A mutations will be eventually incorporated into the full-length Xa21

gene inder the control of the native promoter. It has been established that

XA21 S686A/T688A/S689A mutant is unstable (Xu et al., 2006), so we will test whether the

X2P685A/S686A/T688A/S689A OrX2SG692A/S686A/T688A/S689A mutant protein in the transgenic

lines becomes more stable than XA21S686A/T688A/S689A. These experiments can provide

verification of the putative cleavage motif in the JM domain of XA21. If the residues

indeed provide a protease recognition site, accumulation studies would show higher

levels of protein than the phosphorylation mutant described by Xu et al. (2006).

Additionally, an XA21 P685A/S686A/T688A/S689A/G692A mutant could be created to investigate if

both residues are required for instability. For further analysis, similar mutants could be

made to the putative cleavage motif examined in Chapter 3. Also, simultaneous

mutations of the conserved residues of the conserved proline and glycine residues at both







81


locations of XA21 could be fascinating to examine. Higher levels of XA21 may be seen

as a result of these combined mutations.
















CHAPTER 5
CLONING OF SELECTED ARABIDOPSIS RLKS

Introduction

Arabidopsis thaliana is the model system of plant research today. It is ideal for the

laboratory as it is small, manageable, inexpensively cultivated, while producing

numerous progeny within only approximately one month (Arabidopsis Genome Initiative

2000). Consequently, Arabidopsis genes and proteins related to those studied in other

less manageable plants are frequently investigated first or in parallel with genes and

proteins in those other plants. Also, because Arabidopsis has the smallest genome known

of plants, research of these related genes can also help determine conservation of these

genes through evolution.

Previous investigation of about 600 Arabidopsis RLKs by Dr. Guozhen Liu of the

Song Lab at the University of Florida (unpublished) included alignments of their

juxtamembrane domains. It was quickly noted that the RLK's fell into five separate

categories based on a single amino acid in the JM domain corresponding with Thr705 of

XA21; at the specific site there is a threonine (T), aspartic acid (D) or glutamic acid (E),

histidine (H), serine (S), or other amino acids (0.) Aspartic acid and glutamic acid are

grouped because of their similar structures and similar behavior.

In this chapter, four Arabidopsis open reading frames (ORFs) encoding DE group

RLKs (Figure 5-1) were randomly selected, amplified from a library, and cloned into a

vector.












XA21 ... KGHPLVSYSQLVKA 70sDG

At1 948480 ... KLVF FG NAT KVF DLEDL L

At2 936570 ... SRLVF FE RRKQFE LDDL L

At3g28040 ... TSRSSSSSQEFERNP SL
At5 g01~890 .. .VMFSGEVDVFDTTGADAL


Figure 5-1. Sequence alignment of XA21 and selected Arabidopsis proteins of the DE
group. Threonine705 of XA21 is red and underlined as well as the
corresponding residues in Arabidopsis proteins.

Materials and Methods

Amplification of the At5g01890, Atlg48480, At2g36570 and At3g28040 ORFs

Four Arabidopsis ORFs encoding DE RLKs were randomly selected and primers

were designed for each of them (Table 5-1), including the addition of a BBBBBBBBBBBBBBBBBamH1 site for

cloning. The ORFs were amplified from a hTriplEx2 Arabidopsis total seedling library

(Clontech, Mountain View, CA) provided by Dr. Robert Ferl's lab at the University of

Florida, by PCR using the RoboCycler (Stratagene).


Table 5-1. List of primers used in the amplification of the selected Arabidopsis RLK' s.
"F" and "R" indicate forward and reverse, respectively
Gene Group Primers (5' to 3')
At5g01890 DE F-CGGGATCCCACGCCCGATCTAGTGTTTCACG
At5g01890 R-CGGGATCCTCACTCTAAGTCATGAGAGGGACA
At3284 DE F-CGGGATCCAACGCGTCTGTTAGAAGACGGCTTG
At3g28040 R-CGGGATCCTTAGAAACTATCCATGATACGGTGGG
At2g36570 DE F-CGGGATCCCGTAACGGCGAAAGATCAAAATCCGG
At2g36570 R-CGGGATCCCAGAAACCAAACTTAGCCGTCGGTGG
At1g48480 DE F-CGGGATCCCGGAAAAAGAGTAATAAGAGATCAAGA
At1g48480 R-CGGGATCCTTAATCAGCTTCGTTCACTTGGTCTG









The PCR reaction used to amplify At5g01890, At2g36570 and Atlg48480

contained 2 units of Taq DNA polymerase, lx PCR buffer (Invitrogen), 200 uM each

dNTP, 0.2 uM each appropriate primer, approximately 25 ng of template DNA. They

were successfully amplified under 1 cycle of 990C for 10 minutes (after which the cycler

was paused in order to add the enzyme); 35 cycles of 94oC for 1 minute, 50oC for 1 min

30 sec, 72oC for 1 min 30 sec; 1 cycle of 72oC for 10 minutes.

At3g28040 was amplified under the following conditions: 1 cycle of 990C for 10

minutes (after which the cycler was paused in order to add the enzyme); 5 cycles of 94oC

for 1 minute, 45oC for 1 min 30 sec, 72oC for 1 min 30 sec; 25 cycles of 94oC for 1

minute, 50oC for 1 min 30 sec, 72oC for 1 min 30 sec; 1 cycle of 72oC for 10 minutes.

Amplification of gene At3g28040 was successful.

Molecular Cloning of the PCR Products

Following amplification, the PCR products sizes were checked by agarose gel

electrophoresis. Approximately 60 ng of each product was then applied in separate

ligation reactions into vector pGEM-T (Promega) according to manufacturer instructions.

One microliter of the resulting ligation was added to 19 uL of XL1i-Blue competent cells

(Stratagene) and electroporated at 400 mV. Cells were then transferred to 1 mL of Luria

Broth (LB) and incubated at 37oC at 100 rpm for one hour. Cells were then spread onto

solid LB medium containing 50 ug/mL Ampicillin, isopropyl-P-D-thiogalactopyranoside

(IPTG) and X-galactose, and incubated at 37oC overnight. Single colonies were chosen,

inoculated into 3 mL of liquid LB, and incubated at 37oC overnight with shaking. DNA

from these cultures was purified using the Qiagen miniprep kit. DNA was then digested

overnight at 37oC with BamBBBBBBBB~~~~~~~~~HI to check for the insert.









Results and Discussion

The ORFs of At~g0 1890, Atlg48480, At2g36570, and At3g28040 were PCR-

amplified from an Arabidopsis cDNA library and cloned into the pGEM-T vector. Figure

5-2 shows BamBBBBBBBB~~~~~~~~~HI-dgse recombinant DNA. Expected inserts of At~g0 1890 (FigureS-

2A), Atlg48480 (Figure5-2D), At2g36570 (Figure5-2B), and At3g28040 (Figure5-2C)

are 1008, 1095, 1100, and 1050 base pairs (bp), respectively. The size of the pGEM-T

vector is 3000 bp. The inserts shown do not exactly equal the expected sizes of clones of

At5g01890 and Atlg48480. These clones were sequenced at the DNA Sequencing Core

of the University of Florida at Fifield Hall to confirm accuracy. Because the maj ority of

plant RLKs contain a threonine residue at the position corresponding to Thr705 of XA21

and this conserved residue is essential for autophosphorylation activity of many RLKs,

molecular cloning of members belonging to the DE group of RLKs will provide a basis to

test whether these kinases are capable of autophosphorylation.
















































Figure 5-2. Identification of recombinants carrying the ORFs of At~g0 1890, Atlg48480,
At2g36570 and At3g28040. The DNA was digested with BamH1. A: pGEM-
T-At5g01890, all lanes represent correct clones. B: pGEM-T-At2g36570,
correct clones indicated y triangles. C: pGEM-T-At3g28040, all lanes
represent correct clones. D: pGEM-T-Atlg48480, all lanes represent correct
clones.


3kbl


I I


~I


3kb I)


e
e


1 .6kb
1kb






3kb


1kb


C D














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Full Text

PAGE 1

ANALYSIS OF A PUTATIVE CLEAVAGE MOTIF IN RICE RECEPTOR-LIKE KINASES By GINA MARIE CORY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 By GINA MARIE CORY

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iii This thesis is dedicated to all those who have left footpr ints in my life.

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iv ACKNOWLEDGMENTS I must first thank my high school AP Biology and Independent Science teacher, Anthony Arico. He introduced me to the joys of science and is a true diamond in the rough. I also thank Dr. William Zettler, who reeled me as he did many others into the field of plant pathology with his enthusiasm and hi s ability to make a student feel like a student and not a social securi ty number. Also at the Plan t Pathology Department of UF, I thank Dr. Gail Wisler and all the facu lty and staff who guided me along the way. I thank the members of my advisory committee, Dr. Alice Harmon and Dr. Jeff Rollins, for their assistance and patience. I give special thanks to my advisory chair, Dr. Wen-Yuan Song, for giving me a chance as an undergraduate and then as a graduate student. He never stopped raising the bar of accomplishment. Next, I must thank all the members of th e Song lab, past and present: Dr. Guozhen Liu, Dr. Pranjib Chakrabarty, Anita Snyder-Pin eda, Dr. Porntip Tinjuanjun, Dr. Weihui Xu, Dr. Yongsheng Wang, Dr. Xiuhua Chen, Dr. Yan Zhang, Dr. Xiadong Ding, Yingnan Jiang, and especially Lisa Nodzon and Terry Davoli. I also thank Chris Dardick of the University of California at Davis. All of these people gr aciously provided support and necessary laughter. I thank all of my family, in particular my parents, whose generosity and caring never cease. Finally, I thank my husband, Dr. Juan Jose Suarez. I am so happy to enter the real world with him at my side.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT...................................................................................................................... ...x CHAPTERS 1 LITERATURE REVIEW................................................................................................1 Receptor Kinases........................................................................................................1 Plant RLKs ............................................................................................................2 RLK Structure .........................................................................................2 Comparisons of Animal Receptors and Plant RLKs........................................4 RLKs in Growth and Development............................................................................6 RLKs in Disease Resistance.......................................................................................9 Phosphorylation and Protein Stability in XA21.......................................................15 Receptor Shedding...................................................................................................17 2 SEQUENCE ANALYSIS OF RICE RECEPTOR-LIKE KINASES............................21 Introduction ..........................................................................................................21 Materials and Methods.............................................................................................22 Identifying Putative Proteolytic Cleavage Motifs in the JM Domain of Rice RLKs.............................................................................22 Average Length of JM Domains of Rice RLKs.............................................23 Creating Rice Chromosome Maps Depi cting the Locations of Rice RLK Genes.............................................................................23 Results and Discussion.............................................................................................24 A Significant Number of Rice RLKs C ontain the Putative Cleavage Motif.24 A Significant Number of Select Plant Proteins Contain the Putative Cleavage Motif..............................................................................24 Average Length of JM Domains of Rice RLKs..26 Rice Chromosome Maps Depicting the Locations of Rice RLKs..................26 3 MOLECULAR CHARACTERIZATION OF THE XA21T923A/S925/T926A MUTANT...72

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vi Introduction..72 Materials and Methods.............................................................................................72 Mutant Rice Plants..72 XA21 Detection..73 XA21 Transcript Levels...........................................................................................74 Results and Discussion.............................................................................................74 4 DIRECT MUTATION OF THE PU TATIVE CLEAVAGE MOTIF IN THE JUXTAMEMBRANE DOMAIN OF XA21..............................................................78 Introduction ..........................................................................................................78 Materials and Methods.............................................................................................78 Results and Discussion.............................................................................................80 5 CLONING OF SELEC TED ARABIDOPSIS RLKS....................................................82 Introduction ..........................................................................................................82 Materials and Methods.............................................................................................83 Amplification of the At5g01890, At1g48480, At2g36570 and At3g28040 ORFs.............................................................................83 Molecular Cloning of the PCR Products........................................................84 Results and Discussion.............................................................................................85 LIST OF REFERENCES...................................................................................................87 BIOGRAPHICAL SKETCH.............................................................................................95

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vii LIST OF TABLES Table page 2-1. Distribution of the P/GX5-7P/G motif in the JM domains of plant RLKs................25 2-2. The P/GX5-7P/G motif in the JM domains of predicted rice RLKs...........................27 4-1. Primers used in site-directed mutagenesis.................................................................79 5-1. Sequence alignment of XA21 and selected Arabidopsis proteins of the DE group..83

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viii LIST OF FIGURES Figure page 1-1. Generalized structure of a receptor-like kinase protein with major domains labeled.4 1-2. Proposed model of evolution of the receptor kinase family........................................6 1-3. Model of XA21 mediated resi stance through binding proteins.................................10 1-4. Components of signaling pa thways of innate immunity in Drosophila mammals, and Arabidopsis ........................................................................................................13 1-5. A c-Myc-tagged version of XA21.16 1-6. Intracellular juxtamembrane domain of XA21..........................................................17 1-7. A conserved motif in the juxtam embrane domain of the EGFR family.....................20 2-3. Rice RLKs selected for study mapped on the twelve rice chromosomes..................61 2-4. Motif-positive rice RLKs mapped on the twelve rice chromosomes........................62 2-5. Motif-like rice RLKs mapped on the twelve rice chromosomes...............................63 2-6. Motif-negative rice RLKs mapp ed on the twelve rice chromosomes.......................64 2-7. Rice RLKs in subfamily CrRLK1L-1 sele cted for study mapped on the twelve rice chromosomes............................................................................................................65 2-8. Rice RLKs in subfamily DUF26lc se lected for study mapped on the twelve rice chromosomes............................................................................................................66 2-9. Rice RLKs in subfamily L-LEC select ed for study mapped on the twelve rice chromosomes............................................................................................................67 2-10. Rice RLKs in subfamily SD-2a se lected for study mapped on the twelve rice chromosomes............................................................................................................68 2-11. Rice RLKs in subfamily WAK select ed for study mapped on the twelve rice chromosomes............................................................................................................69

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ix 2-12. Rice RLKs in subfamily LRR-1a sel ected for study mapped on the twelve rice chromosomes............................................................................................................70 2-13. Rice RLKs in subfamily SD-2b sele cted for study mapped on the twelve rice chromosomes............................................................................................................71 3-1. Analyses of XA21T923A/S925A/T926A mutant.................................................................76 3-2. Schematic representati on of XA21 indicating putative cleavage motifs (A and B)..77 3-3. Amino acid sequence of the intracellular domain of XA21......................................77 4-1. The intracellular domain of XA21.............................................................................80 5-1. Sequence alignment of XA21 and selected Arabidopsis proteins of the DE group..83 5-2. Identification of recombinan ts carrying the ORFs of At5g01890, At1g48480, At2g36570 and At3g28040......................................................................................86

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ANALYSIS OF A PUTATIVE CLEAVAGE MOTIF IN RICE RECEPTOR-LIKE KINASES By Gina Marie Cory May 2006 Chair: Wen-Yuan Song Major Department: Plant Pathology Plant receptor-like kinases (RLKs) are a large group of proteins found in many different species. XA21 is a rice RLK conferring resistance to bacter ial blight disease. Previous findings demonstr ate that phosphorylated residue s in XA21s juxtamembrane domain assist in the stabilization of XA21. These residue s exist within a motif identical to the proteolytic cleavage motif, P/GX5-7P/G, originally identified in the animal EGFR protein family. Here, it is shown that appr oximately 47% of 800 rice kinases contain this motif within their juxtamembrane domains. Additionally, a second putative cleavage motif in XA21, P922TDSTFRP, was identified within its intracellular kinase domain. Similar to previous findings, mutation of th e phosphorylated residues within this second putative motif leads to degradation of XA21.

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1 CHAPTER 1 LITERATURE REVIEW Receptor Kinases Perception of signal at the surface in an imal cell is known to be regulated by receptor tyrosine kinases (RTKs) and recepto r serine/threonine kina ses (RSKs) (Hubbard and Till, 2000). Many receptor kinases have a signal-receiving extracellular domain, a transmembrane region, and an intracellula r kinase domain. Based on their greatly different extracellular domains, the animal receptor kinases can be placed into 20 subgroups (Robinson et al., 2000). Recognition of ligands, mostly soluble though some are membrane-associated, often leads to dime rization of the receptors and results in phosphorylation of either tyrosine or serine and threonine resi dues of the kinase (Hubbard and Till, 2000). While one function of this phosphorylation is activ ation of the kinase, the other is to provide a binding surface for in teracting proteins (Burgess et al., 2003; ten Dijke and Hill, 2004; Johnson and Ingram, 2005). Receptor kinases function in numerous impor tant processes in the cell, such as embryonic development, metabolism, and im mune system function (Hubbard and Till, 2000). For instance, effects of insulin on th e body are regulated by the insulin receptor, an RTK (White, 1998). Once insulin is bound to the receptor, phosphorylation of the receptor and recruitment of downstream prot eins occurs. Those recruited downstream signaling proteins are the IRS adaptor prot eins, which are phosphorylated on several

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2 tyrosine residues by the receptor. IRS prot eins then bind and activate PI-3k, which is involved in the movement of glucose transpor ters to the cell membrane (Cheatham et al., 1994; Okada et al., 1994). Nonreceptor tyrosine kinases (NRTKs), l acking extracellular and transmembrane domains, are components within the signal transduction pathways triggered by receptor kinases (Hubbard and Till, 2000). Most NRTK s are located in the cytoplasm, though some are actually bound to the membrane. Th e largest subfamily of the NRTKs is the Src family, whose mutation can cause human cancers (Biscardi et al., 1999). Plant RLKs Plants contain a large number of pred icted proteins called RLKS (receptor-like kinases) with structures similar to anim al receptor kinases. Over 600 and 1000 RLKs have been reported in the model plant Arabidopsis thaliana and rice ( Oryza sativa ), respectively (Shiu and Bleecker, 2001a; Shiu et al., 2004). Much knowledge about the exact function of these kinases is lacking, w ith relatively few bei ng described (Shiu and Bleecker, 2001a). It is clear however that RL Ks are involved in a variety of processes, including disease resistance, self-incompatib ility, hormone percepti on and development. RLK Structure Like animal receptor kinases, many plant RLKs are composed of an extracellular domain, a transmembrane domain, and an intr acellular kinase that contains twelve subdomains with fifteen invariant residues (Figure 1-1) (Walker, 1994; Hanks et al., 1988). Some of the invariant residues, such as the lysine residue corresponding to Lys736 of XA21, are important for catalysis. The extracellular domain is thought to perceive signals (also known as ligands ) through binding. The transmembrane domain anchors the protein on the cells membrane, while the kinase domain juts into the

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3 cytoplasm of the cell. It is important to note the juxtamembrane (JM) domain, which is located just after the transm embrane domain. A correspond ing extracellular region is termed the extracellular JM domain. Phosphoryl ation of residues in the JM domain often acts as binding sites for other signaling proteins in animal systems. In plants, such phosphorylation has also been observed in XA21 in vitro and BRI1 in vivo (discussed in more detail below). Plant RLKs can be classified into 45 subgroups based on their extracellular structures (Shiu and Bleecker 2001a). Some major subgroups are described below. The leucine-rich repeat (LRR) s ubgroup contains proteins with a core tandem sequence of Leu-x-x-Leu-x-Leu-x-x-Asn-xLeu. LRRs have been imp licated in protein-protein interactions. The S-domain RLKs, the first clas s of RLKs to be described in plants, share similarities with the S-locus glycopro teins involved in incompatibility of Brassica One distinct characteristic of the S-domain RLKs thought to be important for folding of the extracellular domain is a group of ten cystei ne residues found next to the transmembrane region. Another subgroup is the lectin receptor kinases, which was defined by the Arabidopsis kinase Ath.lecRK1 which has an ex tracellular domain homologous to the legume lectin family (Herv et al., 1996). Lectin receptor kinases may interact with oligosaccharides or cell wall fragments (B uchanan et al., 2000). Epidermal growth factor-like RLKs share repeat s in the extracellular domain with their animal protein namesake, and may perceive signals at the cell wall-plasma membrane interface (Braun and Walker, 1996; Buchanan et al., 2000). There are several ot her groups of RLKs including thaumatins and WAKs (summarized in Shiu and Bleecker, 2001b).

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4 In a phylogenetic study of RLKs, a group of RLKs lacking extracellular and transmembrane domains existed within th e same monophyletic group as other RLKs (Shiu and Bleecker, 2001a). These were so te rmed the receptor-like cytoplasmic kinases (RLCKs). PBS1 is a representative of this class (Swiderski and Innes, 2001). This Arabidopsis serine/threonine kinase along with another prot ein RPS5, recognizes the avirulence gene ( avrPphB ) product from Pseudomonas syringae More recently, PBS1 was shown to be cleaved by a bacterial type II I effector molecule, and that this cleavage was necessary for the induction of defense agai nst the pathogen (Shao et al., 2003). It is thought the PBS1 cleavage product binds w ith RPS5 and subsequently activates resistance pathways. Animal NRTKs and pl ant RLCKs can be considered corresponding soluble forms of their respective groups, as some members of both have been shown to form complexes with receptors and help in the transduction of signals downstream. Figure 1-1. Generalized struct ure of a receptor-like kinase protein with major domains labeled. Comparisons of Animal Receptor Kinases and Plant RLKs Despite similarities between RLKs and animal receptors, major differences can be seen (Johnson and Ingram, 2005) It could be possible that plant and animal receptors

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5 evolved independently, and their similar struct ure organization being due to their inherent suitability to the processes they regulate. To date, most plant RLKs appear to be serine/threonine kinases, while animal receptors, except the mammalian TGFreceptors, are tyrosine kinases. And while both RLKs a nd animal receptors show a great variety of extracellular domains, the sequence and st ructure of the extracellular domains vary greatly between them. Thirdly, increasing evidence shows that downstream interacting proteins of plant RLKs differ from those of animal receptor kinases. Shiu and Bleecker (2001a) phylogenetically analyzed Arabidopsis RLKs in relation to animal receptor kinases using the conserve d kinase domains. This analysis included more than 900 candidate RLKs and relate d kinases from plants. Among the animal receptors, which included representatives from 16 subfamilies, data supported a strong monophyletic origin for the RTKs. Raf kinases, a family of kinases which can elicit or arrest the cell cycle in mouse fibroblasts (W oods et al., 1997), were more closely related to the animal RTKs; the plant RLKs formed a separate monophyletic group within the eukaryotic protein kinase superfamily (previ ously, RLKs were placed into a separate family based on their serine/threonine specifici ty). This indicates that RLKs have a distinct origin from Raf kinases and RTKs. In contrast, plant RLKs were found to be related to the Drosophila and Caenorhabditis Pelle kinases, and to three human IRAKs. Based on these results, a sepa rate group was defined as the RLK/Pelle kinases gene family. Shiu and Bleecker then proposed a model for the evolution of receptor kinases (Figure 1-2) chronologically beginning with (1) divergence of RLK/Pelle from RTK/Raf. Next (2) the divergence of RT K from Raf occurs followed by (3) the divergence of plant

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6 and animal lineages. The split of Raf and RTK from RLK/Pelle is attributed to an early gene duplication event which probably occurred before the divergence of plants and animals. The fact that only five RLK ho mologs were found in animals supports this notion. Figure 1-2. Proposed model of evolution of the receptor kinase family. (1) Divergence of RLK/Pelle from RTK/Raf. (2) Di vergence of RTK from Raf. (3) Divergence of plant and animals. Repr inted with permission. Shiu, SH and Bleecker A (2001a). Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. PNAS 98:10763-10768. RLKs in Growth and Development A number of RLKs have been implicated in plant growth and development. Several examples are br iefly highlighted here. CLAVATA1 (CLV1) is an Arabidopsis RLK with twenty-one LRRs in its extracellular domain (Clark et al., 19 97). CLV1 mutants show a surplus of undifferentiated cells (Clark et al., 1993). Cell prolifer ation and differentiation is regulated within the shoot apical meristem of plants. Consequently, CLV1 mutant plants have enlarged floral meristems, as well as inflorescence and vegetative meristems. Also, mutant plants show an increase in the number of floral orga ns and additional whorls in

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7 their flowers. In the CLV1 pathway, a dditional components, CLV2 and CLV3, have been identified (Jeong et al. 1999; Fletcher et al., 1999). CL V2 is a receptor-like protein with LRRs, whereas CLV3 appears to be a sm all protein that is hypothesized to function as a ligand for the CLV1 receptor. HAESA, previously known as RLK5, is an LRR-RLK from Arabidopsis (Horne and Walker, 1994; Jinn et al., 2000). Antis ense strategy produced HAESA mutants which exhibit delayed floral organ abscission. Jinn et al. (2000) also demonstrate that the intensity of this phenotype is directly related to the level of HAESA within a particular plant; that is, the less amount of HAESA, the greater delay in floral organ abscission. Therefore, HAESA may be involved in floral organ abscission in Arabidopsis The gene Crinkly4 ( cr4 ) encodes an RLK whose ex tracellular domain has a cysteine-rich region quite similar to mammalian tumor necrosis factor receptors ligand binding domains (Becraft et al. 1996). Maize mu tants of this gene show affected leaf epidermis, which in turn compromises t hose cells functioning. It has also been implicated in the differentiation of internal ti ssues as well as the epidermal tissues (Jin et al. 2000). It is suggested in the same study that cr4 is also involved in regulatory pathways of cell prolifera tion, fate, and pattern. The Arabidopsis CRINKLY4 is a involved in the development of sepals and ovuoles (Gifford et al. 2003). The SRK ( S R eceptor K inase) gene is located at the S locus of Brassica oleracea (Stein et al., 1991). This locus controls self-recognition of pollen and stigma in Brassica oleracea Pollen germination is halted if both pol len and stigma come from plants having identical S-locus genotypes, preventing se lf-fertilization. SRK is capable of in vitro autophosphorylation (Stein and Nasrallah, 1993). Confirmati on of SRKs involvement in

