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SUPPRESSORS OF CYTOKINE SIGNALING-1 (SOCS-1) MIMETIC AND ANTAGONIST
PEPTIDES: POTENTIAL AS THERAPEUTIC AGENTS FOR TREATMENT OF
LILIAN WANGECHI WAIBOCI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
O 2007 Lilian Wangechi Waiboci
To the memory of my grandmother Mrs. Priscilla Wangechi Matu and my grandfather Mr.
Richard Mwaniki wa Nyangi
I wish to thank my maj or supervisor Dr. Howard M. Johnson for funding this research, for
helpful suggestions and discussions, and for providing a conducive environment for learning. I
also wish to thank the members of my graduate committee Drs. Ayalew Mergia, Edward
Hoffmann, Peter Kima, and Janet Yamamoto for their helpful suggestions, and time.
I wish to thank member' s of the Johnson laboratory who helped with various aspects of
this research. I wish to specially thank Drs. Chulbul Ahmed and Mustafa Mujtaba for teaching
me many of the techniques used in this study. I wish to thank Mr. Mohammed Haider for
synthesizing all the peptides used in this study, Dr. Levy Omara-Opyene, Dr. Lawrence Flowers,
James Martin, Ezra Noon-Song, Rea Dabelic, Lindsey Jager, and Lauren Thornton for helpful
suggestions and assistance.
I also wish to thank Janet Lyles and Mary Ann Soncrant for helping make sure that my
studies and stay at the Microbiology Department ran smoothly and the staff of the International
Student Center for ensuring that my stay in the USA went smoothly.
I wish to thank my parents Mr. Francis Waiboci and Mrs. Esther Waiboci for all they have
done for my education, for instilling the love for learning, and discipline in my siblings and I. I
also wish to thank my siblings for their constant encouragement. Last, but not least, I wish to
thank my family, my husband Dr. George Kariuki for his love, understanding, and constant
encouragement through out the rough tides and my son Victor Kariuki for his love,
understanding, and patience.
TABLE OF CONTENTS
ACKNOWLEDGMENT S .............. ...............4.....
LI ST OF T ABLE S ............ ..... .__ ...............7...
LI ST OF FIGURE S .............. ...............8.....
AB S TRAC T ............._. .......... ..............._ 10...
1 INTRODUCTION ................. ...............12.......... ......
Rationale ................. ...............14.................
Specific Obj ectives ................. ...............16................
2 LITERATURE REVIEW ................. ...............18................
Janus Kinases ............... ... ... ... ....... .. .. ....... ........................1
Signal Transducers and Activators of Transcription (STAT) Proteins .............. ................. 19
Interferon Signaling Through the JAK/STAT Signaling Pathway............_._. .........._._. ...21
Regulation of the JAK/STAT Signaling Pathway ........._._........__. ......__. .........2
Suppre ssors of Cytokine Signaling (SOC S) ................. ...............23........... ...
Physiological Role of SOCS-1 Protein ................. ...............25...............
SOC S- 1 Mimetic Peptides ................. .. ........ ... ...... ........ .......2
Inhibition of SOCS-1 Activity and Immunological Relevance ................. ......._._. ........28
3 MATERIALS AND METHODS .............. ...............33....
Cell Culture............... ...............33
Peptides ........._.___..... ._ __ ...............33.....
Binding Assays ........._.___..... .__. ...............34....
In vitro Kinase As says ........._.___..... .___ ...............35....
Immunoblot Analysis............... ...............36
Macrophage Activity .............. ...............37....
Tkip Cellular Targets ................... ............ .. ...............37.....
Antiviral Assays for SOC S- 1 Antagoni st Function ................. ...............39........... .
Transfections of LNCaP Cells with SOC S- 1 DNA ................. ...............40.............
GAS Promoter Activity .............. .... ............ ...... ...............4
Primer Design and PCR Amplification of Murine SOCS-1 DNA ................ ................ ...42
Cloning SOC S-1 into pBlueB ac4.5/V5 -Hi s TOPO TA Expression Vector .........................42
Expression of SOCS-1 in Sf9 Cells............... ...............43.