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8 self-incompatibility was shown through studying self-compatible plants. These plants were determined to have mutated SRK genes (Goring et al., 1993) Later, an SRK-related protein SFR2, an S-locus family member, wa s shown to be rapidly induced by wounding and bacterial infection of Xanthomonas campestris pv. campestris ( Xcc ) (Pastuglia et al., 1997). SFR2 mRNA accumulates af ter bacterial infection by Xcc and Escherichia coli This evidence points towards a role in plant defense. BRI1 is an Arabidopsis LRR-RLK and is involved in the perception of brassinosteroid, a growth-promoting hor mone (Li and Chory, 1997). Dwarf Arabidopsis mutants, unresponsive to exogenous brassino steroid treatments, mapped to the same region as a previously-described brassinostero id mutant (Clouse et al., 1996). BRI1 is autophosphorylated, which has uniquely been shown in vivo in response to brassinosteroid; six phosphoryl ation sites of BRI1 are located in the JM domain (Wang et al., 2001; Wang et al., 2005). Also, BRI1 is one of few RLKs with a known ligand (Kinoshita et al., 2005). Recently it has b een shown that BRI1 exists as a homodimer even in the absence of applied steroids; this state of the protein is thought to autoregulate the activity of the protein (Wang et al., 2005). The proposed model of BRI1 activation begins with this homodimer going through a conformational change upon ligand binding. The subsequent transphophorylation of the kinase domain separates the homodimer, leading to activation of BRI1 through furt her phosphorylation. Afterwards, BRI1 likely then forms a complex with BAK1 (BRI1-associ ated receptor kinase 1) and transduces the brassinolide signal (Nam and Li, 2002; Li et al., 2002). The tomato BRI1, also known as SR160, was shown to bind systemin in tr ansformed tobacco (M ontoya et al., 2002; Scheer and Ryan, 2002). Systemin is a mo lecule which triggers defense responses

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9 against insects after wounding. Additionally, a mutant tomato line with non-functional BRI1 showed reduced production of proteas e inhibitor in response to systemin. RLKs in Disease Resistance Plant RLKs also function in disease re sistance. Flor (1971) first described the gene-for-gene theory of disease resistance. This states that two corresponding genes are required for the onset of disease resistance re sponses in the plant: one avrirulence gene ( avr gene) from the pathogen and one disease resistance gene ( R gene) from the plant. If either of those genes is absent in a partic ular host-pathogen intera ction, disease resistance does not occur. Instead, dis ease of the plant begins. R gene products, a number of which are RLKs, recognize directly or indirectly Avr gene products (Jones and Dangl, 2001). Reviewed below are RLKs to date implicat ed in pathogen or insect resistance. The rice gene Xa21 confers resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae ( Xoo ) and is a member of a small gene family (Ronald, 1992; Song et al., 1995; Song et al. 1997). The protein XA 21 is a serine/threonine transmembrane kinase with an extracellular domain of twen ty-three LRRs (Figure 1-3). The cloning and characterization of this gene was a milestone in the understanding plant disease resistance genes ( R genes) because at the time of its di scovery, it represented a new class of R genes as well as a possible evolution betwee n previously described classes of R genes. The LRRs of the extracellular domai n of XA21 is similar to tomatos CF-9, while its kinase domain is reminiscent of another serine/thre onine tomato kinase, PTO (Song et al., 1995). XA21 is thought to bind a polypeptide signal from Xoo activating the kinase through autophosphorylation (R onald, 1997). It is hypothe sized that XA21 forms a homodimer which activates the intramolecu lar phosphorylation upon pathogen infection, after which it binds with several other prot eins (Figure 1-3) (Liu et al., 2002). The

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10 resulting protein interactions are believed to be the beginni ng of possibly several defense responses. Figure 1-3. Model of XA21 mediated resist ance through binding prot eins (courtesy of Dr. Wen-Yuan Song, University of Florida). Upon ligand binding, it is believed XA21 forms a homodimer. Subsequent phosphorylation recruits binding proteins (blue spheres, purple s quares, and green ov als). This then may activate several pathways lead ing to resistance responses. P: autophosphorylated residues. XA21 binds with several prot eins in the yeast-two hybr id system (Wang et al., unpublished). One XA21 binding protein, XB3, has been studied in depth to date. XB3, whose interaction with XA21 has been confirmed through co-immunoprecipitation assays, contains a RING finger domain capable of autoubiquitination. Downregulation of XB3 in vivo yields greater susceptibility to Xoo as well as lower steady states of XA21.

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11 ERECTA is an LRR-RLK which was orig inally implicated in developmental control of aerial organs (Torii et al., 1996). Plants carrying a mutated ERECTA gene show compact flowers, shorter internodes, and shorter floral organs. More recently, ERECTA has been implicated in resistan ce against bacterial wilt disease caused by Ralstonia solanacearum (Godiard et al., 2003). Arabidopsis thaliana Landsberg, an accession susceptible to bacter ial wilt, transformed with ERECTA exhibited an increased resistance to R. solanacaearum ERECTA, more specifically its LRR and kinase domains, is also implicated the defense against the fungal pathogen Plectosphaerella cucumerina (Llorente et al., 2005). The Xa26(t) gene confers resistance to Chinese Xoo strain JL691 (Yang et al., 2003). This gene was cloned by using a map-based strategy. Xa26(t) encodes an LRRRLK and shares the most homology with XA 21 out of reported LRR-RLKs (Sun et al., 2004). Their different resistance spectra are suggested to be a ttributed to their different structure and variations in the LRR region. Interestingly, Xa26 confers resistance to Xoo from seedling to adult stag es, which differs from the Xa21 -controlled resistance that is only present in adult plants (Xu et al., 2006). Resistance to Puccinia graminis f. sp. tritici causing stem rust of barley, is conferred by RPG1 (Brueggeman et al., 2002). The Rpg1 gene was cloned using a mapbased cloning strategy and subse quent characterization identifi ed it as an RLK. RPG1 has two predicted tandem intrace llular kinase domains, repres enting a novel structure of disease resistance genes. One of these tande m kinase domains does not contain all of the highly conserved residues seen in most kinase s, suggesting it may not be functional. The membrane-targeting sequence of RPG1 is not clear and a ligand binding sequence is not

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12 identifiable. Additionally, Thre204 of PTO, wh ich has been implicated in the interaction of PTO and AvrPto, is conserved in both predic ted kinase domains of RPG1 (Frederick et al., 1998). FLS2 is an LRR-RLK which is involved in the perception of bacterial flagellin. Flagellin is a surface protein on numerous bact erial propellants, also known as flagella (Moens and Vanderleyden, 1996), has been impli cated in the pathogenicity of bacteria (Tans-Kersten et al. 2001). The FLS2 gene was identified by screening flagellininsensitive Arabidopsis thaliana ecotype Ws-0 plants (Gmez-G mez and Boller, 2000). The Ws-0 plants did not respond to the inje ction of flg22, a synthetic peptide mimicking the conserved N-terminus of flagellin. The ability of the plant to perceive flagellin was further illuminated by Zipfel et al. (2004). In their study, fls2 mutant plants not only showed susceptibility to the infiltrated bacterial pathogen Pseudomonas syringae pv. tomato but showed a greater susceptibility when the bacteria was just sprayed on the leaves, which bypasses natural infection suppor ted by flagella functioning. Flagellin perception then could possibly inhibit bacterial infiltration of the plant. FLS2 signaling is often compared to i nnate immunity of animals and insects (Figure 1-4) (Gomez-Gomez and Boller, 2002). In animal and insect innate immunity systems, the perception of a pa thogen leads to rapid responses describing a first line of disease defense. In Drosophila the Toll receptor, an LRR transmembrane protein with an intracellular domain of high similarity to the interleukin-1 receptor, mediates innate immune responses (Hoffmann and Reichhart, 2002); in animals, innate immunity is mediated by the Toll-like Receptors (TLRs) of which TLR5 recognizes flagellin.

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13 Figure 1-4. Components of signaling pathways of innate immunity in Drosophila mammals, and Arabidopsis Reprinted with permission. Gomez-Gomez, L and T Boller (2002). Flagellin percepti on:a paradigm for innate immunity. TRENDS in Plant Science 7(6):251-256. The perception of pathogens in innate i mmunity is dependent upon the recognition of pathogen-associated molecular patterns (PAMPs). PAMPs are highly conserved, general elicitor molecules including chitin, lipopolysaccharide, glycoproteins, as well as flagellin (Lamaitre et al., 1997; McDermott et al., 2000). After pathogen perception in Toll and Toll-like systems, receptor dimers then form and signaling proceeds through adapter molecules (dMyD88 and Tube in Drosophila and MyD88 in animals) (Gomez-

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14 Gomez and Boller, 2002). This leads to th e activation of downstr eam kinases Pelle in Drosophila and IRAK in animals. Eventually, the pathway results in the degradation of Cactus in Drosophila and I B (inhibitor of nuclear factor B (NFB)) in animals, which in turn leads to the release of Di f (dorsal-related immunity factor) in Drosophila and NFB in animals. The released molecules ar e transported to the nucleus, where gene expression results. The mitogen-activated-pro tein kinase (MAPK) pathway is activated in animals also. There is no direct evidence in plants fo r a pathway like Toll and Toll-like systems, but many parallels can be seen. It is known that the MAPK cas cade is activated in plants in response to elicitors as well as gene-for-gene interac tions (Ligterink and Hert, 2001; Romeis, 2001). This supports the notion of a common system for the recognition of and response to elicitors. Additionally, Toll from Drosophila now implicated in innate immunity, was originally implicated in dorsove ntral development. This is much like the duality of plant RLKs ERECTA, BRI1, and SRK described earlier. A proposed model for FLS2 signaling in Arab idopsis begins with the recognition of flagellin, or degraded form s of flagellin (like the fl g22 synthetic peptide), by the extracellular domain of FLS2. The activati on of the FLS2 kinase possibly occurs via dimerization and autophosphorylation. Subse quently, an unknown pathway proceeds to the activation of ion channe ls and the NADPH oxidase complex, while simultaneously the activation of AtMEKK1, which activat es AtMAKK4/5, which then activates MAPK3/6. This activation may in turn activate the WRKY type transcription factors involved in expression of defense genes.

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15 Molecular cloning of plant disease resi stance genes and thorough understanding of the molecular mechanisms of plant defense can have global impacts. Historically, plant pathogens have greatly affected humans, espe cially with reference to our food sources. The late blight of potato cause d by the oomycete fungal pathogen Phytophthora infestans led to the Irish potato famine of 1845 (Ristai no et al., 2001). Identi fication of molecular components involved in plant disease defense would allows us to manipulate them and create transgenic plants with broad spectru m and durable resistance, thereby reducing the usage of pesticides, fungicides, and the like. Phosphorylation and Protein Stability in XA21 Protein stability has emerged as a common regulatory step of plant R gene products. As known in Arabidopsis the disease resistance pr otrein RPM1 (recognizing Psuedomonas syringae pv. tomato carrying avrRPM1 or avrB ) requires the presence of RIN4 and HSP90 to accumulate (Mackey et al, 2002; Hubert et al., 2003). RPM1 interacts with both RIN4 and HSP90. Add itionally, RAR1 regulates the accumulation of MLA1 and MLA6, which provides resistance to powdery mildew, a lthough their direct interactions with RAR1 have not ye t been shown (Bieri et al., 2004). In addition to the proteins that directly or indirectly interact with resistance gene products, protein phosphorylation has also been implicated in the st abilization of plant resistance proteins (Xu et al., 2006). An XA21 dead kinase mutant, XA21K736E, is unstable in rice extracts. In XA21K736E, glutamic acid was substituted for Lys736, which corresponds to a highly cons erved lysine required for the kinase activity XA21. Additionally, the simultaneous mutation of the phosphorylated Ser686, Thr688, and Ser689 to alanine implicates these resi dues in the stability of XA21. Both XA21S686A/T688A/S689A and XA21K736E transgenic plants exhibit lower steady-state protein

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16 levels than plants containi ng wild-type XA21. Because wild -type XA21 is stable for at least six hours in plant extr act, this suggests phosphoryla tion is a key factor in the stability of this protein. Western blot analysis illustr ates that the 140 kD XA21 pr otein can be cleaved to a 100 kD product in the microsomal fraction. Because XA21 is tagged at its N-terminus with c-Myc for convenience of antibody detec tion and the calculat ed weight of the extracellular domain of XA21 is approximate ly 100 kD, this dete cted 100 kD cleavage product should represent the extracellula r domain and transmembrane domains. Subsequently, it is hypothesized that the mi ssing 40 kD corresponds w ith the intracellular portion of the kinase, which wa s calculated at approximate ly 36.6 kD (Figure 1-5). Western blot analysis showed a significan tly increases accumulation of the 100 kD cleavage product for both XA21S686A/T688A/S689A and XA21K736E, further supporting that autophosphorylation of these three residues pl ays a role in the st abilization of XA21. Figure 1-5. A c-Myc tagged version of XA21. A blue triangle marks the N-terminal tag c-Myc used for immunodetection of XA 21. At the top of the figure, bars indicate the portion of th e protein of the corresponding kD weight: the full-

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17 length protein is predicted to weigh 1 40 kD, and the intrac ellular portion is 40 kD. Further investigation illustrates that XA21 stability is developmentally controlled, with younger plants containing hi gher steady states of the prot ein. This suggests that an unknown proteolytic activity incr eases with the age of the pl ants. Finally, inoculation with Xoo demonstrated that XA21S686A/T688A/S689A and XA21K736E mutations lead to decreased disease resistance in rice, indicati ng that accumulation of XA21 is required for resistance against Xoo Ser686, Thr688, and Ser689 are located in the JM domain of XA21, more specifically within a region id entical to a cleavage motif identified in the epidermal growth factor receptor (EGF R) family of animal receptor kinases (Figure 1-6). Figure 1-6. Intracellular juxtamembrane dom ain of XA21. The putative cleavage motif is underlined, and the autophosphoryl ated residues are shown in red. Receptor Shedding Many receptors in animal systemscan be cleaved in the extracellular JM or transmembrane domain. This phenomenon is kn own as receptor shedding or ectodomain shedding (Hooper et al, 1997; Dello Sbarba and Rovida, 2002). Shedding of the

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18 ectodomain is often regulated by a protease, also known as a sheddase, acting upon a regulatory protein(s) at the surface (Dello Sbarba and Rovida, 2002). These proteases are usually metalloproteinases belonging to the groups known as MMPs (matrix metalloproteinases) or ADAMs (a disinteg rin and metalloproteinase domain). One example is the regulation of Met, a hepatocyte growth factor receptor, which is involved in cell growth amongst other processes (Nath et al., 2001). Shedding of Met by a metalloproteinase is activa ted through EGF (epidermal growth factor) and LPA (lysophosphatidic acid) bi nding to their receptors. Possible biological purposes of receptor shedding include cessation of signaling after the initial signal transduction resulting from a ligand-receptor interaction (Dello Sbarba and Rovida, 2002). Overexpression of re ceptors has been shown to be deleterious to the organism. Another significance of receptor shedding is that the resulting transmembrane-cytoplasmic domains of the pr otein may act differently in signaling than their full-length precursors, and are not c ontrolled by ligand-bindi ng (Dello Sbarba and Rovida, 2002). Much is still unknown about the regulation of receptor shedding. In the case of Her-2 however, a particular cleavage site wa s identified and implicated in its receptor shedding (Yuan et al. 2003). Her-2 is a receptor tyrosine kinase of great sequence homol ogy to the EGFR family of proteins (Coussens et al., 1985). The cleav age product of Her-2 has been suggested to be involved in the aggressiveness, metastasis and morbidity of breast tumors and it is suggested that the ectodomain released duri ng shedding of Her-2 may act as a decoy for

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19 treatments (Christianson et al., 1998). Recen t advances have led to the use of EGFR inhibitors as treatments (Shelton et al. 2005). Yuan et al. (2003) identified a cleavage site in the ex tracellular JM domain of Her2 at which the receptor portion of the protei n is released. The cleaved extracellular domain of Her-2 was detected in supernatan ts of the cell cultur e through Western blot analysis. The Her-2 extracellular domain was then immuno-purified from soluble fraction of a conditioned cell cu lture. Identification of the cleavage site was performed via C-terminal sequencing and MALD I-TOF mass spectroscopy. Sequence analysis of the JM domains of eight other EGFR family members led to the hypothesis that this cleavage site rests within a particular motif, P/GX5-7P/G (Figure 1-7) (Yuan et al., 2003). Prot eases may recognize this short, unstructured motif flanked by proline and glycine. The P/GX5-7P/G motif is seen in EGFR family members originating from very different species s uggesting the motif is highly conserved. In contrast, Her-4, which does not carry a comp lete motif, is not cleavable. Proline and glycine are commonly associated with proteolyitc in cleavage sites because they can locally disrupt the secondary structure of the protein, possibly leaving that portion exposed to cleavage (Hooper et al., 1997). Therefore, it cannot be assumed that protease(s) recognizing P/GX5-7P/G in EGFRs are identical to the suggested protease(s) recognizing P/GX5-7P/G in XA21.

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20 Figure 1-7. A conserved motif in the juxt amembrane domain of the EGFR family. Several EGFR family members from di fferent organisms are represented. Reprinted with permission. Yuan CX, Lasut AL, Wynn R, Neff NT, Hollis GF, Ramaker ML, Rupar MJ, Liu P, and Meade R (2003). Purification of Her-2 extracellular domain and iden tification of its cleavage site. Protein Expr Purif 29(2):217-22.

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21 CHAPTER 2 SEQUENCE ANALYSIS OF RICE RECEPTOR-LIKE KINASES Introduction Bioinformatics studies have become crucial in modern molecular biology, particularly when the genomes of a nu mber of organisms, including rice and Arabidopsis have been sequenced. Through analyzing the rice genomes sequences, 1429 protein kinases have been predicted (Rice Kinase Database, http ://rkd.ucdavis.edu). Genomewide comparative analysis suggests that ri ce has significantly mo re numbers of RLKs that are involved in resistance and defense than Arabidopsis (Shiu et al., 2004). To support this hypothesis, three qualitative resistance genes, whose protein products are RLKs, have been identified in rice (Song et al ., 1995; Sun et al., 2005; Chen et al., 2006). Therefore, rice appears to be an excellent model system to study RLK-mediated defense signaling. As describe din Chapter 1, a putative prot eolytic cleavage motif (P/GX5-7P/G) was identified in the juxtamembrane (JM) domain of XA21 (Xu et al., 2006 ). This motif was originally described in the extracellular JM re gion of animal epidermal growth factor receptors (EGFRs) (Yuan et al., 2003) and has r ecently been implicated in the stability of XA21 (Xu et al., 2006). In this study, rice RLKs are se arched for this motif. Chromosome maps depicting locations of the RLKs are then created to investigate the distributions of rice RLKs carrying this motif.

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22 Materials and Methods Identifying Putative Proteolytic Cleavage Motifs in the JM Domain of Rice RLKs Raw protein sequences files were downloaded from the University of California at Davis Rice Kinase Database, which includ ed a total of 1429 recep tor kinases (January 1, 2006, http://rkd.ucdavis.edu). The Rice Kina se Database incorporates and shares genomic and proteomic data resulting from a collaborative rice proteomics study, and enables the user to easily view this data. The database creators have additionally made a phylogenetic tree of these kinases using the nearest neighbor method. To quickly determine if each kinase qualified for this study, its ID was entered into the PlantsP website (http://plantsp.genomics.purdue.edu/ ) to obtain detailed motif and domain structure information. Because the JM dom ain is loosely defined as being located between the transmembrane domain and the P-lo op of the kinase, proteins which did not clearly carry both of these we re simply eliminated and the resulting number of rice RLKs totaled 796. Each proteins JM domain was then searched by eye (u sing Microsoft Word and Excel) for the putative cleavage motif, as well as additional proline and glycine residues which may also contribute to the expos ure of the cleavage motif to proteases. All proteins were subsequently placed into one of three categories: motif-positive, motif-like, and motif-negative. Motif-positiv e proteins are those c ontaining at least one perfect motif, while motif-negative protei ns contain none. The motif-like category contains proteins having a cleavage motif(s ) with +/one amino acid outside of the normal range in the variable region of the mo tif. That is, these cleavage motifs may be P/GX4P/G or P/GX8P/G. This allows for the investigation of a group of proteins which may be recognized by less specific interactions and can also allow for some sequencing errors.

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23 Additionally, 27 previously de scribed plant RLKs are investigated in the same manner as described above. These included RLKs functioning in both growth and development and defense responses origina ting from various plant species. These published data provide an opportunity to quick ly examine the putativ e cleavage motif in species other than rice without examin ing whole kinomesmany of which are unavailable. Average Length of JM Domains of Rice RLKs Upon the collection of the rice RLKs sequences and identification of the JM domains, a marked difference in the lengt h of the JM domain was noticed between the motif-positive RLKs and the motif-negativ e RLKs. Each motif category was then analyzed separately for an average JM dom ain length. All JM domains of proteins making up a group were compiled, with carefu l elimination any ch aracter except those representing an amino acid, and counted usi ng Microsoft Words character count. The total was then divided by the number of proteins belonging to the category. Creating Rice Chromosome Maps Depicting the Locations of Rice RLK Genes To view the distribution of the rice RLK genes in this study with respect to their motif categories across the rice genome, rice chromosome maps are created. To further analyze this relationship, seven different s ubfamilies of rice RLK genes are also mapped with respect to their motif category. All chromosome maps of rice RLK genes were created using the freely availabl e Genome Pixelizer software (http://www.atgc.org/GenomePixelizer/Genom ePixelizer_Welcome.html ). Genome Pixelizer was created to he lp visualize relationships between genes and following footprints of evolution. Using data provided by the pr ogram user, Genome Pixelizer rapidly creates a chromosomal map, indicating the location of each gene with a colored

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24 square. Each square is interactive, provi ding all input informati on when clickedthough this feature is only available when using th e program. The maps shown in this chapter are snapshots only and are not interactive. Fo r the RLKs studied here, protein IDs, their subfamilies, 5 end positions of genes, and other necessary information were obtained from http://rkd.ucdavis.edu. Technical assistance was generously provided by Chris Dardick of the Ronald lab at Univ ersity of California at Davis. Results and Discussion A Significant Number of Rice RLKs Co ntain the Putative Cleavage Motif Of 796 rice RLKs analyzed, 47.1% were cat egorized as motif-positive. This represents a significant portion of the tota l proteins analyzed. Motif-like proteins comprised a group of only 5.9% of the total proteins analyzed, while motif-negative proteins represent 46.9%. Table 2-2 shows all rice RLKs involved in this study and their JM domains. Residues involved in putative cleavage motifs have been highlighted in red, while proline and glycine residu es flanking the putative cleavage motif are highlighted in blue. As discussed in Chapter 1, these flanking proline and glycine residues may contribute to exposure of the site by di srupting the local secondary structure. A Significant Number of Select Plant Prot eins Contain the Putative Cleavage Motif Table 2-1 shows the previously described pl ant proteins implicated in development and disease defense chosen for examination in this study. Putative cleavage motifs have been highlighted in red, while flanking pro line or glycine which may contribute to exposure of the site are highlighted in blue ; ellipses indicate amino acids which have been left out for convenience of space in the figure and have no bearing on the immediate study. The majority of defense -related RLKs contain the P/GX5-7P/G motif although some RLKs that function in developm ental processes also carry the motif.

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25 Table 2-1. Distribution of th e P/GX5-7P/G motif in the JM domains of plant RLKs. Perfect motifs are highlighted in red, wh ile motif-like sequences are in blue. Proline and glycine residues which ma y contribute to the disruption of secondary structures are al so highlighted in blue. Protein Function Reference Juxtamembrane Domain BAK1 development Li et al. (2002) RRKK PQDHFFDVP AEED PEVHLG QLKRFSLRELQVASDN EXS development Canales et al. (2002) RRWAMTKEESRLKGFVDQNLYFLS GSRSREP LSINIAMFEQ PLLKVRL G ... HAESA development Jinn et al. (2000) RKITRSKNIGYVWILLTIFLLA GLVFVVG IVMFIAK...LAASKWRSFHKLH NORK development Endre et al. (2002) RKTKRADKGDSTETKKKGLVAYSAVR GGHLLDEGVAYFISLP VLEEAT DN PRK2 RRNKNL G GSSLTSSSP TSQDQKLIP GQSSAASTPDRACNDGGKRA EVAG QKL SERK3 development Hecht et al. (2001) RRKK PQDHFFDVP AEED PEVHLG QLKRFSLRELQVASDN SYMRK development Stracke et al.(2002) RYRQKLI PWEGFAG KKYPMETNIIFSLPSKDDFFIKSVSIQA CLAVATA1 development Clark et al. (1997) RQMNKKKNQKSLAWKLTAFQKLD HAR1 development Krusell et al. (2002) RKRRLHRAQAWKLTA ERECTA Devel.,defense Torii et al. (1996) RPHNPPPFLDGSLDK PVTYSTP KLVILHMNMALHVYEDIMRMTEN BRI1 Devel.,defense Li and Chory (1997) REMRKYAE GHGNSG DRTANN...NLAAFEKPLRKLTFADLLQATNG XA21 defense Song et al. (1995) HKRTKK G A PSRTSMKG H P LVSYSQLVKATDG XA21/A1 defense Song et al. (1997) EV PATTSMQG H P MITYKQLVKATDG Pi-2(t) defense Jiang and Wang (2002) KRKRH PPPSQDDAGSSEDD GFLQTISG A P VRFTYRELQDATSN RKL1 defense Ohtake et al. (2000) CRKKSNKRSEI P GDKEAVDNG NVYSVSAAAAAAMTGN GKASEGNG P ATKKLV RKS1 defense Ohtake et al. (2000) RERRSIEVF GKLRPVP FDFDESFRFEQDKARNRELPLFDLNTIVAATNN SFR1 defense Pastuglia et al. (1997) RFWKRLSGDMKTEDLELPLMDFEAIATATHNFSSTNKL GQGGFG IVY K SIRK defense Robatzek and Somssich (2002) RRFKKKQQR GTLGERNGP LKTAKRYFKYSEVVNITNN WAK1 defense He et al. (1997) RMKHLKDTKLREQFFEQN GG GMLTQRLSG A GP SNVDVKIFTED GMKK ATNG WAK2 defense He et al. (1997) KIKHRKNTELRQKFFEQN GG GMLIQRVSG A GP SNVDVKIFTEK GMKEA TNG WAK4 defense He et al. (1997) EHKMKNTKDTELRQQFFEQN GG GMLMQRLSG A GP SNVDVKIFTEE GM KEATDG LRK10 defense Feuillet et al. (1997) RTRYNEEIHLKVEMFLKTYGTSKPTRYTFSEVKKIARR PBS1 defense Swiderski and Innes (2001) HTFAFRELAAATMN RKC1 defense Ohtake et al. (2000) KRRKQKQEIELPTESVQFDLKTIEAATGN SFR2 defense Pastuglia et al. (1997) WKRKQKRSILIETPIVDISSRRHISRENNTDDLELPLMEFEEVAMATNN XA26 defense Sun et al. (2004) RKKANHQNTSAGKADLISHQLLSYHELLRATDD

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26 Average Length of JM Domains of Rice RLKs The average length of the motif-positive RLKs JM domains was 75 amino acids, while motif-like and motif-negative categories were 41 and 40, respectively. This is interesting to note and suggests some correl ation between length of the JM domain and containing cleavage sites. Rice Chromosome Maps Depicting the Locations of Rice RLKs Analysis of the chromosome maps show s that the rice RLK genes are welldistributed within the genome (Figure 2-3,) even when separated into their respective motif categories (Figures 2-4, 2-5, and 2-7.) Many of the genes exist within clusters, which may have originated from duplication events. To address what types of motif categories of RLKs any one subfamily contai ns, chromosome maps were created showing seven large subfamilies of rice RLKs (Figures 2-7, 28, 2-9, 2-10, 2-11, 2-12, 2-13). Generally, many proteins within a subfamily are clustered. However, for subfamily LLEC and LRR-1a the clusters c ontain proteins of the same motif category. This further supports similarity and evolution within a subfamily. Subfamily CrRLK1L-1 and WAK, though not having significant clusters, is com posed of proteins of a particular motif category. Subfamily DUF26lc contains one majo r cluster which is interspersed with both motif-positive and motifnegative proteins. Subfamily SD-2b though, appears to have no clear significant clus tering or motif category preference. These findings are crude and the significance of the phenomena noted within separate subfamilies is better determined through further examination of each subfamily.

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27 Table 2-2. The P/GX5-7P/G motif in the JM domains of predicted rice RLKs. Proline and glycine residues within a motif are show n in red, while pr oline and glycine residues possibly contributing to the di sruption of local secondary structure are shown in blue. Protein ID Category Juxtamembrane Domain 9629.m00042 motif-positive RRSKRRRRSREGSSTSATALV PPLWKVFTSEELRSITKNFS E G NRL PG NAKT GG TYSGIL P D G SRVAIKRLKRSSLQRKKD FYSEI G RVAKLYH P NLVAVK G CCYDH G DRFIVYEFVAN G P LDVWLHHV P R GG RCLDW P MRMRVATTLAQGIAFLHDKV KPQVVHRDIRASNVLLDEE 9629.m00043 motif-positive RRSKRRRRSREGSSTSATALV PPLWKVFTSEELRSITKNFS EGNRL PG NAKT GG TYSGIL P DGSRVAIKRLKRSSLQRKKD FYSEI G RVAKLYH P NLVAVK G CCYDH G DRFIVYEFVANGP LDVWLHHVPR GG RCLDW P MRMRVATTLAQGIAFLHDKV KPQVVHRDIRASNVLLDEE 9629.m00111 motif-positive WYKKKRRRAT G YHA G FVM P S P ASS P QVL G YS G KTNYSA G S P DYKETMSEFSMGNCRFFTYEELHQITNG 9629.m00112 motif-positive WYKKKRRRAT G YHA G FVM P S P ASS P QVL G YS G KTNYSA G S P DYKETMSEFSMGNCRFFTYEELHQITNG 9629.m00153 motif-positive P SVEDIE P SC G YLAMT P I GGG WNSVNL P EYTSYAEVVKSM R GG FAVKF P T P WLNWF G LIKKCLNKSVSHRTMSTNGGLE HLDSLFAANSLL P I PP TS PP FA G TRRTSSAVQL P RDLLCL G Q SYMA G LVVRR G QG P SAAWRR G EG PP LVA GGGG DVDRA G DD GGG E G D G QATKSTMDGVRRLRMAKVCVRTVGRDD 9629.m00167 motif-positive KWIADDPYCYILVLDCLAFYPQVSCTLAAFTNEICSGPTKS NSRYRYFTLGMTGIVRSNDRKQFGAVKYRYLEV G GGGG A P LVVMTFLAHKCWKKRITIDAV 9629.m00191 motif-positive KDKDFFSAC P AKQC G KVEIRY P FRFEPSNTSSSCGLPCMKL TCSDRQETILDIKNYLGRPYKVTAIDYKRATLTIV P LADDS SL PP TPGC P L P NLISE G ALDHRCEPYAMWLLKSINIEFPFRR AVHVSESLQTRYNEEIHLKVEMFLKTYGTSKPTRYSFSEIK KIARR 9629.m00341 motif-positive PRLSGRRSARRLRE G EKIVYSSS P YGAT G VVTAA GG TFER G KMVFLEDVSS GGG KRFELDDLL 9629.m00363 motif-positive RRKRKRSASFE G LIH GG T P L P SLTKEFSLAGLAYTHIFTYEE LDEATDG 9629.m00513 motif-positive RRRK P VHRKK G QSLT P VVEEQFERVSYQELSNGTKG 9629.m00554 motif-positive RRKEEKEELRTPTTSQ P STAWM P LL G RISFRSA PP SAV G SR S P SFTIDTNANT PGGGA T PG MAAAASSS P SYRF P FAALQDA T G N 9629.m00555 motif-positive RRKEEKEELRT P TTSQ P STAWM P LL G RISFRSA PP SAV G SR S P SFTIDTNANT P GGG AT P GMAAAASSS P SYRF P FAALQDA T G N 9629.m00688 motif-positive RRRQ P LF G R G STS P AD G D G KDADML PP WDVTLYQKLEIS VGDVARSL

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28 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9629.m00937 motif-positive RFR G SD G LAQRF P HSAL P KFSRSS G T G QTLLA G RYSS P S GP S G SL G SSIATYA G QAKTFKFAEIEKATNS 9629.m01024 motif-positive QDKD G EDEFAEL P G M P TRFSFQMLKLATKD 9629.m01041 motif-positive MARM G ISK GG S G KEAKK PP LLL G R 9629.m01044 motif-positive RKR G RAAI G P WKT G LS GP LQKAFVT G V P KLNR P ELEAACE 9629.m01045 motif-positive RKR G RAAI GP WKT G LS GP LQKAFVT G V P KLNR P ELEAA 9629.m01193 motif-positive KLFATR P LDARS G LMVFSYAQVKNAT 9629.m01202 motif-positive CNRS GGGG DEEVSRVVS G KS G EKK G RES P ESKAVI G KA G D G NRIVFFE GP ALAFDLEDLL 9629.m01205 motif-positive RYRRDLF G SSKFVVE G SLVVYSYAQIKKAT 9629.m01240 motif-positive FKKKKRHHHHH PPPPP P PHLLHYY G H PPPPPPPPPP FK G DH Y GG VYQNWQQN GPPPP P DHVLKKV P SH P S PPPPP A P LNVH S GG S G SNYS GG DNSQ P LVS PG AAL G FSRCTFTYEDLSAAT DG 9629.m01429 motif-positive KKWARKKRIIENSLTGGKMVMFRSAAMQSLS P KSFLTMI M G 9629.m01474 a motif-positive KLRK G KRKV P P VET P KQRT P DAVSAVDSLPRPTSTRFLAY DELKEATNN 9629.m01474 b motif-positive SNKRSKKL GGGG ADSHTSAWL P LYHSHTS G KSS G HITANI A G MCRHFSFAEIKAATKN 9629.m02103 motif-positive RSRKRVRRASANI P ITQI P AISKEIKEVRVEQV P TSDFAAHD G VLMTIQDKSSEKESDKVMVHL G VSKSKR G DESHS G SFR YMDKDL G FQSADE GG S G TFRHNSAHAITA P SPLV G L P EFS YL G W G HWFTLRDLEVATSR 9629.m03098 motif-positive WRAVSSKEK G N GG AA G SK G SRC G KDC G CFSRDESATPSE HTEQYDLVPLDQQVRFDLDELLKASA 9629.m03450 motif-positive DS P GGSLASL P EWQPM P CKSVSVN P LCSSYLYVT P EGRNL SKVASDFSGNASLFQRITRLS G SEDLLVNV P CVCEAINATM TGLFHDTNYRVKDGDM G DIINSKTFS G LALNVGD G QILHK EEKLIIHL P CGCSSTA P E G VLSYAVQD G DTLGNIASLFRSS WKDILDLN P RVAN P DFIK PG WILFI P M G VA GP SNKKID P FQ TER P VIFSLRAIEDATSN 9629.m03772 motif-positive RWRKQRVVA G S P AAV GG RCSTDAA G KDSFRKSASSTLVS LEYSN G WD P LAD G R GG I G FSQEVAQSFRFNMEDVESATQ Y 9629.m03992 motif-positive RKR G NRF G DTAE P EI P DITKEIAVDEARNRVAAENVQRQE SYTLSLKERQTNKGSRKMLAHFLSCKSS G SHNLV G CSSMY QNDKAQCSYSSDEGTSGHNEREYSQYATMSTS P QI G L P EF SHL G W G YWFTLRDLEDATNG 9629.m04252 motif-positive RRKTKRTMDNLRQTQI P IFSKEI P VDRV G G RSLAQTMHER EQPSFP P QDKHTNRE P GKTL G HMALSKSSE P DNMSQ G SSV CNVDRA G SVHS G ED G ST G H G RK P YS P AAFVSAS P LV G L P E FSHL G WGHWFTLRDLELATNR

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29 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9629.m04552 motif-positive MRRKKKTAVGFD G RSHMEI P IVSKDINVDRVDAQSLHDS GTPIMPVQDKYEQMKGVSHLAESRSVDVDAFSQCSSVYNI EKAGSSYSEDYSSSGP G RKGSSSY G YASAS P LVGL P ELSHL G WGHWFTLRDLEYATNR 9629.m04594 motif-positive FLFKMHNI P KSMEK G YKMITSQFRRFTYRELVEATGK 9629.m04595 motif-positive KKHNI P KSMED G YKMITNQFRRFTYRELKEATGK 9629.m04597 motif-positive SKHNI P MSMEA G YRMVTSQFRMFTYRELREAT 9629.m04603 motif-positive SSKQSI P SSLQA G YKMVMTSQFRRFTYRELKGAT 9629.m04613 motif-positive SSKQSI P SSLEA G YRRVMTSQFRRFTYRELKDVT 9629.m04615 motif-positive LRSKQNI P KSVMD G YELMTEHFRKFSYRELKEAT 9629.m04657 motif-positive CRNKSVATI GP WKTGLS G QLQKAFVT G VPKLQRSELEGA C 9629.m04784 motif-positive RKRKHKKVNSSSKLLKYSGS GG TPRSM GG DMES G SVKDL QTHLFSYEELEEATDS 9629.m04795 motif-positive P ENID P ITCSFI G LI G PSYVL P KDQVP PG NWSQFCKTFEVPV VKYQQMDPK G DAWRK GG Y G QVLRQ G FLLSVNDSRRPPN CTQCEESK G RVFKLK G ENETFLKKYRHRRISKGTPRIESFL QRNGTLHPKRYTYTEVKRMTKS 9629.m04796 motif-positive CHKKHHGQQP P VQELTT PP KAEPSQKKQRAQHLKRYSYS EVERMT 9629.m04824 motif-positive KFCT G KKLK G L P ITMSLESNNNHRAISYYELVRATNN 9629.m04969 motif-positive FKIRNRNHLTEES P M PP K P A GPG SAVV G SRL G SR P ISAS P SF SSSIVTYKGTAKTFSLIEMERATQR 9629.m05220 motif-positive KKKRRRIE P P ASL P TQQ P A PPPPP NYF P SS GG SSLTSDAFFIS PG YH P VRLFSA G SH G Y P YS P ADSAI G YSRMLFT P ENLAEFT N G 9629.m05240 motif-positive KLSQYN P QA P NNQ G KS P DQSISHKFQLLKS G SFCY G S G RY LCCQF G NVKQSRTD G SDHHMNT P K G VVVDVFDREKPIVF TYQEILASTDS 9629.m05252 motif-positiv e RDIRRRRKRV AQREDWKIT P FQTDL G FSEAAILR G 9629.m05253 motif-positiv e RDIRRRRKRV AQREDWKIT P FQTDL G FSEAAILR G 9629.m05335 motif-positive SKRRRRRQH PPPP HHP G Y PP F P AEFYDPHR PP SQQQSHALS P SPSST PP LLLQ P HSFVSS GG ASEAASAV PG IAMM GG AF G Y DELAAAADG 9629.m05510 motif-positive KRRSRV G KDT G MSD G HS G WL P LSLY G NSHSS G SAKSHTT G SYASSL P SNLCRHFSFAEIKAATNN 9629.m05592 motif-positive FVSQWRKA G ELAD G DIDEEM G YDELADEEFFVESGPRRF RYSDLAAATKN 9629.m05634 motif-positive FWRNRVRTRRNETAAAAA GGG DDVL P FRVRNQQH P ASS VKRDQRLDVKRECDEKDLDLPLLDLKAIVAATDD 9629.m05637 motif-positive RNKARRKQHTEMEKSSDADDL P FRVRKS P ALSPARDQWF DENR G AEDDLDL P LFDLEMIFNATDR

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30 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9629.m05640 motif-positive TRKKKRARKT G SSKWS GG SRST G RRYE G SSHHDDDLEL P I FDL G TIAAATD G 9629.m05897 motif-positive RQKKIAKEAVERTTN P FASW G Q GG KDN G DV P QLK G ARYF AFEELKRCTNN 9629.m05964 motif-positive RRARATTSRSAPATALSDDYLSQS P ENDASS G KLVMF G K G S P EFSA GG HAL 9629.m06449 motif-positive RRRMVK G TTQVE G SLISFTYRDLKSMTKN 9629.m06547 motif-positive WRRRQDSIRSKSRRLS G ERRLSR P RPNV G SVLFSL G ELAKA TC G 9629.m06610 motif-positive G RRRTI G INRDD G KLITFKYNELQFLTRN 9629.m06844 motif-positive RRKLVRSRPLAFE SASKAKATVE P TSTDELL G KKSRE P LSI NLATFEHALLRVTADDILKATEN 9629.m06926 motif-positive G A G EVQLAA P Q P EKIAAVQKDES G W P LWLSSAA G DALA G WA P RSADAFHKLEKR 9629.m06939 motif-positive RVAILHAKYA P AIVR GP VSFT P QIS P KNLHSA 9629.m06940 motif-positive RVAILHAKYA P AIVR GP VSFT P QISPKNLHSA 9629.m06957 motif-positive RHRRRASDSSESSSSS P AQ P ELQ G ARCMTLEELSSATRN 9629.m07015 motif-positive RVARRSRRNSDT G SSET P P TLVEW G RC G RTLSA P EYQGAR QFSLEELAHATKN 9629.m07193 motif-positive RRRA G D GG TTT P EKELESIVSSSTKSSKLAT G KMVTF GPG NSLRSEDFV GG ADAL 9629.m07395 motif-positive RKQLQ G ASA GG NSKSKSIVVSAEQKKKAT P VA GGG GG EI DNMMAAMAAR GP LEFEVRELEEATS 9630.m00072 motif-positive RHCLSGLRRRHRAAAAADVDAEAAE GG A G ARQLM PP CS PPP QQEL P LLR P AAKLV P STKEE G E P WKLTWREVEALT GG 9630.m00103 motif-positive RRKDALSPHSHAYSFDKYTSWSSRSNLVSHRSS P L P Q P K P K P RISVLKEFLCSCN P IC G NE GGP L PG VIVRFSYSELEQATG K 9630.m00104 motif-positive RRKDALSPHSHAYSFDKYTSWSSRSNLVSHRSS P L P Q P K P K P RISVLKEFLCSCN P IC G NE GGP L PG VIVRFSYSELEQATG K 9630.m00105 motif-positive RRKDALSPHSHAYSFDKYTSWSSRSNLVSHRSS P L P Q P K P K P RISVLKEFLCSCN P IC G NE GGP L PG VIVRFSYSELEQATG K 9630.m00115 motif-positive YAYRRWYVD G A G CCDDENL GG ES G AW P WRLTAFQRLGF TCAEVLAC 9630.m00168 motif-positive DDGVY P E G QILEA P NLRTFTFIELRTATKN 9630.m00356 motif-positive KRRRETSLH GG FLR G VEMST P DWS G K P S G QSAVVKVDKE QSTVAEEKDTKGSISSYQKNVQESLQNHPLQFKFTIFTVAS LQQYTNSFSEQNLMRQTL 9630.m00518 motif-positive LTLLRSTSFLSKNRRYSND G TEA P SSNLNSEQ P LVMV P Q G K G EQTKLTFTDLLKATKN 9630.m00525 motif-positive SLRDAIPKIENKSNTS G NLEA G SFTSD P EHLLVMI P R G S G EA NKLTFTDLMEATDN

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31 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9630.m00733 motif-positive KR GG RRDEH G RLVFVQESRKRFEIEDLL 9630.m00752 motif-positive LSKYQERQSRRDYTTSQV G RVHQRVEE P KVKQASVQSRN DAKK G STEV P ERRQMYKITIQLLAAALEKPPEKRKEHQYT NS 9630.m00890 motif-positive RNAQKRKDDTSSNSKDFVGPLSVNIERASNREIPEQSPENT SVATMKISPAEKMT P ERIY G KT G SMRKTKVPITATPYTVA SLQVATNS 9630.m00912 motif-positive RRRR G V G EESVRP G KVV G DVSSSAEY G ALAS G KQTTTAT SMSSLSAARSLMASEVREALESLTVYK 9630.m01346 motif-positive RRRKRGKKAAAAWEDDEVAV G AVKVAATA P VVLVER P L MELTLADLAAATSGFG 9630.m01660 motif-positive RRKLAKK G KEII P G DFVSIWNFDAKVAFQDALYATEN 9630.m02590 motif-positive RN RSSLKRRQSSCSEEVEDIKSVLLD P SVIRSAT G N 9630.m02923 motif-positive K G KFKTRKEL P A P SSVI GG INNHILVSYHEIVRATHN 9630.m03277 motif-positive RRRRRRR GP NTS P VQQL P VSA PP KN P QKVKA P KDIQEV P A QATAAAAAKT P LAQVLQM P A PPPPPPP MAAAA PPP ETVQI AT G KEHRITY P E PP HRS G SSSH G SGEA P SV P EVSHL G W G H WYTLKELEAATEM 9630.m03316 motif-positive KRKKY G EQSEN G V G DAKVFSVWNFEGGEACRQIFETTKY 9630.m04006 motif-positive KSSFRRQDYIVKAVADTTEALELAPASLVLLFQNKDD G KA MTI G DILKSTNN 9630.m04684 motif-positive RWRKRNAVRRAQIESLR P LSNSDL P LMDLSSMYDATNQ 9630.m04997 motif-positive KQYEHLE PG NQLHSL P S P SE G NESIEKSADEFWDKQNFEID HGQDNTLDQEKDSAEVRQDAERTSDKSSGTESAKSEITLD DVAEFEIQWEE 9630.m04998 motif-positive KQYEHLE PG NQLHSL P S P SE G NESIEKSADEFWDKQNFEID HGQDNTLDQEKDSAEVRQDAERTSDKSSGTESAKSEITLD DVAEFEIQWEEI 9630.m05216 motif-positive RKQERRAGRRASRSRDVQLRRHRSPASDSAHLVY G NDDD G NDIVI PG L P TRFTHEEIEDMTNS 9630.m05315 motif-positive P HWAQV P KDVSLCK P DIHAL P SSNV PP KLVILHMNMAFLV YEDIMRMTEN 9630.m05589 motif-positive KRKLIRTKQRFFEQNGGVLLQQQMSSY GG TS GG A GG FKIF SKEELEKATNS 9630.m05597 motif-positive QKRKLIRTRQKFFEQNGGIFLQQQMRSY GG A GGG V GG FKI FSTEELKNATNN 9630.m05673 motif-positive KWYKLRQLQEAVSPATT P AVNRRY GG RSTLSVSRVSSAS ASMLSTVATCTTSVKTFSLSQLEKATDG 9630.m05830 motif-positive RR G RNKRVA G DVDK G AM P EEEEEQQQQQPQAQPREEINA SASASASVASERR GG REFSWERE G I G KLVFC GG VAEMYSL EELL 9630.m05880 motif-positive KSCTYSPKSTANNAKS PP ANVEKV P KANEVLYSWNSLMNDCEA SSSDVIKPERAMKTKVWAKTSKNFLTAKQFQAVDILAATRN

PAGE 42

32 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9631.m00239 motif-positive RRRRQA P T P A P EEKESTQL P WSQHTQD G SSWVDMSNAS G AGMT G G LHRMSMQLNISLADITAATEN 9631.m00251 motif-positive SRRRRRRDDLASNLYPADTKIL KQHLQQPTPPKDIQEIVRR QQRQQQT P TPT P P Q PPPP AAQH G VQLAKAET PPPP QRTQ PP VL P A G STRSTAAS G MSATTS GG SERD G AT P RSTAS G SA GP EVSHL G W G HWFTLRELEEATDG 9631.m00268 motif-positive AMNIKAYMRRSKEEQEGKEEDEVLESEST P MLAS P G RQ G S NAII G KLVLFSKSL P SRYEDWEA G TKA 9631.m00800 motif-positive RRRKRRAALEEHFEQHQ P FTSF P SNEVKDMK P IEESTTIDV ESL P S P ASFSLK PPP KIERHKSFDDDDLSNK P VLKKTNVA P I KATVYSVADLQMATES 9631.m01136 motif-positive RMRRRR G KVTAVQ G SLLLLDYHAVKTATRD 9631.m01146 motif-positive RKKSSSST P ATAVEK G RDLQMA P MDME P K G QN G SAA G N G AHV G AAAAA P AAATSAAVAAAAAAAKT GG AT G G SKK LIFF G PMAAA PP FDLEDLL 9631.m01182 motif-positive CCSKKKKR P P HMHM P YYTDEN G KVYYANSM P RWQNSV DQ GGG WHAQYS P G QA PP SSEMS G SH G A G P L PPP S PG MAL G FSKSSFSYDELALATGG 9631.m01193 motif-positive CARRRRGAKHLSMSRVE HAPSSGSLRQASSSSA P KEKDHA EA G A G T G T G TSSSDVASSSAAASYLES P VRRK P ERISCAAA MDMGWGRWYDLEELEAATGG 9631.m01412 motif-positive RR G RKRGA G A GGG AAARM G V G V G V G ARTQ P A P ALRRLS CQQLRRATGG 9631.m01555 motif-positive KNF G KKDIH G FRVELC GG SSIVMFH G DL P YSTKEILKKLET 9631.m01585 motif-positive RRCRRQRRRRRQAQ P F P L PPP IYN P N P YYK G DL PP Q P FVAQ Q PP SDHYFIQHQH P T PP QTS G TFSDA G SER P HSIDILTELPT GGSLSYDQLAAATDG 9631.m01698 motif-positive RMLRRKKKPVKQ P SNTWV P FSASAL G ARSRTSF G RSSIVN VVTL G QN G A G A G A G YRF P FAALQEAT GG 9631.m02117 motif-positive RRRAQKAREEL GGP FASWKRSEER GG A P RLK G ARWFSYE ELKRSTNN 9631.m02118 motif-positive RRRAQKAREEL GGP FASWKRSEER GG A P RLK G ARWFSYE ELKRSTNN 9631.m02148 motif-positive SKRKEKKDD G LDNN G K G TDNARIEKRKEQVSSGVQMAE KNKLVFLDGCSYNFDLEDLL 9631.m02151 motif-positive CKRRQRAGKDSGMSDGHSGWL P LSLY G NSHTSSSAKSHT T G SHASSL P SNLCRHFSFVEIKAATNN 9631.m02173 motif-positive RVRT PG SHSAAELELSD G YLSQS P TTDVNS G KLVMF GGG N P EFSASTHAL 9631.m02769 motif-positive KRKSTRHQH GG D P EKNE P LTLR P IASGKFNQLRTISIIS P TA KE G LQKTVSMNLKPPSKIDLHKSFDENDLTNKPVLAKNV DLSSIRATAYTVADLQMATES