Statistical Analysis............... ...............44
4 RE SULT S .............. ...............46....
Tkip Family Members Bind to JAK2 Autophosphorylation Site.............__ ..........___.....46
Tkip Family Members Inhibit JAK2 Kinase Activity ........._._ ......... ... ........._.....47
Tkip Inhibits Superantigen-induced Proliferation of Mouse Splenocytes ......................47
SOCS-1 Kinase Inhibitory Region (SOCS1-KIR) Binds to JAK2 Autophosphorylation
S ite ......................... .. .. .. ........ ..... .. ... ..................4
Tkip and SOCS1-KIR Bind to JAK2 Autophosphorylation Site ............. ..............49
JAK2 Kinase Activity and STATlot Activation .............. ..... ... ..............5
Tkip and SOCS1-KIR Inhibit IFNy-induced Activation of Macrophages ................... ...52
Tkip and SOCS1-KIR Inhibit Antigen-specific Lymphocyte Proliferation ................... .52
An Extended SH2 Sequence (SOCS 1-ESS) Peptide does not Bind to pJAK2 (1001-
1013) .............. ...............53....
Tkip Cellular Targets ........._._........... ..._._............._._ ............5
Effect of Tkip and SOCS1-KIR on CD4' T Cells ................ ...............54...........
Effects of Tkip and SOCS1-KIR on CD8' T Cells .....__.___ ..... .._. __ ........_.......55
Effect of Tkip and SOCS1-KIR on B Cells .....__.___ ..... ........ ...............56
Effect of Tkip on Macrophages .................. ... ......__............. ........ ....... 5
SOCS-1 Antagonist Activity of pJAK2 (1001-1013) Peptide .........__......... .._.... .........57
Expression of SOCS-1 Protein .............. ...............59....
5 DI SCUS SSION ............ ..... ..__ ............... 0...
6 FUTURE WORK............... ...............86..
APPENDIX: Vector Map of the Transfer (Cloning) Vector ................. ............................87
LIST OF REFERENCES ................. ...............88................
BIOGRAPHICAL SKETCH .............. ...............94....
LIST OF TABLES
2-1 A table showing the JAK kinases and STAT proteins utilized by some cytokines,
growth factors, hormones, and oncogenes. .............. ...............32....
3-1 List of peptides used in this study. ................ .......... ......... ........ ............45
LIST OF FIGURES
2-1 A schematic representation of the domain structure of the JAK kinase family. ........._.....30
2-2 A schematic diagram showing the domain structure of SOCS-1 protein. .........................31
4-1 JAK2 autophosphorylation site peptides JAK2(1001-1013) and pJAK2(1001-1013)
bind to SOCS-1 mimetic peptides............... ...............61
4-2 Both soluble DRTkip and soluble Tkip inhibit the binding of biotinylated
JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip. ........._......62
4-3 DRTkip and Tkip but not the control peptide, MulFNy(95-106), inhibit JAK2
4-4 Tkip, but not DRTkip inhibits superantigen-induced splenocyte proliferation. ................64
4-5 JAK2 autophosphorylation site peptides bind to SOCS1-KIR. A) JAK2(1001-1013)
peptide binds to both SOCS1-KIR and Tkip. ................ ...............65.............
4-6 Both soluble SOC S1-KIR and soluble Tkip inhibit the binding of biotinylated
JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip or
SOCS1-KIR. ............. ...............66.....
4-7 Differences in the kinase inhibition patterns of SOCS1-KIR and Tkip in JAK2
autophosphorylation, STATlot phosphorylation, and EGFR phosphorylation. ...............68
4-8 SOCS1-KIR and Tkip inhibit IFNy-induced macrophage activation. ............. ..... ..........69
4-9 Both SOCS1-KIR and Tkip inhibit proliferation of murine splenocytes.. ................... ......70
4-10 Biotinylated pJAK2(1001-1013) binds to SOCS1-KIR but not to SOCS1-ESS............_...71
4-11 Tkip and SOCS1-KIR inhibit antigen-specific CD4+ T cell proliferation and CD4+ T
cell-induced IFNy production. ............. ...............72.....
4-12 Tkip and SO C S1-KIR inhibit CD 8 T cell-induced IFNy product on............... ..... ........._.73
4-13 Tkip and SOCS1-KIR inhibit antigen-induced B cell proliferation and antibody
production. ............. ...............74.....