PAGE 43

33 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9631.m03402 motif-positive KAKKNRKAI P SALNN G QVT P F G QRNHTASALNNWEITPF WQRNHVAASNDAQDNNSMR P A G Q G NHQDLDL P SFVIETI LYATNN 9631.m03557 motif-positive KNRWKRRRRPAQVMNLARRRTLVV P ERVAS P EVYQ P SN G PTAS P S G TSSYEFS G TTSWFTYDELAAVTGG 9631.m03714 motif-positive RRWRRRRRRQQAQ P L P L PPP MLYN P N P YYK G DQ PP L P FVF MQQHHHH P TA P QTS GG TFSDA G SER P HSISID GG SLSYDQL AAATGG 9631.m04965 motif-positive KRKQK P F G RVQS P HAMVVH P RHS G SD P DMVKITVA G G N VN G G AAASETYSQASS G P RDIHVVET G NMVISIQVLRNVT NN 9631.m05382 motif-positive RRRHRPGWQKTNSFQSWFLPLNS TQSSFMSTCSRLSSRNR F G STRTKS G FSSIFASSAYGLGRYFTFVEIQKATKN 9631.m05526 motif-positive SRKTA P RRKRKKK P HNPVTHFDADTS G SK GGGG RDTS GP K PPPPPP WLAE P RAAPSTSDAAGMSKGTFTYEQLAAATGG 9631.m05779 motif-positive RRYR G R GP A P AA G PTNAAARAAAFLRRN G LHQHR P SFTY EQLRAATAG 9632.m00032 motif-positive WRNRFKWC G V P LHRSQ GG S G IIAFRYSDLDHATKN 9632.m00033 motif-positive RNRFEWC G A P LHD G EDSS G IKAFRYNDLVHATKN 9632.m00260 motif-positive KWR G T G R G FDK G TR G VRRFKYHQLVSATNQ 9632.m00342 motif-positive RAR G M G IV GG K G GH GGG SSDSES GG DLAW P WQFT P FQK LSFSVEQVVRNLV 9632.m00847 motif-positive RKS G KFQLRII G KNSN P KENIEELLDNYGSLAPKRYKYSQL KDMTGS 9632.m01083 motif-positive RRKTTKTMI P P DNT G NEEDNDYVDPPTLNLTVLRAATRN 9632.m01131 motif-positive RKKTQLSQL P SNSGL P SKIFTYRELEKATGG 9632.m01135 motif-positive RKDVQ P LQ P SRD PG L P LKAFSYAELEKATDG 9632.m01392 motif-positive KRRMISFSWFNYGHRQCTDVLRQFS G MLNMLCSSN P KRR EV P TT P INNETLKKVSYGDILKATNW 9632.m02040 motif-positive RRARKRRRRDDGVSYDDS IDDDDEEDMES G T GP RRI P YA HLAAATGG 9632.m02719 motif-positive R G NKFKCC G A P FHDNE G R GG IIAFRYTDLAHATKN 9632.m02858 motif-positive RHKQKIKRQALLRQTDEFFQQHGGQILLEMMKAD G ND G F TLYKR G EIETATNN 9632.m02868 motif-positive KYKQRIKRQDIMRKR G EYFHLH GG QLLTDMMNIENNISFK LYDRDDIELATKG 9632.m02875 motif-positive KYKQRIKRHDLMRKR G EYFNLH GG QLLTDMMNIENNISF KLYDRDEIELATKG 9632.m02879 motif-positive RHRQNTKKQALLRQTDEFFQQHGGQLLLEMMKVEGNV G FTLYER G QIETATNN 9632.m03307 motif-positive WRIK G KWIIAH P LEKSEDSI G IIAFRHIDLRRATKN

PAGE 44

34 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9632.m03373 motif-positive SRRRRRRR G VAVAT P VLHLATAVA P PKHPGK PP KDIQEV P SRAAAAAAAPKAQ P AQVIQA PP PQPP P SESIQIETGKEHRIT FREQQHQPPQPP P YHQRS GGP SSR GG S G ESRGG GGGGGGG G AE P GV P EVSHL G WGHWYTLKELEDATAM 9632.m03459 motif-positive RRHR G SSSPVS G RIHAEPT G TAPRVERRLSALLSKG P NTTV EQF P LVALRAATDC 9632.m03631 motif-positive WWRRRKPEEHFFDV P AEED P EVHL G QLKRFSLRELQVAT DN 9632.m03678 motif-positive QRKPRPHHSRDVAAELALHSKEAMSPSVYT P RVSDAR PPP P PAAVV P AIQ P AVAANVA G KKKLFFF G RVPRPYDLEDLL 9632.m03707 motif-positive SCHLRRRAHNLKRSKKDIEVTAVSVEYEEVTCKQMCTKEI YDATEN 9632.m03789 motif-positive KKSKKIDP P KQSYST GG LPLKSFTYEELHEATGG 9632.m03791 motif-positive KKTDL P KQSSST GG LPLKSFTYEELHEATGG 9632.m03956 motif-positive GSGKVQQTTDVDVKVKLDNGSYHGVAYMHNSFDLYSQL YD GG SWSMQ GP D P ATDSA G KIVGSQVKSNLKSKRKTIIEK SRVSSDEVNVAK G LLPDNA G QKNIMKHDVIRETVPSLHV VAEETENDSKTVSTSNRENTSGTPERSFSSVHQLEDSDLSD EDWNDEDSGS G SGFSNT P SFDMFDDASRNKKKDLILVHLL RLACASKDSLSASLPAISSELCNIGILSEWAKDLISKS P AVF G ETF G HFF GP QMTSSECSLFWRADNSSSRPNSRYLND 9632.m04082 motif-positive HMRK P LETVA P LQDKQIFSAGSNMQVSTKDAYTFQELVS ATNN 9632.m04117 motif-positive WKYK G FEKSR G TGRVSNSSATRKTGMRSSFSSMTSSTASF VSTIATCPPTVKTFSISELEKATENFS 9632.m04727 motif-positive RWRRRFIEKRD G VKKRRRS P GRGSGSS G TSTDSVSQPKLI MFNSRITYADTVEATRQ 9632.m04923 motif-positive SR G RENQW PP LEGNSQLTTTKNLSCDEV P YPF G LK G KSAP G FRVTCRKNDSAAMLRIGHQKFRIDQVSSQE G FVVIFA GP I YRLCYDRN G R P VVGST G I G PTNLTDTPFFFSKRNTLVATG CYSNFTATFTSSLHHHGWSTN G SCTTN G RVNSD G LCP G TA CCDAY G M P LDDAQEVTFEFNKTSASVAGTCSAAFILYQKE QIFKVSGNSKPMHLHQEEHIFRA G GGDSK P VHLEDVLV P L GERRMVLDWVIGRATCEQARNNSFKTQYRCNNESSCMD RFMGE G YVCRCKA G YDQHN G N P YEA GG CQAYSVITL P ED ILALA P IIRLVMAT G Q G Q G ALT P I G HLA P L P TTNRKKRKVE RNRAELFRKNGGLLLQQRFSMMTSQGEDSSAKIFSAEELK DAT 9632.m05098 motif-positive WRQNRRKRKLSLEQQELYSIVGR P NVISY G ELRSATEN 9632.m05100 motif-positive WMQKRRKLSLEQQELYCIVGR P NVFSY G QLRSATENFN 9632.m05101 motif-positive WRKKRRKLSLEQQELYSIVGR P NIFSY G ELRSATEN 9632.m05116 motif-positive RRYRRKRRAADIAGDS P EAAVVV P ELRRFSY G QLAAATN S

PAGE 45

35 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9632.m05247 motif-positive KCRVLSGKRHQNKVVQKRGIL G YLSASNEL G DENLEL P FV SFGEIAAATNN 9632.m05252 motif-positive RKCRGKRQNKVVQKRML G YLSALNEL G DENLEL P FVSFG DIAAATNN 9632.m05258 motif-positive RRRQRPRVSDDDAGV PAATAAVHAR P NPALAA P SINLSSV KEATGN 9632.m05263 motif-positive NYTGVVERLVWVASSRAWQRFFQGPRD P CDSYARC G PFG LCDADAAATSFCGCVD G FTAAS P SAWALRNTSGGCRR G V ALDCA GGGGG SRTTDKFKVVRGVKLPDTRNASVDMGAT AAECERRCLGNCSCVAYAAADINGGGCVIWTDDIVDLRY VDRGQDLYLRLAKSEFDVI P DNPSM G VASVNLATIKSITEN 9632.m05264 motif-positive RRIQA P RLITKEA G PPADEAIFRSDSVKSAVLSSPLVEFSTIY SATNN 9632.m05414 motif-positive MKRGKKRRR P SSAAY P SPKKSAAMSEVSRDNTDL G YVEC V P DEETAAMMMPEEKARRLERS G CLTFCA G EGASYSLEQ LM 9632.m05461 motif-positive RADSVAA G RVGDE G YSLVFSHFRRFTYDELSDATCG 9632.m05466 motif-positive KCAKIAIKMWYSSSRDHHT P IANGG G SSSSR GG IGGADAD VVEM G SMSHFIE G LQNERPVRFSARQLRA 9632.m05467 motif-positive MRCVEAKHAERARRREEEAV P VSPPAS G TYSSVDVRVEM GSVDRFLDDILREKPARFTPENLREFTG 9632.m05492 motif-positive WKFLR P DIMRRLMR P KRAPSEV P EYFSGNMS G NLRTITYF DYATLKKATRD 9632.m05758 motif-positive RKKRGSVP P NAASVVVH P RENSD P DNLVKIVMVNND G NS SSTQ G NTLSGSSSRASDVHMIDTGNFVIAVQVLRGATKN 9633.m00004 motif-positive TTKKKKKQRRRDN G YRAGFMS P TS P LSSHH P SSGS G ASAN V G SSLDPSFKTNYSAGSPKLKACMSDISMGNSRFFTYQEL YQITDA 9633.m00288 motif-positive FGFCSHKKVHNSVQ P NIASNNN G GG GGG AAAAV G SGA P S P YGS P N G SLGRLRRQLSRVMTRQRS GP SSFKD P AEEFTFA QLAAATKD 9633.m00622 motif-positive RRKKKMKK P QT P LTSR P SSSWT P LSLNALSFLSTGTRTTSR TTYTSGTNSDTSYRIPFVVLQEATNH 9633.m00690 motif-positive HRR G GG AR GG EDKD G EMS PP WDVTLYQKLEIGVSDVARS 9633.m00702 motif-positive APAAPPDVLSS P AAAAGE G EAEALLAVKAALHDTANVLA DWNAGSGGVVVAGGGGGGGPCNWSMVTCSKTGHVSVL DLAHRNLSGTLSPAIGKLRRLRLLFLQHNAISGPIPDTIGRL KVLQTLDLAYNHFTGTIPSILGHSKGIFLMDLSFNNLSGPA PVFSANSVLFSALTSVQKVILRG SETFVSRYSGHIFPYQRPE IYLGHLKQFMIKEIKEATNN 9633.m01041 motif-positive TRKVSRRLHSLDLPKHHRRSSSSS P PPMP PP L PPPPP SANA P TL G KES P SSNSASD G AAAAVVV GG ERGAVQVFSYRQLHA ATGG 9633.m01042 motif-positive TRKVSRRLHSLDLPKHHRRSSSSS PPP M PPP L PPPPP SANA P TL G KES P SSNSASDGAAAAVVVGGERGAVQVFSYRQLHA ATGG

PAGE 46

36 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9633.m01043 motif-positive TRKVSRRLHSLDLPKHHRRSSSSS PPP M PPP L PPPPP SANAP TL G KES P SSNSASDGAAAAVVVGGERGAVQVFSYRQLHA ATGG 9633.m01831 motif-positive QSKKRKVLNNSASHSS G WL P VY GG NSHTSTSKSS G G RSA ALIN P NITAMCRHFSFGEIKSATKN 9633.m02137 motif-positive RQKKRAQKLVSIND P FASW G SM G QDI G EA P KIKSARCFTL EDLKLSTND 9633.m02285 motif-positive RRQQRIRLAKEKLAKEREEILNANNSS G RTAKNFS G RELR RATAN 9633.m02291 motif-positive RRKKKVAKDT G KSDEGRWT P LTDFTKSQSATS G KTTNT G SHSML P ANLCRHFSFAEIQAATNN 9633.m02806 motif-positive P FYFFL P SLFCLQ GP WWLT G HDLEGS G CVWEWGLHPWLP KSKLEGGKAFLAVGTSSVAAEFVINGTSLGLGEEREVQQL SRLEYLVDHGSVPSGMVAPNTRSFSLDKSKSQGGLDSRKD AFIPRDANGQPIAAHTFTFRELAAATKN 9633.m03039 motif-positive TRRWKP G TVD G A GG ASCN G DK PGG A P ASSC G SSVRGYN NSRYYAAAAA G CIY GG RL G FSVQ P RNRGAQVFTYRELES ATDG 9633.m03361 motif-positive RRRKPAAKRASSAAAAAAA P GGAAV P LSPATI PP VSKEIQ EVAVHVGSLRHYLEAGATFLKEGGGVGGAVVDGDSLGG STVYGSQRVHIEAGKGRRMVAYADGEVGPVASDLAASA QAAVGVGVGPEVSHLGWGHWYTLRELEEATAA 9633.m03735 motif-positive RRVRSAASHSAVPTALSDDYDSQS P ENEAN PG KLVMF G R G S P DFSA GG HALLN 9633.m03811 motif-positive RQKRRAKELKERAD P FASWAA G QKDSG G A P QLKGARFFS FDELKICTNN 9633.m03812 motif-positive RQKRRAKELKERAD P FASWAA G QKDSG G A P QLK G ARFFS FDELKICTNN 9633.m03875 motif-positive KRNRK P HKHMMI G SVDL G DEDEMR G SESLLYDLSTLRAA TAN 9633.m04105 motif-positive RRRRRRRRRQ P TS PG DFL GP L P VTSRHHQQSQFIK P TVTYP P QLNAHS P LQSSSNSD PP S P LLQ P S PPPP AAS GG TVSY G DL VAATN G 9633.m04180 motif-positive KRRKRLARTE P AWKMT P FQPLDFSEASLVRG 9633.m04201 motif-positive KKKQ G SMNTSIK P QNEANYV P TNDSD G H G SSMQLENRRF TYKDLEKITNN 9633.m04202 motif-positive RKKKQ G SMNTSVK P QNETASYV P TNGSH G H G SSMQLENR RFTYNDLEKITNN 9633.m04204 motif-positive RKKKNKSK G AVK P QIL G N G VQSHSQN G SGGSLLELHNRQ FTYKDLAVITNN 9633.m04448 motif-positive KKFWH G LLSSM G KSKEA P NIESFLQKHEAQHPKRYSYSEV KTMTKS 9633.m04450 motif-positive RKRRQYKMTSSSRLLKYTTS G RTPRSK G SSDKFVESGSFH YLQTHHFAYEELEEATDG

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37 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9634.m00267 motif-positive KWYKRPQDWERRNSFSSWLLPIHT G QSFTTSK G GSSKS G Y TFSSTL G LGRFFSFAEIQAATKN 9634.m00625 motif-positive RKWETR P EITDE G YAIISSQFRRFSY KELQKATNC 9634.m00626 motif-positive KWGRR P EIRDE G CTIISSQFRRFSYKELEKATGF 9634.m00628 motif-positive KWGRR P EIQDE G YTIISSQFRRFNYKELEKATDC 9634.m00665 motif-positive KKKYFQQHG G MLLLQEI G LKQGQSTAFTIFTEAELIEATN K 9634.m00812 motif-positive KKLTTTSVS P HLSSG G VMDERNSEHPRISHRELVDATGG 9634.m00813 motif-positive RRDARRSMLLA GG AGDE PG ERDH P RISHRELAEATGG 9634.m00996 motif-positive RPHSPPVFKDVSVSK P VSNVP P KLVILHMNLSLLVYEDIMT MTEN 9634.m01240 motif-positive NKQYEYLE PG CQLLSL P SSS G ANELI P K G RHDFWDNQLEI DHGQTSVPEKEKDLVEVPQEAERVSDKSVGTESSRSDIAL DGVAE 9634.m01323 motif-positive RCVKKNGL P AVNINTN P TAAAAMYAVVPDSQIRDATVER FLKEIAGEKPIRFTAQQLAGFTNN 9634.m01742 motif-positive RRRRASASVAAPARSPESSTETLR ANGSLNSSVSLSVASD WDHHPPPAKRAAAFWAWR G G ANN G SHS PPP VSVS G I P KY HYKDLQKATNN 9634.m01764 motif-positive SIRKLIELTSRKNA G FLPELVK G PRKFSYKELSAATRG 9634.m02243 motif-positive RKKKTLEKQHSKTWM P FSIN G LTSLST G SRTSY G TTLTS G L N G SY G YRFAFSVLQEATNN 9634.m02283 motif-positive PRKWLRNRKPKKL G SFIKKSH P LVSYEELNQVTSS 9634.m02784 motif-positive RRKKKMD G LVYHYD G NNYFVPSSQF GG SSRNHH P P P SAI MLNS GG ASAD GGG YYNS G TFS GG E G T G P A G SKSRFSYEE LTGITSN 9634.m02811 motif-positive RRRRRQAERYY PG FAVPSYT P QHMS G EA P FLR PP SAS G SM NFSA G QSQ G VS P MMSS G QAY G QSTSY G QQQRLTSANYST G SQGG G AARSVAAS G ELSV G NTKAFTFDELYDITAG 9634.m02861 motif-positive KRKRH PPP SQDDA G SSEDD G FLQTIS G APVRFTYRELQDA TSN 9634.m03396 motif-positive RMRTRRERVD G ENIEHL P GMPRKFSFEELKVVTGD 9634.m03685 motif-positive RSRKRVPQNSRKSMQQE P HLRLFN G DMEKITYQDIVKAT NG 9634.m03690 motif-positive RIY G MKEMQAN P HCQQINDHVKNITYQDIVKATDR 9634.m03723 motif-positive RSRTLAGKKAMSMSVA GG DDFSH P WTFTPFQKLNFCVDN ILECL 9634.m03838 motif-positive RME G RQLT G VW P AES G YEMITSHFRRYTYKELQRATRK 9634.m04131 motif-positive RKSKDGGISESKDVASTFAVNIDRASNREIWDHTQQDA P V SSSVL PP MGKMT P ERVYSTNSSMSKKMKVSVTANPYTVA SLQVATNS 9634.m04643 motif-positive RRTSSVHQNKSSNNGDIEAASLSSVSEHLHDMIK G TILVM V P QGKGGSNNLKFKDILKATNN 9634.m04648 motif-positive RKVMSN G AVRDG G K G VEVSLFDSMSELYGDCSKDTILFM SEAAGEAAKRLTFVDILKATNN

PAGE 48

38 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9634.m04650 motif-positive RRVMSN G AVHDG G R G VGASLFDSMSSELYNDNDSSKDTI FFMSEVAGEAAKAVTFVDVLKATNN 9634.m04652 motif-positive RKLMSNAAVRDGGKGVDVSLFDSMSELYGDCSKDTILFM SEAAGETAKSLTFLDILKATNN 9634.m04653 motif-positive RRVVSN G AVRDG G KCVESTLFDSMSEMYGDSSKDTILFM SEAA G EAAS G VTFVDILKATNN 9635.m00383 motif-positive RRRSATQRWQNHHAAAFGY QGNTTAYYYHHTGGAR P Q WAATKT G APSTP P NMMMH P TNMTG P HVVVRPPLVPPPPP PVPAGLDENAFGYDELAAATGG 9635.m00413 motif-positive RCVKKNGL P AVNINTN P TAAAAMYAVVPDSQIRDATVER FLKEIAGEKPIRFTAQQLAGFTNN 9635.m00473 motif-positive RWKKKTSANKTKDNPP G WR P LVLH G ATT P AANSRS P TLR AA G TFGSNRM G RQFTVAEIREATMN 9635.m00841 motif-positive NKSKWC G VPLY G SQ G ND GG IIAFRYTGLVRATKC 9635.m01226 motif-positive RRRRRSTTTAGDVSDDESVASL P PLPRE G LYIFTKSELKQA TNG 9635.m02031 motif-positive RKARRCKKVRVLDET G DFITGC P TYPFEIIRAATNG 9635.m02803 motif-positive KYCRQFVIRFLKPFMRDEKLMD P RGKSE G TSKRRKARKK DGLINSTQIFSASDKEGNGTGGSTEAQSNKAHDSTNVELP N G LNGRQI G KL 9635.m03088 motif-positive RRRRSGEVNCTAFSSSL GGG GNNTN G RQLVVK G SARREF RWEAIMEATAN 9635.m03453 motif-positive KQLPFCSNANTITHMPEGTYKTNLLQLAKNLITNVNQTQL HSAN G TAGAAG P DTVYGAVLCRGDSSAESCATRLQRVLD TASINGTSGDDSGYFQNQKNVTLYDHDFQALLSFSDKDFI SSFSNAPECTVSAYLNPPPDADRAQFSQLFSELMEKIAAAV VSRRPVNYLTGRGWFDLKSQTVYALAQCTDGMPPENCRS CLDGIIDEGKKMVGGGLTGGAVLGMRCSLWYQTDVKFF AGDPEVSLHMPTQQARFELRLLSMAVQNVINLWRIEEGNS GFSLYDFSQIKEATQN 9635.m03481 motif-positive KRKTERARK P SIADPTD P ADIESIDSLILSISTLRVATNN 9635.m03483 motif-positive KAWHFC G SSGDVFA P RSTYQSNLALLSAGLAKNASASPA LFAAGGVGDPPDTVYGLALCRGDTTNATACGACVAAAF QDGQQLCAYAREATVFYDPCYLRFSGRNFLAADGDNFAA YFSKVRNVTAPAEVFDAAVVALLNATADHAAASSPRRFA TGVEAFRGWGVRDIYALVQCTPDMSPAGCRSCLAGIISWV NDPDYFSGSPTGRVLGVRCNYWYDVHPFFPGSPLLRLDAP AFDVSPPAPSPAPVAADTTPPA DRAVDRDLKFFSYTFSVSQ CSVFICLKRRKASKNQNTPIIPAPNKIKRGNCAIFDLPTLQI ATDN

PAGE 49

39 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9635.m03495 motif-positive YPWGLCNDTAGEFPARRSSYLASINLIAATL PG NASAS P DL FATAEGVGAPPDQVSALALCRGDANASTCLACLTQAFLD LPNACAYHKVAAIFYDSCLLAYSNATIAAGDFSSEKIPIYG FYSNANATTEQARFNRLV AALVNATADYAARNSTRRRYA SGEADFNAEFPKVYSWAQCTPDLTPASCRSCLAQIIGTYIG FFENRVGGFVRAVWCSFQYSTTPFLDGPMLVRLQGTSGAS PAPSPAAVVPAVNQTPPAATPTLEGDANYSTEAEDIENLDS MLIDISILRSATGD 9635.m03516 motif-positive Q P WEIC G ENGNYTANSTYQANLKQLAAALHKNVSSGTGG GRLFAS G AVGAV P DAVYALALCRGDINASACADCVGTIF QDAQQLCPYRKEVSIVYDSCYLRFSNLDFLSSADNSGVVD LYNTGTVSGDVGRYDRAVTGLLNATARYAAGNTNASSRL FATGVMVGFDAQFPKIYAMAQCSPDLSPAQCGLCLGAMV ARWWQTFEPNTQGARSVGARCNMRVELYSFYNVPSMLQ LQAEAVAPSPSPAPAPAGKPPAVPGTTGAASRSEDFESIES LFLDLSTLRIATDN 9635.m03524 motif-positive RRRT P ARKAS P V P YSTN P DDIQSIDSLLLDLSTLRAATDN 9635.m03526 motif-positive RRKRT P ANKASSL P FSTN P DDIQSIDSLLLDLSTLRAATDN 9635.m03530 motif-positive DAQPMPWHRCNTSSGNYTANSTYQSNIQYLATSL P AYASS S P SLFAS G SSGAP P DAIYALALCRGDTTNASSCATCVAAAI QAAQKHCALVKTVAIYDDPCIVRFSNLVFPVSPPYNKGMF VAWDDNNVSAAAAAAFDAAFARLANATAEHAAADSVR RFATGEEAALAVAGEVYPKIYSLAQCTPDMSADACRSCLE DILVRMVPTYLAGRKGGRVLGVRCNFRFETYPFFFGQPLL QLPGSPASSSAPVNGVPTESSTDDMQSIGSLILDLSTLRVAT DD 9635.m03531 motif-positive RKKRRR G KAEHFT GP DAAEDFESVKSTLLSLASLQVATDN 9635.m03539 motif-positive CGTS G GNYTA G STYESNLLRLASTLRANASASPTLFASGV RGVGPDAVYGLLLCRGDMNPSDCFDCGTNVWRDAGPTC NRTKDAILVYNQCYAQFSDRGDFLAATNNSGEVSLLISGT NITSTDVAGYDRAVTELLNATVRYAVENSTKLFATGQRV GNDTGFSNIYSMAQCSPDLSPAQCRSCLDGLVGQWWKTF PLNGKGARVAGPRCYLRSELGPFYTGNPMVRLPVKADEL TQRRLAKAERHPGTDTNEDFESVKSTLLSLASLQVATDN 9635.m03613 motif-positive DKLFRNKVAN P VRFQS P QRFTSFDSSIPLNQVQDRKMEDE TRHSNELNVTLFDFNTIAFSTDN 9635.m03616 motif-positive KCRRRIKEKL G IGRKKAQL P LLRPARDAKQDFSGPAQSEH EKSEE G KNCEL P LFAFETLATATDN 9635.m03618 motif-positive KRGRNIKDVMHKSWRSMHTSTRSQQNS G MLDISQSI P FED DTED G KSHELKVYSFDRIKAATCN 9635.m04060 motif-positive RARFPVDQDPD P ESLSCEN P KCGGGGGGGGKCGAFHMSA TSSPPSGCSSSCVTGCSSSSEGVKVFRLDKTAFTYRDIVAA TSG 9635.m04226 motif-positive KKKS GG KKVR P R GGGGGG LRRLSFTDLTGAADQDLSVSL VGSNLHVFTVAELRDATRG

PAGE 50

40 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9635.m04270 motif-positive KKKYWSLPR GG D P EQKE P LS P IVS G FKDSLKQMKSIKIISTI GKEELQKTVSMNLKPPTRIDLHKSIDENDVTSKSFTRKISFS SIRTPAYTVADLQVATGS 9636.m00204 motif-positive RRRHQRKKMREAEEANDDDDDTEGD P IMEIEN G MGPRRF AYHVLVNATKS 9636.m00205 motif-positive RRRHQRKKMREAEEANDDDDDTEGD P IMEIEN G TGPRRF AYHVLVNATKS 9636.m00208 motif-positive RRRQRKKMREEEEDDSEGD P IVEIEM G TGPRRF P YHILVN ATKS 9636.m00210 motif-positive RRRRIKNRKEAEDEQD ISSDSEDNDGE P IVEIEM G TAPRRL P YYELVEATKN 9636.m00214 motif-positive RRRRSKKRREVEEAEEARHVGLARDDDDDDDGE P IVEIE M G MGPRQIPYQDLIEATNS 9636.m00216 motif-positive RRRRIKNRKEAEDEQD ISSDSLDDDGE P IVEIEM G TGPRRF P YYELVEATKS 9636.m00218 motif-positive RRRQSKKRREAQDGSWH G SDDDDD G ELIMEIEM G TGPRR F P YHKLVDATKS 9636.m00223 motif-positive RRRQSKKRREAEDGGWH G SDDDDD G E P IVEIEM G MGPRR F P YHELVDATKS 9636.m00224 motif-positive RRRQSKKRREAEDGGWH G SDDDDD G E P IVEIEM G MGPRR F P YHELVDATKS 9636.m00225 motif-positive RRRQRKKMREEEEDDSEGD P IVEIEM G TGPRRF P YHILVN ATKS 9636.m00229 motif-positive RRRRIKKRREAEDEENASTDSDNGE P ITEIEV G TGPRRL P Y YELVEATKN 9636.m00233 motif-positive RRRRSKKRREAEEAEEARHVGLA G DDDDDDD G E P IVEIE M G MGPRQI P YHELVEATKS 9636.m00365 motif-positive RYRRSQAQIRSSSSRRASTIPIRANGVNACTILSNSTTGQES P REVEDR G ASMWLE GP GRKSVISASGIPKYAYKELQKATS N 9636.m00457 motif-positive RWLQWRGGDDTAAADAL G PGGARH P DLVVG P WRMTAF QRLSFTADDVARCV 9636.m00764 motif-positive RGKQR G DEIYG G LMLGDISTSRELSDRKVDFPIFSFREIASA TNN 9636.m00975 motif-positiv e TQKRKRLEVEMEELLSIVGT P NVFSY G EIKSATDN 9636.m00986 motif-positive RQKRKKMEAEM G ELLSVV G R P DVFSY G EIKSATNN

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41 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9636.m00990 motif-positive RNCKISDNLRTVNFSKLGR LTLLDLSFNNITGEVPQSILNLN NL G YLFL G NNSLTGSL P DAKSSSLTNLDFSYNQLTGSFPSW VTNNNLQLNLVANKFNIRENNNSILPSGLNCLQQDTPCLL GSPEYYSFAVDC G SNKSMK G SDNTIYEVDAANLGVASYY VTRNTRWGVSNVGIFNDASSRNYVINSSQQFQNTLDSELF QTARMSPSSLRYYGLGLENGNYSVKLQFAEFAYPDSKTW ESTGRRIFDIYVQGVLKEKNFDIRKAVGGKSFTAVNKIYNT IVSKNFLEIHLFWAGKGTCCIPTQGYYGPMISALSVTPNFT PTVRNGEPKKKSKAELYNLAGRPNVFSNAELKLATEN 9636.m00993a motif-positive KKRRRLAQQQ G ELYNLV G RPDVFSNAELKLATNN 9636.m01372 motif-positive KRR P FLRYTRA P QHHETEFDEESIGIRPYSFHDLELSTDG 9636.m01496 motif-positive KTRMK P NIVDNEN P FLYETNERISYAELQAATES 9636.m01743 motif-positive RSRRARRRS P TLPFS PPP APAR P LRRYSRRALRRATGG 9636.m01856 motif-positive KKTRQALDSKDKKLSSTTK G HMLLPMF G KLNSMKTSKKE VVAMMDFSVLDSATGK 9636.m02427 motif-positive KKHKCLL P WQRSTTA P RLHSLLRSQLKSYTYSEVRKMTKS 9636.m02489 motif-positive KFHKNKTKEIQA G CSESL PG SSKSSWKLSGIGEPLSINMAIF ENPLRKLTFSDLHQATNG 9636.m03449 motif-positive RRRRRHRCLLS G PSSVL G ILEK G RDVED GGGG EVMARL G NVRQF G LRELHAATDG 9636.m03477 motif-positive K P KINSDL G ILFQGSSSKLNEAVEVTEN 9636.m03886 motif-positive RSRRSDSS G AIHGA G EAWEVTLYQKLDFSVDEVV 9636.m04075 motif-positive KRRRKVS G GDDGS G IT G TMIRSLA G GPREFEYRELRKATN N 9636.m04332 motif-positive TEAAGDGCSAGCDLALASFYVTPNQNVTNMADLFGIGAA NYRSLA P YNPNI P NLDFINVGGRVNVYFTCGCRSLPGSPGA TYLAGAFPFQMSRGQIYTSVAANYNNLTTAEWLQATNSY PANNIPDTAVINATVNCSCGDASISPDYGLFLTYPLRAEDT LASVAATYGLSSQLDVVRRYNPGMESATGSGIVYIPVKDP NGSYLPLKSPGRRKAKQATLLQSSEDSTQLGTISMDKVTP STIVGPSPVAGITVDKSVEFSYEELSNATQG 9637.m00068 motif-positive RKKRQREEAAKQRM G VVFKK P E P DES P D G IGRSLSCCLRK KAGDESDSTEEVTDTSASKEGVVAAKAKT 9637.m00536 motif-positive KKW G S G FKK G L G AKAAV G K P RQYTYQHLFSATKG 9637.m00673 motif-positive RRKTK G SANNTIN P HNE P TSHSH G S G SYGH G SMQFENRRF TYKDLQMITNN 9637.m00942 motif-positive KLRMNQKTEEVRT G YVESL P TS G TSSWKLS G VREPLSINV ATFEKPLRKLTFAHLLEATNG 9637.m01166 motif-positive RRWRRRRRRQQAQ P LPLP PP MLYNPNPYYKGDQPPLPFVF MQQQHHHPTAPQTSGGTFSDAGSERPHSISIDGGSLSYDQL AAATGG 9637.m01453 motif-positive KCRRKRMALSTKHQ HIKAAATHESEP NELRDAEAGALE P VASSA GP NHGKEY GG D P AAAAG P RQYEY G ERVVSDGPR HGAAYNELVAAGPRLYEYGELAAATRD

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42 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9637.m01534 motif-positive PFVSSVNLRTL G SGVYH P VLAANQSMCLFDRRNMGSNVSI LRYPDDPYDRYWWKMRSDPTWKNLSTASTIEQNDNFVVP LPVMQTAIEASNNDTIIKVTRKDKTAHKCMIFAYLADFQN SQLRQFNITLSDTKPLLYSPPYLSAGIVDISDWDMPNNGM YTITLEPTSASKLPPMLNAFEIYTLIPSDNPMTFPRDSWDGV KCSNPSDNTSRIISLDLSNSNLHGPISNNFTLFTALEHLNLA GNQLNGPIPDSLCRKNNTGTFLLSFDSDRDTCNKSIPGINPS PPKSKLVFVGIVSADVPHSEPELEIAPASRKYHEDGLQRVE NRRFTYKELEKITNK 9637.m01540 motif-positive RQKRKPKILSYNLYFLE P VSTDD PP RE P ELDIA P ASRNNH G G TLLKVENRQFTYKELEKFTNN 9637.m01545 motif-positive RAKRKLNTSSTDLAMV P ELMGA PG HITNHWDHLQKPENR RFTYQELEKFTEN 9637.m01547 motif-positive RIVHDNPTTFSQDFDAIMAIKYEY G IKKNWM G DPCF P HEY VWD G VKCSDAVIFPTANCMGRY P ILSHCLL P SSTSSSTDLA MV P ELRGA PG HITNHWDHLQEPENRRFTYQELEKFTDN 9637.m01568 motif-positive GTPFVNTVELRQLDSMLHFRKIMGNSSIYLYERRNM GP SS RDN P IIRYPNDTYDRFWYPWGSEDDPTYSNLSAPSTLIIPPS PSYAVPSPVLETAVVPADNNKSVLSIIQTNDKEIHEYLVLV HYADFQSTLQRQFQAYSNGDPIQGTGGPYVADYTGQTVG TIDWISAETSGKYNITLAATDSSQLPPIVNAFEVYGRIPLDN PSTFPTDYTCKIISLAYNKLNRWIKELRLIKVPHKVDAIMTI KFEYGIKKNWMNDPCFPSNLVWNGVRCSTGSDNTMRIISL DLSNSNLHGSISNNFTLLTALEYLNLSGNQLSGTIPSSLCEN NAGSFVFRFSYLFNVDIGDNFVHLDSTYGPEFLNAPGSTK NHWDHMQKTENRRFTYEELEKYTDN 9637.m01571 motif-positive RAKRKHNND PP TVLELT G APGHKTNHWDRLQKPENRRFT FEELQKFTDN 9637.m01581 motif-positive RAKGKHNVSTFD P PRVPD P KKAPGSTTDHWSHLPINGSRQ FTYEELKNFTLN 9637.m01604 motif-positive KKQAIVKSRGQEQY G DHIHI P ENREFTYEELVKITNN 9637.m01629 motif-positive RVKRKSNIFAYN P PRVPE P TNASRNEKYHWDHLQENENR QFTYKELEKITDN 9637.m01633 motif-positive RAK G KSNISI PG SEKYHWDRLQKNENRHFTYDELKKLTDN 9637.m01645 motif-positive KAKRK P NTSAYN PP RVPE P MNAPVSEKYHWDHLEKNENR QFTYEELEKFTNN 9637.m02208 motif-positive RKIRERLAAVREEFRR G RRR GGGG SS P VMKYKF P RITYRE LVEATEE 9637.m02488 motif-positive RNKMSKN G RKKK G KSSTMKVYL G RQKS P SRDT G YNADA DDD G G G DDDDIVI P GMPARFSYQEITTMTSN

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43 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9637.m02629 motif-positive CQTRCGDVDI P FPFGI G DHCAIHE G FRLECDNATKGTSNQK PFWGDFEVIKISMEDGKVWVKAYMSRQCYDQSTGGMSY SDASANLSGSSFWLSDTDNKITVIGCKTLAYMTTDSYVIG CSSACDNKVNKLTPKNGSCSGAGCCQANVPKSIQYYQGY FNEGYNTTKIWMSSPCSYMAVMETAAFNFSTSYLTSSVFY DTYKGGVPVVYDWAITSKTCTESRRNKTSYACISNNSQCI DNLTNAQGYRCKCSNGYEGNPYIKDGCKDIDECLNNATY PCKGICTNTLGNFTCSCSPGSYMMNGDCMPKKKLRFDSVP VVVELQEATNR 9637.m02693 motif-positive RMRR G GGAGG GG RVVH G E G AWEVTLYQKLDISMDDVL RGLTSAN 9637.m02882 motif-positive AI P YPLPL P RGGGDTYDAVAANYADLTTAAWLEATNAYP PGRIPGGDGRVNVTINCSCGDERVSPRYGLFLTYPLWDGE TLESVAAQYGFSSPAEMELIRRYNPGMGGVSGKGIVFIPV KDPNGSYHPLKSGVGIVLLF CELLCIYAKVAKVQEGHIASI SRRNQPPCCYYLCDDASQAEGIKVERSIEFSYEEIFNATQG 9637.m02917 motif-positive FVSLVHQVAVDTEHDDSI G VTDES G W P CIELDRRMRRMN GRNPEKNIKMEHENDSSLAWVFHAPYREHENEFPDIPEEK EGNGFAPKSDDPTKAPPPIEVPELSFDELKEKTDN 9637.m03043 motif-positive EKIMTGKLTDFTSAYAYKCRLFFIRILSAWPAGSIDCCEEM INRCFCCVT G GDSDPE P AATSSRRRTNPARASKNRTSVDY PWETYTLKELLQATGNFSESN 9637.m03212 motif-positive PKFKEEMIVKHDGKNNKKRALRVLSVSDEFGKEI P AQDLD F P FVEYNEIATATENFSDAAMN 9637.m03219a motif-positive KIKGKKRNREKHRKLIFD G ANTSEEI G QGN P VQDLEL P FV RFEDIALATHN 9637.m03220 motif-positive KIKGRYIQCSFIYLDAYGKKRKREKHRKLFLDGACTSEEIE DGS P IQDLEL P YVRFEEIALATHN 9637.m03224 motif-positive KACKKRNREKHRKQILFGMSAAEEVGEGN P VQDLEF P FV TFEDIALATNN 9637.m03295 motif-positive RRNHSSPVSSHYYTDESGRRNSSAVNMKSLEHS P SMGCKT PP AVPRKSMSDNEFENKLNHSRRSTDPISLMNHSSSDLQA ATGN 9638.m00012 motif-positive RKYSQYREMARTL G LEYLPA GG PRRFSYAELKAATKE 9638.m00059 motif-positive KITKKKKRPPPPNM P FFTDEK G NVYYATGGLPPMWQQHG SSNYSIPPPPPPGWHMSSSAG GFSGEMGMGYSSGPYGPAL PPPSPNVALGFSKSSFSYEELAAATSG 9638.m00828 motif-positive KQHLKLKKFYEQN G GPVLK G VRNIKIYTKKELKQITSN 9638.m01519 motif-positive RKLQAKQCSPLAATAQQLIVKQAASKQIKQQSTYKSKQA MLAVAD G DGAAL G EDVGEV G VLDECAVAVVEGEAVVA ALSVARGPGDDVGTRSALVLRHLMVAPRKSSATASWSST ARAMRPPPRPTAASPSHRLGPAAASPDRRHLTPADTADQD VEAGSLLFDLATLRKATAN 9638.m01657 motif-positive RKQKWFSR G VENAQE G IGIRAFRYTDLQCATKN 9638.m01661 motif-positive RRK G KLFAR G AENDQ G SIGITAFRYIDLQRATKN 9638.m01937 motif-positive RKKLR P SSMRTTVA P LPDGMY P RVSYYELFQSTNGFNVN N

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44 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9638.m02114 motif-positive KRKRKGTRQEHVEQRQPFNSY P SNEVKDVK P IPESTKIEVE PLPS P VAVSLK P PPKIERNQSFDDDDDDFSNKPVAKKSNSA SVKATVYSVADLQMATDS 9638.m03092 motif-positive HHSKKSRVVRLGGGDIKDKAAEQA G KKVSS G SGNGSRST TESGKGAADQLQFFRPEKATFSLDELF 9638.m03136 motif-positive RRKPKKSFDKIPVSQIPDVSKEIAVDEVREHAVVENFRVQE SHAISVQEKHYEKDSGKMLAHLVRSKSSDADNLSQCSSV YQCDRAGSSYS G DEGSS G NARRHFSQYATVSASPLVGLPE FSHLGWGHWFTLRDLEHATNR 9638.m03492 motif-positive KAYKNKRRREQQQHDDEEEILVSDSAAIVS PG STAIT G KL VLFRKNSSASRYEDWEAGTKAVLDRN 9638.m03505 motif-positive RRCRRRRCSRLA P APPHH G RSNRSLKQQQSMVSDKDIEEA ARWPPPPSFQPPIEVIKAEQTAPLIMVEAARTSGETATSSGG STRGWSTESGGSDAAEPEASRRGWGRRYTRRELEEATNR 9638.m03516 motif-positive RRRQRRATLPVPEEEEKESV G TPWSPFT P DGEGSFGSAVV TPRRMNMKLHIPLAEIMVATGD 9639.m00075 motif-positive KRSRRTFKEIPITQIPSASKDIKEVRAVDEFLPNDFVVHDGL LLAIQNEPVEPVDKDVNQFAQEDKTIQ G EENSPV P LHYVD NYDVIQSVSTCEQSSSHAPVDSVLLPGLPEFSYLGWGHWF TLRDLELATNC 9639.m00613 motif-positive RHRHRHRVVVK P RCRCLQ P LSSAATL P VTAPSSRSCECVS SWSFYGGGGDAGDRSLKMLSLDDLAGATGG 9639.m00642 motif-positive REKQKRKSVSL P SFDSSF P KVSYHDLARATDG 9639.m00647 motif-positive RGKQKKKCTSLT P FDSKF P KVSYNDLAKATEG 9639.m00648 motif-positive RGKRKRESLSL P SFGTNF P NFSYNNLFKATEG 9639.m00651 motif-positive RAKLKRESVSL P FFGSNF P RISYNALFKATEG 9639.m00653 motif-positive GRGKRKKKSISF P SLGRKF P KVSFNDLSNATDR 9639.m00659 motif-positive RGKQK G HSISL P LSDTDF P KVSYNDLARATER 9639.m00662 motif-positive ANNLRGEV P SNLGNSL P NLQYLILSDNFFHGHFPSSLINSSK LNLIDMAENNFTGVIPSSIGKLAKLNVLSLQLNQFQAGTK KEWEFMDSLANCTELEVFSVARNHLQGQSFPPISYFGDIPN TLSNCESLEDIRLDRNAFTGIIPTSLGNIRSLKVLNLSHNKL TGSIPVSLGNLQLLEQLDLSFNHLKGKVPTNGVFMNETAI QIDGKSWALWRRKHEGNSTSLPSFGRKFPKVPYNELAEAT EG 9639.m00663 motif-positive KGKQRTNSISL P SFGREF P KVSYKDLARATNG 9639.m00664 motif-positive NRKQNRQSISS P SFGRKF P KVSYSDLVRATEG 9639.m00665 motif-positive KRKHKRQSISS P SFGRKF P KVSYHDLVRATEG 9639.m00844 motif-positive GGAVGLEERSDAAEDSLVSVLAVYDGFCNLKQINLELKV CGGSSIRKTLVKEAASY G AAHLIL G VAKNSLSFSRSSSISV AKYCAKRVPTGCSVLAVNNGKILFHKDAVQQEPYHSAST MTETPRRSYRKLLTSVIGEKLRDECEQDNRSIFRAVTMPPS SPAPTREVSLALVPMKVHRRESPEVATGWSFLRKKFLPDR KPASHDRSKMSVVQWAMRLPSRYSSASPVCSEYRTTTPD GITSASRILRDRVAVPSRSNSGKSSVVIEELDNSSDKEIPEEL IALREKFPSVYSTFSHSELAKITSD

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45 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9639.m00968 motif-positive RIKFNCCGV P LHHNQ G NSGIIAFKYTDLSHATKN 9639.m00970 motif-positive QNKLKCCGM P LHHTQ G NSGIVAFRYTDLSHATKI 9639.m00973 motif-positive EKKHKKAKAY G DMADVI GP QLLTYHDLVLATEN 9639.m01092 motif-positive RRRRRRGRGGQADQAAAMSLML P RHGSSK GP GSVVEHF ALEALQAATDG 9639.m01134 motif-positive LYSDMDIDLASYFKITGSPFDTFSSSKIKMHVTTILKSGEFS RQNNKSEIKSQLTSK P TLQSS QG ITDNF P SEQDFEHGAFKG PYVGFSDFGQSEMKGESSTQNKAGNKSQYPITIPKNPTLDS LKVITDN 9639.m01209 motif-positive RARSCR G GGGPD GGG AWRFTAFHKVDFGIAEVIESMKDG N 9639.m01301 motif-positive RRNKR P KLQPQ P RSPSYASWDIKSTSISTPHLQGARVFTFD ELKKITNS 9639.m01600 motif-positive D G SSSVAN G VTISSEMT P SGESFNSSYNSSTTPVDISLAAIE ACTDG 9639.m01817 motif-positive KLRDRR G DGCGGD G DDDEERKRGLFPFPCMRADDSSDDG SDAGDDVKRNNTTTTTTASGGGG GGEEGQLVAIDKGFKM ELDELLR 9639.m02341 motif-positive HHRKKKNVEKFR P VSTKTS P AESEMMKIQVVGANGISNGS SAFPTELYSHVSAANSSNISELFESHGMQLSVEVLLKATNN 9639.m02342 motif-positive RYMSKKSKADETIDSTRSSQDNKVH G EVINRWS G LYKFSK G EIEKAIN 9639.m02532 motif-positive TRQSKK P LKPRR G DIL G ATELQ GP TSFYYKDLKVATNN 9639.m03250 motif-positive RKNIKTNI P STTSME G HPLISHSQLVRATDN 9639.m03256 motif-positive HKKIQTEI P STTSMR G HPLVSYSQLVKATDE 9639.m03257 motif-positive HKRRKKEV P AMTSIQGH P MITYKQLVKATDG 9639.m03259 motif-positive HKRTKKGA P SRTSMK G HPLVSYSQLVKATDG 9639.m03260 motif-positive KLLARYKKIKSKI P STTCME G HPLISYSQLARATDS 9639.m03261 motif-positive RKPMSKL P SATSMQ G YPLISYQQIVRATDD 9639.m03532 motif-positive RRYKK G EVSLQ G DMNMQTDEEALAWGREACSSEFTSFKL SQVLDATNN 9639.m03591 motif-positive QRMRHR P EIYVDV PG QHDHNLEFGQIKRFSLRELQIATNN 9639.m03592 motif-positive QRMRHR P EIYVDV PG QHDHNLEFGQIKRFSLRELQIATNN 9639.m04143 motif-positive DVITLVKVIKSRVETVRQNKEDCELLAERADMILDLLRRV QASKVIED P DMWKPTE G LKSTLCRAGAIVKSCQEEWSYA YRFCKGGRIARELRKVLKDLKFYILHLIGMITIINHDQNTR YYYIPETDVVKPQDATASNAGKPVALEETGLKKFTLSELE VATDN 9639.m04147 motif-positive DVASLVKEIKERVQTVSQNKEDCELLAERAELILDLLGRL QKSKVIEDPDMWKPTERLRSTLRRACEVIEFCRERSCTYRF CKSDHTAKELRKQTPDVVQLQDGVQVPALGLPAQHFKYN DRNDR G ETLGIS G KAQLVTEPSSVNEPGLKRFAFSQLEVA TDN

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46 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9639.m04236 motif-positive ICGSSKYTANSIYQSNLDSLLSSSFLVVSGDSSS G ALFAK G S RGAA P DTVYAVALCRGDANASACSGCVDAAYAAATARL CPLSKDAAVFYDECALRFSDEDILNMDAFGRVNTSAAVG VAPLVLMNITSEPMLSGWNTNIQGTKNFTQFFIKTMNYIV AQALSTTKHYAAIRVDMDDADASNTVTLPRRLFCLAQCA PDLVEDICYNCLQNFSDLATANFAGRQGGRILALRCNLRY DTDKFFAGKTNADEDEALIWGLQGRSSEFTIYDFSQVLEA TDN 9639.m04368 motif-positive KWRRRWFFDNN GG RLLE G M G ITIFTEKELDS 9639.m04369 motif-positive GNESFRLPSLWFFSIDANNFTGPIPQGFAACQQLQVFSLIQN LFE G ALPSWL G KLTNLVKLNLGENHFDGGSIPDALSNITM LASLELSTCNLTGTIPADIGKLGKLSDLLIARNQLRGPIPAS LGNLSALSRLDLSTNLLDGSVPSTVGSMNSLTYFVIFENSL QGDLKFLSALSNCRKLSVLEIDSNYFTGNLPDYVGNLSSTL QAFIARRNNISGVLPSTVWNLTSLKYLDLSDNQLHSTISESI MDLEILQWLDLSENSLFGPIPSNIGVLKNVQRLFLGTNQFS SSISMGISNMTKLVKLDLSHNFLSGALPADIGYLKQMNIM DLSSNHFTGILPDSIAQLQMIAYLNLSVNSFQNSIPDSFRVL TSLETLDLSHNNISGTIPEYLANFTVLSSLNLSFNNLHGQIP ETVGAVACCLHVILKKKVKHQKMSVGMVDMASHQLLSY HELARATND 9639.m04384 motif-positive RMEYEKRKLRDHFNKN GG QLLKNI G IKIFTKEEVGKITNN 9639.m04387 motif-positive RKKWKLK G CYDRN GG QMLEKTSVKIFTKQELDKITNN 9639.m04402 motif-positive KKMKN P DITASF G IADAICHRLVSYQEIVRATEN 9640.m00074 motif-positive KRSRRTFKEIPITQIPSASKDIKEVRAVEEFL P NDFVVHD G L LLAIQNEPVEPVDKDVNQFAQEDKTIQGEDNSSSVPLHYV DNYDGIQSVSTCEQSSSHAPADSVPLPGLPEFSYLGWGHW FTLRDLELATNC 9640.m00282 motif-positive DDLYIYVHLVFFRLAEQMPTADAETVTSESTAVIQQDSTM VNVDAA G LHEQDEL P HWMSQLFEKLDLEVDEDVVDEDIC SIGNMTHEEEEADLERGIHSILKNHPIMKKLNKGIHPVVLA LHVLLEEEEEEEVVQEEDMAKGLAELDEYLSRHTYHTIEE ATAS 9640.m00283 motif-positive G CWLFSSK G LFRHSRVYAIDQEGYKLITSHFQRYTYADIK KATAN 9640.m01285 motif-positive KVKDRKKQ G RSSTVAA G DENESRHGLCRCIWGHRGVDS DTDTDDSSASENGGGGGKYG EGELVAIDRGFRVELDELLR SS 9640.m01512 motif-positive KRKPRE GG IRRSVS PG ITSIDRVTLQNATEN 9640.m03313 motif-positive NQSIGKTVVWTADRDVPVNGR G SRIELRD G NMVLLDFNS RLVWSTGTTSGQVRSAKLLDTGNLVLLGHDGSRIWQSFD SPTDTLLPTQPIAANLKLVSGKYMLSVDNNGSLALTYDTP EGHSKYWPRNINATPFSGDQPQGLDMLGCISAGNHIRKFT LKELVAATAK 9640.m03756 motif-positive HRYKNRDVVE P WELDY GP HRYSYAELRRATRG 9640.m03953 motif-positive RCRRS G GGGGRS G FDRLAAKRLLSEAASSSGVPVYSYHEV ARATNS

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47 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9640.m04040 motif-positive HRRRRDSKIRPVKLPSSSRDESVE P DLEHG GGP RRFSYGEL AAATND 9640.m04064 motif-positive RRRRKRAGEARELEMDEGD FFDDEADDFEKGTG P KRFHY G ELAIATDD 9640.m04066 motif-positive RFVLRRRRKHAGLTKEQEMEEG GIFDDETAMEDDFEKGT GP KRFRF G ELAIATDD 9640.m04068 motif-positive HRRRKHAGLTMEQEMDE G DFFDDEA G DFEKGT GP KRFR YGELAIATDD 9640.m04069 motif-positive RRRQWRRNNAKLTVKMARKH LPKDARFFRGKPIEDELEL EAA GP RRFHY G ELAAATAN 9640.m04089 motif-positive EQRSALCLFHATSQSCNQLKSIPVGHLLVDSHIFRDSGQRA IHTLSSSFSSSPRVLCWVLAVLGYSYRLQASSLSA G ELTDL Q G KFFMETFFRKDTPIIRNVKIYSSKELRKATKN 9640.m04300 motif-positive KKRRRAEAEAEEAA G GKVFGKK G SWDLKSFRVLAFDEHE VIDGVRDEN 9640.m04302 motif-positive RRWVLRARQD G EHDGL P TSPASSSSYDVTSFHKLSFDQHE IVEALIDKN 9640.m04368 motif-positive KSRQASNKKKKKKQGGSRSWFKLPMLSSQQASYASEEQQ G EEDDGD G DEVLI PG LPARFTYAELEEATEG 9629.m05927 motif-positive FKRKKSTEPTTASSSK G KTVA GG R G EN P KEEYSS G VQEAE RNKLVFFEGCSYNFDLEDLL 9634.m03594 motif-positive KQLR G VWPAEA G YEMIANHFRRYTYRELVLAT 9629.m00145 motif-like KRRIRRHQEMQEEEQEFEEL P LQGM P RRFTFQQLQEATDQ 9629.m00370 motif-like HRRKQTL G FIIHHKYT G NESNTEEELKRYQSLSPKRYRYSD LKKITKC 9629.m00524 motif-like HRKLK G RQNSQEIS P VIEEQYQRISYYALSRGSNE 9629.m00695 motif-like RHRRNQQIFFDVNDQYD P EVCL G HLKRYAFKELRAATNN 9629.m06510 motif-like RYENYKLEQFHSK G DIES G DDSDSKWVLESFHPPELDPEEI CN 9629.m07224 motif-like RRRSRYSSKRRSAKRI P MKID G VKDFSFQELSHGTND 9630.m01244 motif-like RHQKRQQDLA G WKMT P FRTLHFSECDVLGN 9630.m01729 motif-like KGRRKSHLREVFVDVA G EDDRRIAF G QLKRFAWRELQIA TDN 9630.m01864 motif-like RKRYNHGELREDWEVEF GP HRI P YKDLRRATER 9630.m05595 motif-like QKRKLIRTKQRFFEQNGGVILQQQMHS GGG T GG FKIFSTE ELKKATNN 9630.m05599 motif-like KRKLIRTKQRFFEQNGGVILQQQMHS GGG A GG FKIFSTEE LEKATNN 9630.m05621 motif-like QKRKLIRTKQKFFEHNGGVILRQQMHS GGG TH G FRIFSTE ELKRATHN 9631.m00434 motif-like KQLYHRWYVHGGCCDDAAVEEE G SGSW P WRLTAFQRLS FTSAEVLAC 9631.m04841 motif-like NGRRKSHLREVFVDVS G EDDRRIAF G QLKRFAWRELQLA TDS

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48 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9632.m00264 motif-like QKRVRNTF G KGTG G ARRFEYDDLAIATGN 9632.m00511 motif-like KRIRHTF G KGTG G TRRFEYDDLAIATDN 9632.m01387 motif-like RKSRGNQ P SKKVQSKY P FQHMNDSNEVGSENVELSSVDL DSVLTATNN 9632.m02841 motif-like RHKRSIKRQALLRQNDEFFQQHGGQLLLEMMKVEGNA G F TLY G RQEIETATNN 9632.m03289 motif-like RRHRKKLHCQALNSIYA G TGVI P FRYSDLQRATKN 9632.m03297 motif-like KWRNKTKLSGGTRKDYQFCN G IIPF G YIDLQRATNN 9632.m03298 motif-like KIPRNKSWLL G HRRKNFHS G SGVIAFRYADLQHATKN 9632.m03302 motif-like WRRKGKWFTRTLQK P EGGI G VVAFRYINLQRATKA 9632.m03306 motif-like WRRKGKWFTLTLEK P EVGV G IIAFRYIDLQRATKN 9632.m04086 motif-like RRNKRNCSSV G RIIC G TVAFRYKDLQHATKN 9632.m05124 motif-like RRKRKADEKEA P PGWH P LVLHEAMKSTTDARAAGKSPLT RNSSSIGHRMGRRFSISEIRAATKN 9632.m05259 motif-like WCRRKHKISE G IPHN P ATTV P SVDLQKVKAATGN 9632.m05801 motif-like RRKNRELKNADLHAQNPENAFCQSQSWRC P EGQS P MFQR YSYKETMKATNN 9633.m01394 motif-like RRRKTN P DVLPEAD P YKSRRFKYKELQVITN 9633.m02300 motif-like TLWRRSRRSTGGKVTRSSDAAK G IKLV P ILSRFNSVKMSR KRLV G MFEY P SLEAATEK 9633.m04198 motif-like RRKKQGSMNNSVKRQNETMRY G PTNN G SGHNSSLRLEN RWFTYNELEKITNK 9634.m01190 motiflike RRKPHDQFFDLLEEET P EVHL G QLRRFTLRELQVATDN 9634.m03596 motif-like RRE G KLAR G ISEV G YEMVTNHFRRYTYRELMIATRK 9634.m03679 motif-like RSRK G MKLK P QLLPFNQHLEQITYEDIVKATKS 9636.m00717 motif-like RRRK P QEHFFDV P AEED P EVHL G QLKRFSLRELQVATDT 9636.m04116 motif-like RKKR P ADVT G ATNPFENRRFKYKELKLIADS 9637.m01434 motif-like RQRKRRRKNSPPPA NNDSDQYSSD G QRQH G TADLERAVT GGGPRRYQFHELAAATRD 9637.m01597 motif-like HKKRKQQMTL G LVHQYSVQ P TGISNSVSHVDIKGHVLMS DDHEFTYEELVKITNN 9637.m02621 motif-like ERNKLHSIKQKYFRQHG G RLLFEEMK G TAFKIFTEEELQK ATNN 9638.m00203 motif-like HNKRK P QESTTAK G RDMFSVWNFDGRLAFEDIVRATED 9638.m00382 motif-like RWSRRFRKDRVRLREKRSRRFR G DELICEME G EISEFSVFE FREVIKATDN 9639.m00021 motif-like KRKKKSSDETVVIAA P AKKL G SFFSEVATESAHRFALSEIE DATDK 9639.m00293 motif-like NKGVFR P SQVSVLEE G YRIVTSHFRAYRYSKLERGTKK 9639.m00297 motif-like G CWLFSSK G LFRHSRVYAIDQEGYKLITTHFQRFTYVDIK KATAN 9639.m02693 motif-like RKKPKAQSKKTI G FQLIDDKY P RVSYAELVQGTNG 9639.m03758 motif-like KKRS G PERI G INHSFRRLDKISYSDLYKATYG

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49 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9640.m00020 motif-like KRKKKSSDETVVIAA P AKKL G SFFSEVATESAHRFALSEIE DATDK 9640.m00279 motif-like KGVFR P SQVSVLEE G YRIVTSHFRAYRYSELERGTKK 9632.m05428 motif-negative KITNKIKQRRAKKLRRKFFKKNHGLLLQQLISSNKDIAER MKIFSLEELDQATNK 9629.m00140 motif-negative KTKNDDEIQLKVEMFLKTYGTSKPTRYTFSEVKRITRR 9629.m00141 motif-negative KYRKTRISIDAVEKFLRMQQAHGPKRYAYTEITAITGH 9629.m00142 motif-nega tive KRRTRRRREIREEEQELEEIT LQGMPRRFTFQQLQEATDQ 9629.m00143 motif-negative WKTRITIDAVEKFLRMQLMLGPTRYTYTDIIAMTSH 9629.m00144 motif-negative KVEMFLRTYGTSKPTRYTFSEVKKIARC 9629.m00147 motif-negative KYWKTRIKIDAVEKFLQMQLMLGPTRYAYTDIIAMTSH 9629.m00148 motif-negative KQRYNEEVHLKVEMFLRTYGTSKPTRYTFSQVKKITRR 9629.m00151 motif-negative RNKITIDAVEKFLQMQLTLGPTRYAYTDLTAITGH 9629.m00152 motif-negative KSRYNEEIHLKVEMFLKTYGTSKPTRYTFSEVKKIARR 9629.m00154 motif-negative SLKSRYDEEVHLKVEMFLRTYGTSKPTRYSFSDVKKITRR 9629.m00155 motif-negative KYWKARITIDAVEKFLRMQEMLSPMRYGYTDIIAITSH 9629.m00156 motif-negative KTRYNE EIHLKVEMFLKTYGTSKPTRYSFSEVKKITRR 9629.m00168 motif-negative SLKSRYNKEIHLKVEMFLKTYGTSKPMRYTFSDVKKITRR 9629.m00169 motif-negative AHKYWKTRLAIDAVEKFLQMQQVLGPTRYAYTDLTAVTS H 9629.m00170 motif-negative KIRITTDAVEKFLRMQLMNGPTRYAYTDLIAITGH 9629.m00171 motif-negative WKTRVTIDAVEKFLRMQQMLGPTRYAYTDITAITGH 9629.m00172 motif-negative KSRYNEEIHLKVEMFLKTYGTSKPTRYTFSEVKKIARR 9629.m00173 motif-negative EKFLRMQQMLGPTRYAYTDIIAITGH 9629.m00180 motif-negative KTRIAIDAVEKFLRMQDMLGPKRYAYTDIIAITSH 9629.m00181 motif-negative YISLKSRYDEEVHLKVEMFLRTYGTSKPTRYNFSDVKKIA RR 9629.m00185 motif-negative DTYGYHSICHRYHQALFWLMLAPLVVFIFLAHKYWKTRIT IDAVEKFLQMQQMIGPMRYAYTDIIAITSH 9629.m00187 motif-negative KTRYNEEIHMKVEMFLKTYGTSKPTRYTFSEVKKIARR 9629.m00188 motif-negative KYWKTRITIDAVEKFLRMQQMIGPMRFAYTDIIAITSH 9629.m00189 motif-negative LSLRTRYN EEIHLKVEMFLKTYGTSKPTRYTFSEVKKIASR 9629.m00190 motif-negative KYWKTRIAIDAVEKFLRMQEMLGPKRYAYTDIIAITSH 9629.m00192 motif-negative KYWKTRITIDAVEKFLRMQQMIGPTRFAYTDIIAITSH 9629.m00193 motif-negative LSLKTRYN EEIHLKVEMFLKTYGTSKPTRYTFSEVKKIARR 9629.m00195 motif-negative KTRITIDAVEKFLRMQQMIGPTRFAYTDIIAITSH 9629.m00196 motif-negative LSLKTKYN EEIHLKVEMFLKTYGTSKPTRYTFSEVKKISRR 9629.m00252 motif-negative RRKKQGPMNNSLEQQNEMSTSTSHVLINSGYGDNVSLRL ENRRFTYKELEKITNK 9629.m00358 motif-negative HKRKKRKQTRDLKDLMHSSSSMQSYSKDLELGGSPHIFTY EELEEATAG 9629.m00364 motif-negative RQRQKFRSAICGVYSGNTKNEEEMLKKCESLALKRYKYS ELKKITKS

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50 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9629.m00366 motif-negative TCRQKEKGSLLTLQKYVANESKIEEALKEYDSLAPKRYNY SELKKITRS 9629.m00367 motif-negative HRKGQILCYLLCNKTRSRNESNIQKLIVSYGSLAPKRYKYS EVAKITS 9629.m00375 motif-negative HGKGKQLRYFLYTKTSSTSERNIEALIISYGSIAPTRYKYSE VTKITS 9629.m00376 motif-negative KCRRRMQNRFSFLNAMDGASRTDTAKVEKLLQSYGSLAP RRFRYSELKKITKS 9629.m00490 motif-negative HQRKFKQRQNRQATSLVIEEQYQRVSYYALSRGSNE 9629.m00520 motif-negative HNRKLKRRQNRQATSLVIEEQYQRVSYYALSRGSND 9629.m00522 motif-negative RKLKRRQNSRATIPGTDEHYHRVSYYALARGSNE 9629.m00550 motif-negative ILMFIIRRNKDKNRSENYGSLVAFRYKDLRSATKN 9629.m01025 motif-negative RKRCNRQRADESDFADLPGTITRFTFKMLKAATND 9629.m01192 motif-negative WRGKRKLFTEKPVNSDSRLMIFSNSQLKNATKG 9629.m01203 motif-negative LIYKKRSSCVASQAKMEGFLAVYSYAQVKKAT 9629.m01204 motif-negative MYKRSSCVARQTKMEGFLAVYSYAQVKKAT 9629.m01206 motif-negative CRYRRDLFASSKFEVEGSLIVYTYAQIRKAT 9629.m01247 motif-negative MIRRKSKNKKEDSSHTPGDDMNHLIVTYHELARATDK 9629.m01357 motif-negative WFYWRY RSFNNSKLSADRSKWSLTSFHKLSFSEYEILDC 9629.m01868 motif-negative RRKKYKIHDAGDLQQENMFSVLNFDGGNAYEQIIEATEN 9629.m01991 motif-negative EGVAIGALIQLHTKGFLWAGNHLLLSNSSAGLRMDCQEN NLKSFLQTNGHVVLQRVDNNYSLRYFTKNEVWHITNG 9629.m02541 motif-negative RRKLTDVKKKYIQEHGGLLLFEK MKSDQGLA FKVFTQAE LEQATNK 9629.m02553 motif-negative RLMFERRKLTDVKKKYFQQHGGLILFDKMKSDQGLAFKV FTQAELEHATNK 9629.m02555 motif-negative RRWKRGIQKKIRRAYFRKNKGLVLERLISSDESVAHSTKIF SLEELERAPDH 9629.m02564 motif-negative RRGRKGEVNMQNNIAAVNRLEEDALVWRLEERSSEFSLF EFSELLEATDN 9629.m02756 motif-negative RRRRRRAAAAALAKAAADSRSKRSQAMESISASTTLVQFT YDEIKAATGG 9629.m03184 motif-negative KGRKQTDRKNATSDLMLNEKYPRVSYHELFEATDG 9629.m03474 motif-negative WKKKIFRNKKRSKSFIDIYGDGVPVRIAQSSLNFKYEELRK ATNY 9629.m03865 motif-negative RSRRA KALRRLEDSSSFLTVFTYRDLQLVTNN 9629.m04349 motif-negative RKRNLSEANKVEGSLVVFRYRFLQHVTKN 9629.m04404 motif-negative KPYQALLSCIERRLRRKAAAAATAAEPLPPEKKEDAGRPL VQAERLRAAFRIDGLLREYSHGEIQAMT 9629.m04617 motif-negative SIRNDIQNSAEGGYMMIRNQFRGFTYQELKEAT 9629.m04791 motif-negative KKSRYRRISKGTPRIESFLQRNGTLHPKRYTYTEVKRMT 9629.m04980 motif-negative KNKKPE RPRASSRTSSMAREETVRFDGCCVEFDVCTLM

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51 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9629.m05047 motif-negative KRRRLKNEEASTSRDIYIDSR SHSATMNSDWRQNLSGTNL LSINLAAFEKPLQNLTLADLVEATNG 9629.m05632 motif-negative WRQKSKAKGVDTDSAIKLWESEETGSHFTSFCFSEIADAT CK 9629.m05849 motif-negative CKGKPKEHDDYDMYEEENPLHSDTRRFTYTELRTIT 9629.m05851 motif-negative KGKSRKSEEEDYDMYEEETPLHIDIRRFTYAELKLIT 9629.m05922 motif-negative MRRRSKRRTTSRRSLLSRYSV KVDGVRCFTFDEMAAATN D 9629.m06444 motif-negative FRRRMVKETTRVEGSLIAFTYRDLKSVTKN 9629.m06446 motif-negative RRRMVKATTRVEGSLISFTYRDLKSVTKN 9629.m06570 motif-negative TARSRPAT SEQWAAEEGYRVVTDHFRRFTYGELRKATKN 9629.m06607 motif-negative RRSRRLKALRRVEGSLTAFTYRDLQVATKS 9629.m06614 motif-negative G RRRISSMNHTDGSLITF KYSDLQILTKN 9629.m07414 motif-negative KIKMRVDEVEKFLQLQQMLTPTRYSYTDIIAIT 9630.m00079 motif-negative RRKQKPCLQQSSVNMRKISYEDIAKATDG 9630.m00113 motif-negative KRKLMKEKERFFQQNGGMLLYEQIRSKQVDTVRIFTKEEL ENATDN 9630.m00153 motif-negative TWRAWSRWQEDNARVAADDESGSLESAARSTLVLLFAN DDDNGNGDDGERTMTLDDVLKATGN 9630.m00498 motif-negative RRMLKAK DKRRAAGPTYESALLENREFSYRELKHITNN 9630.m00519 motif-negative RGKNFVTENRRCRNDGTEETLSNIKSEQTLVMLSQGKGEQ TKLTFTDLKATKN 9630.m00520 motif-negative ATVKGTDCITNNRSSENADVDATSHKSDSEQSLVIVSQNK GGKNKLTFADIVKATNN 9630.m00522 motif-negative SISGMSFRTKNRCSNDYTEALSSNISSEHLLVMLQQGKEAE DKITFTGIMEATNN 9630.m00523 motif-negative ERSKRFITKNSSDNDGDLEAASFNSDSEHSLIMITRGKGEEI NLTFADIVKATNN 9630.m00524 motif-negative RATKLMRKGELANNRNEETASFNPNSDHSLMVMPQGKG DNNKLTFADIMKTTNN 9630.m00544 motif-negative RRWKKDFDQLAKSMQSLPGVPVKISFADIRKATNN 9630.m00812 motif-negative RHSHRAHDSKNIRSSSDTARVALVPMLNKFNSMKTNKKG LVAMMEYNTLETATGK 9630.m00926 motif-negative RRRRKMLRRRQFVLV PAGDNAMADHETTLSNNLLGRRR MKKREPPSINLATFEHAPVRVTVDEIMRATGN 9630.m01077 motif-negative KRREEKPILTDISMDTKIISYKDIVQATKG 9630.m01086 motif-negative MKRRKEEPNLQHSSVNLRKISYEDIAKATDG 9630.m01088 motif-negative MKRRKEEPNQQHSSVNLRKISYEDIAKATDG 9630.m01126 motif-negative HKRAKKTNANRQTSLIKEQHMRVSYTELAEATKG 9630.m01128 motif-negative RNKKAKPNPQISLISEQYTRVSYAELVNATNG 9630.m01130 motif-negative RLRTKLRRANPKIPLSDKQHMRVSYAQLSKATNS 9630.m01131 motif-negative RNKTQAKSDLALINDSHLRVSYVELVNATNV 9630.m01182 motif-negative RRKKHLQDHLSWKLTPFHVLHFTANDILSG 9630.m01185 motif-negative RRKKLQDHLSWKLTPFHILHFTTTNILSGL

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52 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9630.m01236 motif-negative RRKKGPQDVTSWKMTQFRTIDFTEHDIVSN 9630.m01273 motif-negative KRKGKSNEHIDHSYMELKKLTYSDVSKATNN 9630.m01310 motif-negative KYCRWRLPFASADQDLEIELGHLKHFSFHELQSATDN 9630.m01866 motif-negative KRKF QRYVELREDWELEFGAHRLSYKDLLQATER 9630.m01874 motif-negative KRKF QRYVELREDWELEFGAHRLSYKDLLQATER 9630.m02460 motif-negative RSVRRKNQEHAVASEDMGEATLSMEVARAATKG 9630.m03311 motif-negative RRKMKSVEQSENGAGNTKVFSVWNFDGGDVCKQSFEAT EN 9630.m03878 motif-negative KKTPRRTYLSLLSFGKQFPRVSYKDIAQATGN 9630.m03880 motif-negative KRTSRRTDLLLLSFGKQFPRVSYKDLAQATGK 9630.m03884 motif-negative KMKPREKYISSQSFGENFLKVSYNDLAQATRN 9630.m03885 motif-negative KMKPREKYISSQSFGENFLKVSYNDLAQATRN 9630.m03886 motif-negative KMKPREKYISSQSFGENFLKVSYNDLAQATRN 9630.m03961 motif-negative KRRLAKIKREHFRQHGGLLLFEEMKSRQGLSFALFTQEEL EQATNR 9630.m03963 motif-negative KRRLATVKRRYFNQHGGLLLFEEMKSNQGLSFTVFTKDE LEEATNK 9630.m04029 motif-negative KWKRGIQRRIRRAYFKKNQGLLLEQLIIDENTKDKTRIFSL EELEKATYN 9630.m04033 motif-negative KWRKGIQKRIRRAYFKKNQGLLLEQLISNESATNKTKIFSL EELEEATNN 9630.m04037 motif-negative KWKKSIQKRIRRAYFKKNQGLLLEQLISDESATNKTRIFSL EELEEATNN 9630.m04056 motif-negative W YKNRLTKSPSTMSMRAHQLVSYQQLVHATDG 9630.m04102 motif-negative RRRRRYAELKEEWEVA FGPHRFSYKDL 9630.m04231 motif-negative LRPRLRGLRLDRLTSRLPACLRRSRTANTMLPYFAPIADRL GALQPYLAPIADRL 9630.m04698 motif-negative RKRRRRSGGDPSSAFNAAIDF RKIPGLPKEFDYMELRRGT NN 9630.m04699 motif-negative RKRRKRIGDDPSSVFNTTIDFRSIPGVPREFDYRELRRGTN N 9630.m04857 motif-negative RHRRNHQILFDVDEQHTENVNLGNVKRFQFRELQVATEN 9630.m05594 motif-negative RKIKNRRANMLRQMFFKQNRGHLLQQLVSQNTDIAERMII PLAELEKATNK 9630.m05879 motif-negative KRRKQKPSLQQSSVNMRKISYEDIANATDG 9631.m01172 motif-negative RRRSAALRSQKSTKRLLSEASCTVPFYTYREIDRATNG 9631.m01199 motif-negative KARSLKRSAEARAWRLTAFQRLDFAVDDVLDC 9631.m01470 motif-negative KRWFRHAELREDWKVEFGPQRFSYKDL 9631.m04208 motif-negative RYKLRHCQCSKNELRLAKNTTYSFRKDNMKIQPDVEDLKI RRAQEFSYEELEQATGG 9631.m04253 motif-negative RRKHIQEKQQYFKQNGGLRLFDEMVSRQVDTVRVLTEDE LKKATNN 9631.m04258 motif-negative RRKHTIEKQEYFRRNGGLRLYDEMVSRQVDTVRVLTVDE LKKATDN

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53 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9631.m05490 motif-negative RRRLKY AELREDWEVEFGPH RFTYKDLFRATEG 9631.m05492 motif-negative FRRQRRA IYVELVEDWEVEFGPHRFAYKDLHKATKG 9631.m05502 motif-negative KARSLKKASEARVWKLTAFQRLDFTCDDVLDCL 9631.m05991 motif-negative RSRRYAEEEEEWEIEYGPHRISYKDLHGATKG 9631.m06141 motif-negative WVFCRHSPKYGAASAQYALLEYASGAPVQFSYRELQRST KG 9631.m06166 motif-negative FKRWKRSTRKKIRRAYFRKNKGLLLEQLISSSNNVTPNTRI FSLEDLEKATNN 9631.m06406 motif-negative SRWDLDALEIQAVEQGYKVMASNFRRYNYKELAKATRK 9632.m00228 motif-negative KSKYLRIDATKVGTAVDDSILKRKLYPLISYEELYHATEN 9632.m00291 motif-negative RRWRRHMQRKIRREYFQKNKGLLLEQLMSSDENVAHDP KIFSLEELEKATDN 9632.m00377 motif-negative KWKRVLNFFHKGTAGARRFEYRDLATATKN 9632.m00403 motif-negative KVFHKGTASARRFEYHELATATEN 9632.m00458 motif-negative QKRIRNVFDKGTGGARRFEYRNLAAATDH 9632.m00467 motif-negative WKWRKTNREFDKGTRGACRFNYHRLAAATNH 9632.m01129 motif-negative KKNIPLSQASSKSQLPLKTFTYKELEKATAG 9632.m01308 motif-negative RKKRKLRAYFNRNGGQLLKSIKIDIYTKEKLDQITKNYST 9632.m01335 motif-negative MRKKAKQQDRIISPDMEDVLNNRLISYHDIVRATDN 9632.m01893 motif-negative KYKRRLVRQDLMNKRDAYFRQHGGQLLLDMMKLENQV SFKLYDREEIELATNN 9632.m02006 motif-negative RRWKRDIQRQLRRNYFRKNQGLLLEQLISSDENASDKTKI FSLEELEKATNN 9632.m02009 motif-negative RRWKRDIQRQLRRNYFRKNQGLLLEQLISSDENASDKTKI FSLEELEKATNN 9632.m02077 motif-negative KKRRALAYQKEELYYLVGQPDVFNYAELKLATDN 9632.m02192 motif-negative QKRRKYRERDEELDFDIMPGMPTRFSFQKLRKSTED 9632.m02196 motif-negative RRRRKYQETDEELDFDILPGMPLRLSLEKLRECTED 9632.m02204 motif-negative RRRRKY QKLDEELDFDILPGMPMRFSFEKLRERTED 9632.m02208 motif-negative QRRK YQEIDEEIDFEPLPGMPVRFSYEKLRECTKD 9632.m02260 motif-negative HRWKKDIQKQLRRKHFQKNQGLLLEQLISSDENASENTKI FSLDELEKATNN 9632.m02267 motif-negative RRWKNDIQKQLRRKHFRKNQG LLLEQLISSDENASDKTKI FSLDELEKATNN 9632.m02268 motif-negative RRWKRDIQKQLRRKHFQKNQGLLLEQLILSDQNATDKTKI FSLEELEKATNN 9632.m02809 motif-negative RKRKNDIQKQLRKKYFRKNQGLLLEQLISSDECATDSTKIF TLEELKEATNN 9632.m02818 motif-negative HRRSIKRQRLIRQRDEYFQQHGGQLLSDMMKIDCNLEFTL YRQEDIEVATND 9632.m02835 motif-negative KYKQRIKKQALLRQADEFFQQHGGQLLLEMMKVEGNAG FTLYERERIKIATNN 9632.m02838 motif-negative RHKRSIKKQALLRQTHEFFLQHGGQLLLEMMKVEGNVGF TLYERGEIETATSN

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54 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9632.m02843 motif-negative QHKRSIKRQALQRQTDMYFQQHGGQILLELMKVESSAEFT LYDREKIEVATNN 9632.m02866 motif-negative RHKRSIRKQALLRQTDEFFQQHGGQLLLEMMKAEGNIGFT LYKRVEIETATKN 9632.m03079 motif-negative RGRGNATVKKELSALKDANGNVISAQTFTFRQLAAATRN FREEC 9632.m03291 motif-negative RKGKRYNLTMDNVQGGMGIIAFRYVDLQHATKN 9632.m03294 motif-negative RRSKTKFSGDRLKDSQFCNGIISFEYIDLQRATTN 9632.m03301 motif-negative WKSKGKWFACTQEKPEDGIGITAFRYTDLQRATKN 9632.m03304 motif-negative WWNKSKRYNCTSNNVEGESGIVAFRYIDLQHATKN 9632.m03761 motif-negative CRRLHAAAKLVLFYPIKITADELLAAL 9632.m04193 motif-negative QRRKLTKIKKEYFRQHGGMILFESMKSKKGLAFTVFTEAE LIHATNN 9632.m04316 motif-negative MRRRRM FSELKEEWEVTFGPH RFSYKDLFHATDG 9632.m04317 motif-negative RRRVR YAEVREDWEVEFGPH RFSYKELYQATKG 9632.m04800 motif-negative WRRSTRKRRLAYRNLEKMIDAHGPVKFKLKELRRATAN 9632.m04925 motif-negative WTMKKRKVARKRAELFRKNGGLLLQQRFLMITSQGEESS AKIFSAEELKNAT 9632.m04926 motif-negative KKRRLAKQKQRYFLQNGGLLLQQQIFTHQAPARIFTTSEL EDATNN 9632.m04927 motif-negative KKRKLAKIRQRYFMQNGGMLLKQKMFSQGAPLRIFTSSEL EKATNS 9632.m05095 motif-negative WRQ KKRKILLELEELYNIVGRPNVFSYNELRSATEN 9632.m05097 motif-negative WRQK RRKLTLEQQELYSIVGRPNVFSYSELRSATEN 9632.m05099 motif-negative MWRQK RRKLSLEQQELYSIVGRPNVFSYSELRSATEN 9632.m05246 motif-negative RKWQSKASVLLGKRRNNKNQNRMLLGNLRSQELIEQNLE FSHVNFEYVVAATNN 9632.m05253 motif-negative KWISKGEKRNNENQNRAMLGNFRASHEVYEQNQEFPCIN FEDVVTATNN 9632.m05257 motif-negative RKWQTKGKQRNDENKKRTVLGNFTTSHELFEQKVEFPNI NFEEVATATNN 9632.m05462 motif-negative RVRDDGYSLVFSHFRRFTYDELSDATCG 9632.m05627 motif-negative RIIHSRMQEHNPKAVANADDCSESPNSSLVLLFQNNKDLGI EDILKSTNN 9632.m05838 motif-negative RRRRKYQ ELDEELEFDILPGMPTRFSFEKLRECTED 9633.m00270 motif-negative RRARARRRGTTALA AVADKRDSLASAA ALARSPREFTYK ELSAATRG 9633.m00320 motif-negative KMRNRRNDHHDDMDGSSEIIRTIAASQLSFKYEELCKATD D 9633.m00373 motif-negative KWKRGVQKRIRRAHFKKNQGLLLEQLILDEKAQDKTKIFS LEELEKATNY 9633.m00657 motif-negative RKKLRFCGAQLHDSQCSGGIVAFRYNDLCHATKN 9633.m01194 motif-negative RK KAGPQEDWEMKCRPPSFIYKDLYNATSG

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55 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9633.m01241 motif-negative MRKRARFNHHRLNRKTEILIEMPMEEEVHNTRRFSYAHLL AATEN 9633.m01466 motif-negative KKRRTMAQQKEELYNLIGRPDVFSNTELRLATDN 9633.m01467 motif-negative KKRRTMAQQKEELYNLIGRPDVFSNTELRLATDN 9633.m01492 motif-negative KKRRTMAKQKEELYNLVGRPDVFSNSELKLATDN 9633.m01499 motif-negative KKRRAMAQQKEELYNLVGRPDVFSNVELKLATDN 9633.m01517 motif-negative KKRRT IAEQQEELYNLAGQPDVFSNTELKLATDN 9633.m01570 motif-negative KKRRALAQQKEELYNLVGRPDVFSYAELKLATDN 9633.m01571 motif-negative KKRRALAQQKEELYNLVGRPDVFSYAELKLATDN 9633.m01572 motif-negative KKRRALAQQKEELYNLVGRPDVFSYAELKLATDN 9633.m01594 motif-negative KKRRALAQQKEELYNLVGRPDVFSYAELKLATDN 9633.m02281 motif-negative RRKKVAKHSGKTDKKCLTYQTELYKSPSNLCRNFTFHEM QIATSS 9633.m02283 motif-negative KKVAKHSFMTDKKCMTYRTEFYHSPSNLCRNFTFDEIQV ATRN 9633.m02289 motif-negative RKKRVDHGNTNKELLLATLLS KKSNLCHQFTFLQIQEATS N 9633.m04197 motif-negative RRKKQAMSNSVKPQNETVSNVSSNGGYGHSSSLQLKNRR FTYNELEKITNN 9634.m00350 motif-negative RRQKQKIGSSLEVSDSRLSTD HYQQKEVCRRSASPLISVEY SN 9634.m00424 motif-negative RMKLEKEKQRFYDQNGGHILY QKIISGQVNTVEIFTEEVL KNATNN 9634.m00426 motif-negative AGERRRAARMARRLPSMEDERIRVEYSYFRKVAGLPRKL TLESLAAATDG 9634.m00815 motif-negative RSMAAARAKRQSVRLVDVEDYQAAAEREHPRISYRELAE ATGG 9634.m01052 motif-negative RRRAALADTLEEWELEHPQRIPYKELYKATKG 9634.m01423 motif-negative WRRRRRR HAEVREDWEVEFGPHRFAYKDLVRATRG 9634.m01613 motif-negative RKKVKHQNISSGMLDMISHQLLSYHELVRATDN 9634.m01642 motif-nega tive RHRRNRQILFDVDEQQIE NVNLGNVKRFSFRELQAATEG 9634.m01788 motif-negative RMALNCVRGGYRSKSDTVIFIPKLIKSKEHLAFLEKDQDG 9634.m02185 motif-negative KKRRRRVGDDPESLSSTAAFKFN KSSINLRSLAGTPKEFEY TELRKGTED 9634.m02894 motif-negative RWRKRN AVRRAQMERLRPMSSSDLPLMDLASIHAATDS 9634.m03394 motif-negative RRKRD EPLEDEYFIDQLPGLPTRFSFVDLKSATGD 9634.m03445 motif-negative KVLPRRQNENTTTPRWKLTAFHNINFNYQDIICGLADNN 9634.m03602 motif-negative QREDKQ LRELAEVGYEMITNHFRRYTYRELVTATRR 9634.m03654 motif-negative KRKKAKNPTDPSYKKLEKLTYADLVKVTNN 9634.m03671 motif-negative KKHKSSSGPTSTRAVRNQLPRVSYTELSMGTNG 9634.m03687 motif-negative RRKEMQANPHCQLISEHMKNITYQDIVKATDR 9634.m03694 motif-negative WRKRMQVTPKLPQCNEHVFKNITYENIAKATNK 9634.m03697 motif-negative RKRIQVKPNLPQCNEHKLKNITYEDIAKATNM 9634.m03700 motif-negative TKRMQAEPHVQQLNEHRNITYEDVLKATNR

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56 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9634.m03716 motif-negative KRMQAEPHVQQLNEHRNITYEDVLKATNR 9634.m03999 motif-negative RRINNNDHDIVRHFTKKELHRATNG 9634.m04004 motif-negative RRIRRRSSSDHDMVMRLFTRKELYDATNG 9634.m04433 motif-negative RWRRKIRSSRTGNAAARSVLNGNYYPRVTYAELAKATDD 9634.m04788 motif-negative RQRRALADVKRKYFERHGGLLLYDELSTRPGNTFTIYMEE QLEQATNG 9634.m04798 motif-negative QRRALADIKRSYFKRHGG LLLYEELNARKSNAFTIYTEEQ LEQATNG 9634.m04920 motif-negative RKGCSA WRSAARRRSGAWKMTAF QKLEFSAEDVVECVK 9635.m00073 motif-negative SKREVSIIDDEEINGSCHDSYDYWKPVLFFQDSAKELTVSD LIKSTNN 9635.m00209 motif-negative RKKMFRKQLPLLPSSDQFAIVSFKDLAQATEN 9635.m00303 motif-negative RRRLKYT EIQEDWEVEFGPHR FSYKVLYDATEG 9635.m00304 motif-negative RRRM RYTELREDWEIDFGPH RFAYKDLFHATEG 9635.m00306 motif-negative RRRSR YAELREDWEVEFGPHRFSYKELFRATDG 9635.m00307 motif-negative RRRKRY TELREDWEVEFGPHR FPYKDLHHATQG 9635.m00308 motif-negative RRRQ RYAELREDWEDEFGPHRFAYKDLLHATDG 9635.m00309 motif-negative RRRM RYTELREDWEVEFGPHRFSYKDLFRATDG 9635.m00310 motif-negative RRK LTYTELREDWETEFGPNRFSYKDLFLATEG 9635.m00311 motif-negative RRNLRYAELREDWEVEYGPRRFCYKDLFDATEG 9635.m00312 motif-negative RRQQRYAELREDWEVEFGPHRFSYKDLFNATEG 9635.m00313 motif-negative RRRLR YVELKEDWEIEFGPHRFSYKDLFHATHG 9635.m00314 motif-negative RRRLR YAEIREDWEVEFGPHRFSYKDLFCATEG 9635.m00315 motif-negative RRHLRYKEVREDWEVEYGPHRFAYKDLFDATKG 9635.m00317 motif-negative RRRLR YAELREDWEIEFGPHRFSFKDLYLATEG 9635.m00318 motif-negative RRKRY AELYEDWEVEFGPYRFSYKYLFDATEG 9635.m00320 motif-negative RRQLVYKEVREDWEVEYGPRRFAYQDLFRATRG 9635.m00322 motif-negative RRRLR YAELREDWEIQFGPH RFSFKDLYFATEG 9635.m00324 motif-negative RKKNAKQREVIMDSAMMVDAVSHKIISYYDIVRATDN 9635.m00325 motif-negative RRWFKYAELREDWEIDFGPHRFSFKNLYFATEG 9635.m00326 motif-negative RRRQMR YAELREDWEVEFGPH RFSYKDLFHATEG 9635.m00329 motif-negative RRCRRYQE LHEDWEVEFGPHR FSFKELFKATNGFV 9635.m00339 motif-negative RWCWKKNARSRENWEAELGPRRFAYRDLRRATDG 9635.m00347 motif-negative KARSLKKASEARAWKLTAFQRLEFTCDDVLDS 9635.m00512 motif-negative KSKIPD LVSNEERKTGFSGPHYFLKERITFQELMKVTDS 9635.m01265 motif-negative KQERKREARFKLRLISMAIQNVINLWRIEEGNSGFSLYNFS QIKEATQD 9635.m01838 motif-negative RRRLRY MELQEDWEVDFGPHRFSFKDMYHATEG 9635.m01839 motif-negative RRHMRYTELREDWEVEFGPHRFSYKDLYHATEG 9635.m02971 motif-negative RRLRNHKSMLRKKDTMAREEVLKLWRLEESDSEFMLFDF SQIEDATSN 9635.m02984 motif-negative RRLRNHKSMLRKKDTMAREEVLKLWRLEESDSEFMLFDF SQIEDATSN

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57 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9635.m03469 motif-negative KKLRKGDGRKSNRQLEAHSRNSSKTEEALKLWRTEESST DFTLYDFGDLAAATDN 9635.m03484 motif-negative RRRKRSRSKQQHSYSMQMGE DIESVDSLFIDLSTLRAATG N 9635.m03485 motif-negative RWRKRSRSKQQSSYSIQMAEDIESTDSLFIDLSTLRAATGN 9635.m03488 motif-negative KRKKAAKKDNLLKKMARGKCTIFDLATLKEATEN 9635.m03489 motif-negative RRRSKVTET DHQLRKITRAQCLIFDLPALQEATDNFSDNN 9635.m03490 motif-negative RRRSKVTET DHQLRKITRAQCLIFDLPALQEATDNFSDNN 9635.m03493 motif-negative RRKRPVITKAKQTNANYYAEADDVDSVDSMLMDISTLRA ATGD 9635.m03494 motif-negative RFRRRTKVT DAVHPLKKITRAQCMI FDLSALQEATEN 9635.m03497 motif-negative RFRRRTK AAETDHPLKKITRAQCMIFDLPTLQEATEN 9635.m03511 motif-negative RFKRRTKAVEADHPLKKITRAQCMIFDLPTLQEATEN 9635.m03523 motif-negative RRRRP EEQTFLPYDIQSIDSLLLDLSTLRAATDD 9635.m03527 motif-negative KRIKKRRPEEQTFLSYSVSSDDIQSIDSLILDLPTIRVATDD 9635.m03528 motif-negative RRRRLARKT LRPKSSEDEMQSFASL VLDLQTLRTATDN 9635.m03532 motif-negative RRKRRSRKAEHFSELDASEDLESVKSTLITLASLQVATDN 9635.m03533 motif-negative RRKRRSRKAEHFSELDASEDLESVKSTLITLASLQVATDN 9635.m03537 motif-negative RKKSRATK AEHLSELDASEDLESVKSTLLTLGSLQVATDN 9635.m03639 motif-negative KCRRD RTLRISKTTGGALIAFRYSDLQHVTSN 9635.m03785 motif-negative FNKREKNPQKKDCSSTRNPVFEECSTHKATNSAVQQLSLK SIQNATCN 9635.m03803 motif-negative FRRIRRTTRSREKEKEKLDCD ESIDSEFEKGKGPRRFQYNE LVVATDN 9635.m03805 motif-negative RRRRISRRRTREEYEMGGSDDF DMNDEFEQGTGPRRFLYS QLATATND 9635.m03867 motif-negative RRWHRQFAEVREDWEVEFGPHRFTYKDLFHATQG 9635.m04367 motif-negative RRRTKATKL SLSYSSRSEDIQNIESLIMDLPTLRIATDN 9635.m04368 motif-negative RKKRLPTKTPLIENTEDLEDFESIFIDLSTLQSATSN 9635.m04760 motif-negative KRRRRRARVRSELRRLSMAVQ NVITLWRLEEGNSGFKLY DFSDIKDATNN 9635.m05024 motif-negative RRRRRF AEVREDWEDEFGPHRFAYKDLFRATDG 9636.m00991 motif-negative KKRRKAARQQEELYNLVGRPNIFSSAELKLATDN 9636.m00993b motif-negative KKRRRT SQRKEELYNMVGRRN VFSNAELKLATEN 9636.m01497 motif-negative KRKM KLNVVDNENLFLNETNERISYAELQAATNS 9636.m01501 motif-negative KTRMKPNIIDNENLFLYETNERISYAELQAATES 9636.m02876 motif-negative SRNTYFAERMIINLEELEKATNN 9636.m03313 motif-negative RRRATAPRSRSTAAAAAAHDVAEPITVTVARTDMDAAVK QSHSPPPP 9636.m03475 motif-negative KFYHPKTKNLESVSTLFEGSSSKLNEVIEATEN 9636.m03956 motif-negative KQRREKKLKEKFFKQNHGLLLQQLISRNTDFGERMIITLEE LQKATNN

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58 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9636.m03957 motif-negative KMKLRKMKRMKETFFRQNHGLLLGRLVSQNADIGQRMI MTLQELEKATDN 9636.m03960 motif-negative RKIKLRKMKKTKERFFKQNHGLLLQQLISQKVDIGERMIIT LSDLEKATNN 9636.m03961 motif-negative RRKFKSRRAKKLKEFFFKQNRGLLLHQLVDKDIAERMIFS LEELEKATNN 9636.m03963 motif-negative RKVKLQRVKKMRDKFFMQNHGLLLQQLISRNTDFAERMI ITLQELEIATNN 9636.m04074 motif-negative RMARARRSMERRRQERLEHTLT NLPGMPKEFAFEKLRKA TKN 9637.m00704 motif-negative RRKLLY AELREDWEIDFGPQRFSYKDLFHATQG 9637.m00705 motif-negative RRHLR YSEVREDWEVEFGPHRFSFRDLFHATEGFK 9637.m01303 motif-negative NKRAIEVDSENSEWVLTSFHKVEFNERDIVNSLTENN 9637.m01504 motif-negative KWRRKNSMHKDLRAFDLQTGSFTLRQIKVATRN 9637.m01544 motif-negative RQKRNSNYSTEDPTRDRSNQLENSLEKSQNHGDVLQIVEN RQFTYIELEKVTNK 9637.m01775 motif-negative RRKLQHIKNNYFQQHGGLILFE EMKSQQGHAFKIFSEEEL QQATKK 9637.m02620 motif-negative REKRKLQYVKRRYFRQHGGMLLFEEIKSQQGISFKIFSEEE LQQATNK 9637.m02623 motif-negative RERRKLQHIKQKYFKLHGGLLLFQEMNSNERKSFTIFSEAE LQHATNK 9637.m02625 motif-negative HERRKLQHIKQKYFKLHGGLLLFQEMNSNERKSFTIFSEAE LQHATNK 9637.m02627 motif-negative HDRRKLQHIKNQYFRRHGGLLLYEEMKSKQGLAFKIFSEE ELQQATNK 9637.m02724 motif-negative TNMIKARRAKKLRAVFFKQNRGLLLLQLVDKVIAERMVF TLEELEKATNR 9637.m02896 motif-negative KVAKVQEGHIASISRRNQPPCCYYLCDDASQAEGIKVERSI EFSYEEIFNATQG 9637.m03211 motif-negative PKIKEIIKKKYGENNKRRALRVLSISDDLGQEIPAKDLEFPF VEYDKILVATDN 9637.m03213 motif-negative RGKKRSVKEHKKSQVQGVLTATALELEEASTTHDHEFPFV KFDDIVAATNNFSKS 9637.m03219b motif-negative KSRENRRKRDSQKTLVPGSRNTSSELLEENPTQDLEFPSIRF SDIVAATDN 9637.m03225 motif-negative KIKGKRRNRQKHRELILDVMSTSDDVGKRNLVQDFEFLFV KFEDIALATHN 9637.m03306 motif-negative RRIKQRRARTLRQKFFKQNRGHLLQQLVSQKADIAERMIIP LAELEKATNN 9637.m03310 motif-negative RKIKQRRARTLRQKFFKQNRGHLLQQLVSQKADIAERMII PLAELEKATNN 9637.m03311 motif-negative KRKVKKQRARMLRQKFFKQNRGHLLQQLVSQKADIAER MIIPLSELEKATNN

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59 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9637.m03317 motif-negative RKQKRRRAKKIRQKYFKQNRGQ LLQQLVAQRADIAERMII PLGELKKATNN 9638.m00128 motif-negative KRKMNKYFKKNGGSVLQKVDNIMIFSKDEVKKILKNN 9638.m00133 motif-negative RRKMNEYFKKNGGSILQNVDNIVIFSKDEMKKIL 9638.m00154 motif-negative RARRREVRSAMMLSSLQDGTRT ATTWKLGKAEKEALSIN VATFQRQLRKLTFTQLIEATNG 9638.m00177 motif-negative RKEKQKMREFFIRNGGPILENAKSIKIFRKEELKRITK 9638.m00383 motif-negative RRGHRKGIMGLQARRTDNLQGEEELVWDLEGKNPEFSVF EFDQVLEATSN 9638.m00436 motif-negative SCQQFKLRPSSIYLKQERSYCIIMDLSESKHKEYIERAQWIE ENISNIKPFTEEDIKRITSDYN 9638.m00516 motif-negative HKKRKMNEYFKKNGGSVLQKVDNIKIFTKDELKKITKNN 9638.m00522 motif-negative QKRKINEYFKKNGGSILQKVDNIMIFSKDDLKKITKNN 9638.m00529 motif-negative HQKRKMNEYFKKNGGSVLQKVDNVKIFSKDELKKITKNN 9638.m00590 motif-negative KRSQRL SANRVSMRNLDSTEELPEDLTYEDILRATDN 9638.m00592 motif-negative KRSQ RLSTNRVSVRNMDSTEELPEELTYEDILRGTDN 9638.m00835 motif-negative HREKRKMREFFEKNGGPILEKVNNIKIFKKEELKPILKASN 9638.m00843 motif-negative KLLFDERRKTKEFFIKNGGPVLEKVDNIKIFKKEELKPIIQS CN 9638.m00886 motif-negative RRWKRHAQKRLQTKYFRKNQG LLLEQLISSDENASEKTKI FSLEELKKATNN 9638.m00929 motif-negative RHRAIKKVALAGPRSYSYEELYTATNG 9638.m01234 motif-negative RKKVKHQKISTGMVDTVSHQLLSYHELVRATDN 9638.m01485 motif-negative RQKRRMNEYFRKNGGSVLQKVENIKIFTKDELKKITKNN 9638.m01489 motif-negative KKLQAKQYAADKNVDSGSLLFDLAIIRKATAN 9638.m01490 motif-negative RKLQAKQYTDENDIYSGSLLFDLATLRKATAS 9638.m01591 motif-negative KDQRTIPKDIDHEEHITSLLIKKYPRISYVELYAATDSLSSE N 9638.m01608 motif-negative KRLREKSSKVNQDQGSKFIDEMYQRISYNELNVATGS 9638.m01814 motif-negative KRKWKSNEHMDHTYMELKTLTYSDVSKATNN 9638.m01940 motif-negative KKRLRPLSSKVEIVASSFMNQMYPRVSYSDLAKATNGFTS NN 9638.m02622 motif-negative RRLRYAELREDWEVEFGPHRFAYKDLFVATAG 9638.m02623 motif-negative RQRLRYAELREDWEVEFGPHRFSFKDLYDATGG 9638.m02876 motif-negative SRKARRAAVVVEKAETSASGGGGSSTAAAVQASIWSKDT TFSFGDILAATEH 9638.m02881 motif-negative RRRPREKKE VESNTNYSYESTIWEK EGKFTFFDIVNATDN 9638.m03509 motif-negative RRRAALADTLEEWELDHPHRLPYRELYMATKG 9639.m00291 motif-negative RKEMRS SQVWIAEEGYRVMTSHFRMYSHRELVKATER 9639.m00654 motif-negative NRKQKRKSVDLPSFGRKFVRVSYNDLAKATEG 9639.m01200 motif-negative RSFKLEELKKRDMEQGGGCGAEWKLESFHPPELDADEICA VG 9639.m03230 motif-negative RRIKHRRKIKLRQKFFILNRGQLLKQLVSQRADIAERMIITL DELEKATNN

PAGE 70

60 Table 2-2. Continued Protein ID Category Juxtamembrane Domain 9639.m03255 motif-negative KYLNRRKKNNTKNSSETSMQAHRSISFSQLAKATEG 9639.m03742 motif-negative KKRNNLGKQIDQSCKEWKFTYAEIAKATNE 9639.m04370 motif-negative RMKVKKHQKISSSMVDMISNRLLSYQELVRATDN 9639.m04371 motif-negative RKKVKHQENPADMVDTINHQLLSYNELAHATND 9639.m04373 motif-negative RATDD 9639.m04376 motif-negative QMTR KKIKRKLDITTPTSYRLVSYQEIVRATES 9639.m04388 motif-negative KRRKRRMFANNNGGRLLKDMNIVLITEKDLNKMTKN 9639.m04389 motif-negative KYKVKHQKMSVGMVDMARHQLLSYHELARATND 9639.m04398 motif-negative RK KIKRKLDTTTPTSYRLVSYQEIVRATES 9639.m04401 motif-negative RKKANHQNTSAGKPDLISHQLLSYHELRATDD 9639.m04403 motif-negative RMTRKKIERKPDIAGATHYRLVSYHEIVRATEN 9640.m00278 motif-negative RKEMW SSEVWAAEEGYRVMTSHFRMYSYRELVKATER 9640.m00281 motif-negative KGVFRRCQ VSALDEGYRMVTNHFRAYSYVELRNGTRN 9640.m00711 motif-negative KTHSARSAIWAAEEGYRVVTDHFRRFTYKELRRATRN 9640.m01015 motif-negative RRKRSPKQTEDRSQSYVSWDIKSTSTSTAPQVRGARMFSF DELKKVTNN 9640.m01616 motif-negative REIKHRRAKRVKQKFFKQNRGHLLEQLISQRADIAERMILP LVELEKATNN 9640.m02587 motif-negative RRCNDHR RRVQQKELELLGIMGPSRFQLQDLVAATGN 9640.m03046 motif-negative RQTR NSRKGEHALLLISDQHVRVSYTELVTSTNG 9640.m03691 motif-negative HRRRNKQDTWITNNARLISPHERSNVFENRQFTYRELKLM TSN 9640.m04130 motif-negative KLQRRKYRKEKEEYFKQNGGLRLFDEMRSRQVDTILILTE KEIKKATENYSDD 9640.m04131 motif-negative RRRHKKEKIEYFKQNGGLRLYDEMISRQVDTIRILTEREIK RATENYN 9640.m04132 motif-negative RKRHKKDKDEYFKQNGGLKLY DEMRSRKVDTIRILTEKDI KKATDNYSED 9640.m04177 motif-negative KPMKQVMQSNETSPRPLLMEQHWKLSYAELHRATDG 9640.m04345 motif-negative SRKKSIGGSYVKMDKQTIPDGAKLVTYQWNLPYSSGEIIR RLELLDEED 9639.m04391 motif-negative RMKVKKHQMISSGMVDMISNRLLSYHELVRATDN 9639.m04405 motif-negative KKMKNPDITASFDTADAICHRLVSYQEIVRATEN

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61 Figure 2-3. Rice RLKs selected for study mapped on the twelve rice chromosomes. Numbers along the top represent length ofchromosome in megabases.

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62 Figure 2-4. Motif-positive rice RLKs ma pped on the twelve rice chromosomes. Numbers along the top represent length of chromosome in megabases.

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63 Figure 2-5. Motif-like rice RLKs mapped on the twelve rice chromosomes. Numbers along the top represent length of chromosome in megabases.

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64 Figure 2-6. Motif-negative rice RLKs ma pped on the twelve rice chromosomes. Numbers along the top represent length of chromosome in megabases.

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65 Figure 2-7. Rice RLKs in subfamily CrRL K1L-1 selected for study mapped on the twelve rice chromosomes. Number s along the top repr esent length of chromosome in megabases.

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66 Figure 2-8. Rice RLKs in subfamily DUF26lc selected for study mapped on the twelve rice chromosomes. Numbers along the t op represent length of chromosome in megabases.

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67 Figure 2-9. Rice RLKs in subfamily L-LEC se lected for study mapped on the twelve rice chromosomes. Numbers along the top represent length of chromosome in megabases.

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68 Figure 2-10. Rice RLKs in subfamily SD-2 a selected for study mapped on the twelve rice chromosomes. Numbers along the t op represent length of chromosome in megabases.

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69 Figure 2-11. Rice RLKs in subfamily WAK sele cted for study mapped on the twelve rice chromosomes. Numbers along the top represent length of chromosome in megabases.

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70 Figure 2-12. Rice RLKs in subfamily LRR1a selected for study mapped on the twelve rice chromosomes. Numbers along the t op represent length of chromosome in megabases.

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71 Figure 2-13. Rice RLKs in subfamily SD2b selected for study mapped on the twelve rice chromosomes. Numbers along the t op represent length of chromosome in megabases.

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72 CHAPTER 3 MOLECULAR CHARACTERIZATION OF THE XA21T923A/S925/T926A MUTANT Introduction The rice disease resistance protein XA21, a receptor-like kinase (RLK), contains a putative proteolytic cleavage motif (P685SRTSMKG) in the juxtamembrane (JM) domain (Xu et al., 2006). Previous studies have suggested that autophosphorylation of three serine and threonine residues within this motif play a ro le in the stabilization XA21. Sequence analysis led to the identification of a se cond putative cl eavage motif (P922TDSTFRP) located in the intr acellular kinase domain of XA 21. Similar to the first, the second motif carries three serine a nd threonine residues (Thr923, Ser925, and Thr926) that can be potentially phosphorylat ed. Therefore, it is hypothesized that autophosphorylation of these three residues also contribute to the stability of XA21. Here, transgenic rice plants with these th ree phosphorylable residue s mutated to alanine are analyzed for the steady-state stability of XA21. Consistent with previous studies, the XA21T923A/S925A/T926A mutant accumulated to lower leve ls than wild-type. This work provides additional evidence supporting that autophosphorylation of XA21 plays a role in the stabilization of XA21. Materials and Methods Mutant Rice Plants Transgenic lines expressing a c-Myc-tagged XA21T923A/S925A/T926A were provided by Dr. Xuihua Chen of the Song lab at the University of Florida.

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73 XA21 Detection Protein isolation. Rice plants approximately three months old were used for this study. Total protein isolation was performe d by first grinding leaf tissue in liquid nitrogen. Ground tissue was tran sferred into a 1.5 ml Eppendorf microcentrifuge tube to a volume of approximately 700 uL. An equa l volume of extraction buffer (50 mM TrisHCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 0.1% TritonX-100; 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma); 2 ug/ml leupe ptin; 2 ug/ml antipain ; 2 ug/ml aprotinin; 5% v/v -mercaptoethanol) was added and the tube was vortexed. The tubes were incubated on ice for five minutes. To pellet cell debris, tubes were centrifuged at 4C, 12,000 x g, for ten minutes. The supernatant wa s saved for application in western blot analysis. Protein concentrations in the extr acts were measured using the Bio-Rad protein assay (Hercules, CA). Western blot analyses. Each protein sample prepar ed above were subjected to SDS-PAGE and subsequently electrotrans ferred to a PVDF membrane (Millipore Corporation, Bedford, MA) for one hour at a constant 265 milliamps. The membrane was incubated at room temperature for three hours in Blotto [5% non-fat dried milk in TTBS (100 mM Tris-HCl, pH 7.9; 150 mM NaC l; 0.1% Tween 20)]. The membrane was washed in approximately 150 ml of TTBS thr ee times for 8 minutes each. Blots were incubated with primary anti body (anti-c-Myc, 1:700) in 3% bovine serum albumin in TTBS or in Blotto at 4C. The membrane was then washed as previously described. Secondary antibody (anti-mouse IgG, Amersham Biosciences) incubation was performed in Blotto at room temperature for one hour. The membrane was then once again washed as previously described. The blot wa s developed using Amersham Biosciences ECL+Plus kit.

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74 XA21 Transcript Levels To address the issues that lower protein leve ls may be due to lower transcripts, realtime PCR was carried out as in Wang et al. (in press). Primers used in this experiment recognize the LRR region of Xa21 and have been scrutinized for specificity. Template RNA was isolated from each mutant plant using the Qiagen RNeasy mini kit. Analysis of raw data, with the assistance of Dr. Li-Ya Pi of the University of Florida, was carried out to obtain values representing re lative amounts of transcripts. Results and Discussion All XA21T923A/S925A/T926A mutant rice plants surveyed exhibit lower levels of XA21 compared to wild-type levels (Figure 3-1). In contrast, Xa21 relative transcript levels in the mutants are comparable to those of wild -type XA21 or higher, confirming that lower steady states of XA21T923A/S925A/T926A is not due to lower transcripts. Ponceau staining of the membrane used in the Western blot ensu res approximately equal amounts of proteins were loaded into the gel. Together, thes e indicate that Thr923, Ser925, and Thr926 are involved in the stability of XA21. It must be noted howev er, that this Western blot analysis only indicates decrea sed level of protein and that degradation product cannot be seen. For future studies, it would be inte resting to purify the membrane fraction of XA21T923A/S925A/T926A mutant rice extract as in Xu et al. (2006) to allow detection of a cleavage product. If the putative cleavage motif examined he re and the one described by Xu et al. (2006) are true cleavage site s acting independently, three cleavage products of XA21 would result: 100 kD, 25 kD, and 15 kD (Fi gure 3-2). However, because of the Nterminus location c-Myc-tag, only full-le ngth XA21 and the 100 kD cleavage product

PAGE 85

75 could be detected on a Western blot. It is possible however that both putative cleavage motifs may act in conjunction when the protein is folded. Instability of XA21T923A/S925A/T926A mutant examined here is possibly due to a lack of autophosphorylation of XA21 on these residu es. Alternatively, au tophosphorylation of these three residues may function as docking sites for downstream proteins. Yeast-twohybrid analysis has led to the identification of 27 proteins that associate with XA21. A complex of proteins could protect XA21 fr om a protease(s) by limiting access to the recognition site. Residues mutated in D1 plan ts are contained within a motif of perfect identity to the proteolytic cleavage motif P/GX5-7P/G found in the animal EGFRs (Figure 3-3). Glycine flanking the putative cleavage mo tif may contribute to the exposure of this motif by disrupting its secondary structure. These findings ar e consistent with previous studies by Xu et al. (2006) and support that autophosphoryla tion of XA21 plays a role in the stabilization of XA21.

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76 Figure 3-1. Analyses of XA21T923A/S925A/T926A mutant. Relative transcripts are illustrated by the bar graph. The Western blot film at center shows amount of full length XA21 in wild-type (WT) plants and in seven XA21T923A/S925A/T926A mutant plants (numbers represent the plant line). Ponceau staining of the membrane was performed to ensure equal loading.

PAGE 87

77 Figure 3-2. Schematic representation of XA21 indicating putative cleavage motifs (A and B). Triangles mark the approxima te location of putative cleavage motifs and the N-terminal c-Myc tag. Bars a bove indicate approximate weights in kD of various portions of the protein befo re and after cleavage at the indicated sites. The transmembrane and JM domains are indicated. Figure 3-3. Amino acid sequence of the in tracellular domain of XA21. The putative cleavage motif characteri zed in the study is under lined and the mutated residues of D1 mutant s are shown in red.

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78 CHAPTER 4 DIRECT MUTATION OF THE PUTA TIVE CLEAVAGE MOTIF IN THE JUXTAMEMBRANE DOMAIN OF XA21 Introduction Previous findings suggest that auto phosphorylation of XA21 in the juxtamembrane (JM) domain plays a role in the protection of XA 21 against proteolytic degradation (Xu et al., 2006). The phosphor ylated residues Ser686, Thr688, and Ser689 exist within a putative cleavage motif flanke d by conserved residues of proline (Pro685) and glycine (Gly692). Herman and Chernaj ovsky (1998) demonstrat ed that Proline211 of the human p75 TNF receptor is required fo r its shedding. Pro211 is located in the juxtamembrane region of this protein. It w ould be interesting to investigate whether the conserved Pro685 and Gly692 of XA21 are important for the instability of this RLK. In this chapter, Pro685 and Gly692 are mutated to alanine. The constructs will eventually be used to confirm the role of the predicte d cleavage motif in XA21 stability. Residues within the putative cleavage motif in the juxtamembrane domain of XA21K, a nonfunctional kinase mutant, are mutated for future research. Materials and Methods The codons encoding proline and glycine residues flanking the putative cleavage motif located within the juxtamembrane dom ain of XA21K were mutated to encode alanine via site-directed mutagenesis (Figur e 4-1). The resulting mutants were named P9A and G16A, respectively. The constr uct used as the template, GST-XA21K, containing the putative intracellular domain of XA21 (XA21K), was used as template for

PAGE 89

79 site-directed mutatgenesis. The primers used are listed in Table 4-1. Each reaction contains 25 ng template DNA, Pfu buffer (Invitrogen), 200 uM of each dNTP, forward primer, reverse primer, and water to total 50 ul. Reactions were placed in MJ Research PTC-2000 Thermocycler and allowed to complete one denaturation cycle for ten minutes at 99C. The cycler was then paused to allow the addition of 2.5U of the DNA polymerase Pfu (Invitrogen) to each reacti on tube. Next, PCR conditions were run continuously: 5 cycles of 94C 1 min, 50C 1 min, 68C 16 min; 16 cycles of 94C 1 min, 56C 1 min, 68C 16 min; 1 cycle of 72C 10 min. The reactions were then subjected to restriction enzyme digestion by Dpn I, at 37C overnight to remove template. Each reaction was checked by agarose gel electrophor esis. Next, 1 ul of each reaction was mixed with 19 ul of ER2566 competent cells (New England Biolabs,) electroporated at 400 mV, and allowed to recover in 1 ml of Luria Broth (LB). Cells were then plated on solid LB supplemented with 50 ug/mL ampicillin and incuated at 37C overnight. Single colonies were picked, cultured, and subjected to DNA isolation by Qiagen miniprep kit. DNA was sequenced by the University of Flor ida DNA Sequencing Core at Fifield Hall. Table 4-1. Primers used in site-directed muta genesis. F and R indicate forward and reverse, respectively. Mutation Primers (5' to 3') P9A F-AGAACTAAAAAGGGAGCCGCTTCAAGAACTTCCATG R-CATGGAAGTTCTTGAAG CGGCTCCCTTTTTAGTTCT G16A F-TCAAGAACTTCCAT GAAAGCCCACCCATTGGTCTCT R-AGAGACCCATGGGTGGGC TTTCATCATGGAAGTTCTTGA

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80 Figure 4-1. The intracellular domain of XA 21. Mutated residues Pro685 and Gly692 are larger and highlighted in red. The putative cleavage motif is underlined. Results and Discussion To confirm that the sequence PSRTSMKG in the JM domain is a true cleavage motif, the conserved Pro685 and Gly692 were individually mutated to alanine. The P685A and G692A mutations will be eventually incorporated into the full-length Xa21 gene inder the control of the native pr omoter. It has been established that XA21S686A/T688A/S689A mutant is unstable (Xu et al., 2006), so we will test whether the XA21P685A/S686A/T688A/S689A or XA21SG692A/S686A/T688A/S689A mutant protein in the transgenic lines becomes more stable than XA21S686A/T688A/S689A. These experiments can provide verification of the putative cleavage motif in the JM domai n of XA21. If the residues indeed provide a protease recognition site, accumulation studies would show higher levels of protein than the phosphorylation mutant described by Xu et al. (2006). Additionally, an XA21P685A/S686A/T688A/S689A/G692A mutant could be crea ted to investigate if both residues are required for instability. Fo r further analysis, similar mutants could be made to the putative cleavage motif exam ined in Chapter 3. Also, simultaneous mutations of the conserved residues of the c onserved proline and glyc ine residues at both

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81 locations of XA21 could be fascinating to ex amine. Higher levels of XA21 may be seen as a result of these combined mutations.

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82 CHAPTER 5 CLONING OF SELECTED ARABIDOPSIS RLKS Introduction Arabidopsis thaliana is the model system of plant rese arch today. It is ideal for the laboratory as it is small, manageable, in expensively cultivated, while producing numerous progeny within only approximately one month (Arabidopsis Genome Initiative 2000). Consequently, Arabidopsis genes and proteins related to those studied in other less manageable plants are frequently investig ated first or in para llel with genes and proteins in those other plants. Also, because Arabidopsis has the smallest genome known of plants, research of these related genes can also help determine conservation of these genes through evolution. Previous investigation of about 600 Arabidopsis RLKs by Dr. Guozhen Liu of the Song Lab at the University of Florida ( unpublished) included alignments of their juxtamembrane domains. It was quickly noted that the RLKs fell into five separate categories based on a single amino acid in th e JM domain corresponding with Thr705 of XA21; at the specific site there is a threonine (T), aspartic acid (D) or glutamic acid (E), histidine (H), serine (S), or other amino acids (O.) Aspa rtic acid and glutamic acid are grouped because of their similar st ructures and similar behavior. In this chapter, four Arabidopsis open reading frames (ORFs) encoding DE group RLKs (Figure 5-1) were randomly selected, amplified from a library, and cloned into a vector.

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83 Figure 5-1. Sequence alignment of XA21 and selected Arabidopsis proteins of the DE group. Threonine705 of XA21 is red and underlined as well as the corresponding residues in Arabidopsis proteins. Materials and Methods Amplification of the At5g01890, At1g48480, At2g36570 and At3g28040 ORFs Four Arabidopsis ORFs encoding DE RLKs were randomly selected and primers were designed for each of them (Tab le 5-1), including the addition of a BamH 1 site for cloning. The ORFs were amplified from a TriplEx2 Arabidopsis total seedling library (Clontech, Mountain View, CA) provided by Dr. R obert Ferls lab at the University of Florida, by PCR using the RoboCycler (Stratagene). Table 5-1. List of primers used in the amplification of the selected Arabidopsis RLKs. F and R indicate forward and reverse, respectively. Gene Group Prim ers (5' to 3') At5g01890 DE F-CGGGATCCCACGCCCGATCTAGTGTTTCACG At5g01890 R-CGGGATCCT CACTCTAAGTCATGAGAGGGACA At3g28040 DE F-CGGGATCCAACGCGTCTGTTAGAAGACGGCTTG At3g28040 R-CGGGATCCTTAGAAACTATCCATGATACGGTGGG At2g36570 DE F-CGGGATCCCGTAACGGCGAAAGATCAAAATCCGG At2g36570 R-CGGGATCCCAGAAACCAAACTTAGCCGTCGGTGG At1g48480 DE F-CGGGATCCCGGAAAAAGAGTAATAAGAGATCAAGA At1g48480 R-CGGGATCC TTAATCAGCTTCGTTCACTTGGTCTG

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84 The PCR reaction used to amplify At5g01890, At2g36570 and At1g48480 contained 2 units of Taq DNA polymerase, 1x PCR buffer (Invitrogen), 200 uM each dNTP, 0.2 uM each appropriate primer, appr oximately 25 ng of template DNA. They were successfully amplified unde r 1 cycle of 99C for 10 minut es (after which the cycler was paused in order to add the enzyme); 35 cy cles of 94C for 1 minute, 50C for 1 min 30 sec, 72C for 1 min 30 sec; 1 cycle of 72C for 10 minutes. At3g28040 was amplified under the following conditions: 1 cycle of 99C for 10 minutes (after which the cycler was paused in order to add the enzyme ); 5 cycles of 94C for 1 minute, 45C for 1 mi n 30 sec, 72C for 1 min 30 s ec; 25 cycles of 94C for 1 minute, 50C for 1 min 30 sec, 72C for 1 min 30 sec; 1 cycle of 72C for 10 minutes. Amplification of gene At3g28040 was successful. Molecular Cloning of the PCR Products Following amplification, the PCR products sizes were checked by agarose gel electrophoresis. Approximately 60 ng of each product was then applied in separate ligation reactions into vector pGEM-T (Promega) according to manufacturer instructions. One microliter of the resulting ligation was a dded to 19 uL of XL1-Blue competent cells (Stratagene) and electroporated at 400 mV. Ce lls were then transfer red to 1 mL of Luria Broth (LB) and incubated at 37C at 100 rpm for one hour. Cells were then spread onto solid LB medium containing 50 ug/mL Ampicillin, isopropyl-D-thiogalactopyranoside (IPTG) and X-galactose, and inc ubated at 37C overnight. Single colonies were chosen, inoculated into 3 mL of liquid LB, and inc ubated at 37C overnight with shaking. DNA from these cultures was purified using the Qiag en miniprep kit. DNA was then digested overnight at 37C with Bam HI to check for the insert.

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85 Results and Discussion The ORFs of At5g01890, At1g48480, At2g36570, and At3g28040 were PCRamplified from an Arabidopsis cDNA library and cloned into the pGEM-T vector. Figure 5-2 shows Bam HI-digested recombinant DNA. Exp ected inserts of At5g01890 (Figure52A), At1g48480 (Figure5-2D), At2g36570 (Fig ure5-2B), and At3g28040 (Figure5-2C) are 1008, 1095, 1100, and 1050 base pairs (bp), re spectively. The size of the pGEM-T vector is 3000 bp. The inserts shown do not ex actly equal the expected sizes of clones of At5g01890 and At1g48480. These clones were se quenced at the DNA Sequencing Core of the University of Florida at Fifield Hall to confirm accuracy. Because the majority of plant RLKs contain a threonine residue at the position co rresponding to Thr705 of XA21 and this conserved residue is essential fo r autophosphorylation ac tivity of many RLKs, molecular cloning of members belonging to th e DE group of RLKs will provide a basis to test whether these kinases are ca pable of autophosphorylation.

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86 Figure 5-2. Identification of recombin ants carrying the ORFs of At5g01890, At1g48480, At2g36570 and At3g28040. The DNA was digested with BamH1. A: pGEMT-At5g01890, all lanes represent corr ect clones. B: pGEM-T-At2g36570, correct clones indicated y triangl es. C: pGEM-T-At3g28040, all lanes represent correct clones. D: pGEM -T-At1g48480, all lanes represent correct clones.

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95 BIOGRAPHICAL SKETCH Gina Marie Cory was born in Ft. Lauder dale, Florida, to Maureen and Samuel Cory. She graduated from Piper High School in Sunrise, Florida, in 1998. She began her undergraduate studies at the University of Fl orida in the fall of 1998, and graduated with a bachelors degree in plant science specializing in plant pathology in the spring of 2002. Gina began her graduate studies in plant pathology with Dr. Wen-Yuan Song the same fall.