4-14 Tkip inhibits LP S-induced macrophage activity ........._.._........_. ......._.. .......7
4-15 pJAK2(1001-1013) peptide has SOCS-1 antagonist properties. ............. ....................76
4-16 SOCS-1 protein was expressed in baculovirus infected Sf9 insect cells. ..........................78
4-17 Recombinant baculovirus containing muSOCS-1 DNA ................. ................. ......79
A-1 A map of the pBlueBac4.5/V5-His vector. ................. ....___.....__ ..........8
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
SUPPRESSORS OF CYTOKINE SIGNALING -1 (SOCS-1) MIMETIC AND ANTAGONIST
PEPTIDES: POTENTIAL AS THERAPEUTIC AGENTS FOR TREATMENT OF
Lilian Wangechi Waiboci
Chair: .Howard M. Johnson
Major: Microbiology and Cell Science
Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by interferon
gamma (IFNy) by binding to the autophosphorylation site of Janus kinase 2 (JAK2) and by
targeting bound JAK2 to the proteosome for degradation. Studies on a tyrosine kinase inhibitor
peptide, Tkip, which is a SOCS-1 mimetic, are described. Tkip was synthesized from the
physiological form of amino acids, L-amino acids. We synthesized two additional SOCS-1
mimetic peptides, DTkip and DRTkip, from D-amino acids, which are potentially more resistant
to degradation and therefore would likely be better therapeutic agents. DTkip and DRTkip bound
to the unphosphorylated JAK2 autophosphorylation site peptide, JAK2(1001-1013) and the
tyrosine 1007 phosphorylated peptide, pJAK2(1001-1013). Further, DTkip and DRTkip inhibited
JAK2 autophosphorylation and JAK2 phosphorylation of IFNy receptor-1 (IFNGR-1).
Tkip was also compared with the kinase inhibitory region (KIR) of SOCS-1 for JAK2
recognition, inhibition of kinase activity, and regulation of IFNy-induced biological activity.
Tkip and a peptide corresponding to the KIR region of SOCS-1, 53DTHFRTFRSHSDYRRI
(SOCS1-KIR), bound similarly to JAK2(1001-1013) and to pJAK2(1001-1013). Dose-response
competitions suggested that Tkip and SOCS1-KIR similarly recognized the autophosphorylation
site of JAK2. While Tkip inhibited JAK2 autophosphorylation as well as IFNy-induced STATla
phosphorylation, SOCS1-KIR, like SOCS-1, did not inhibit JAK2 autophosphorylation but
inhibited STATla activation. Both Tkip and SOCS1-KIR inhibited IFNy activation of murine
macrophages and antigen-specific splenocyte proliferation.
The fact that SOCS1-KIR bound to pJAK2(1001-1013) suggested that the JAK2 peptide
could function as an antagonist of SOCS-1. Thus, pJAK2(1001-1013) enhanced suboptimal IFNy
activity, blocked SOCS-1 induced inhibition of STAT3 phosphorylation in IL-6-treated cells,
enhanced IFNy activation site (GAS) promoter activity, and enhanced antigen-specific
proliferation. Further, SOCS-1 competed with SOCS1-KIR for pJAK2(1001-1013). Thus, the
KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide
corresponding to this site can function as an antagonist of SOCS-1.
In summary, Tkip and SOCS1-KIR recognized the autophosphorylation site of JAK2
similarly and pJAK2(1001-1013) peptide functioned as a SOCS-1 antagonist. Thus, we have
developed peptides that function as SOCS-1 agonists and antagonists, which have potential for
suppressing or enhancing the immune response.
Janus tyrosine kinases (JAKs) are an enzyme family that mediates the biological effects of
cytokines, hormones, and growth factors by tyrosine phosphorylation of signal transducers and
activators of transcription, STATs (Reviewed in Yamoaka et al. 2004; Parganas et al. 1998). The
interferons (IFNs), including types I and type II, hormones such as growth hormone and
angiotensin, and growth factors such as thrombopoietin, are all among 50 related factors
dependent on JAK tyrosine phosphorylation of appropriate STAT transcription factors for their
physiological functions (Subramaniam et al. 2001; Johnson et al. 2004).
The immediate early signal transduction events associated with IFNy and its receptor
subunits involve the obligatory action of two tyrosine kinases, JAK1 and JAK2 (Reviewed in
Kotenko and Peska 2000). The IFNy receptor (IFNGR) system is a heterodimeric complex
consisting of an a-subunit (IFNGR-1) and a P subunit (IFNGR-2), both of which are essential for
the biological activity of IFNy (Kotenko and Peska 2000). JAK1 is associated with the IFNGR-1
chain, whereas JAK2 is associated with the IFNGR-2 chain. The interaction of IFNy, primarily
with the IFNGR-1 subunit, initiates a sequence of events that results in increased binding of
JAK2 to IFNGR-1. This has important consequences for subsequent critical phosphorylation
events. JAK2, in the process of binding to IFNGR-1, undergoes autophosphorylation, and at the
same time IFNGR-1 is phosphorylated. These events occur in concert with JAK1 function,
resulting in recruitment and tyrosine phosphorylation of the IFNy transcription factor, STAT 1
(Reviewed in Kotenko and Peska 2000; Bromberg and Darnell 2000).
A family of proteins called suppressors of cytokine signaling (SOCS) negatively regulates
JAK/STAT signaling (Starr et al. 1997; Endo et al. 1997; Naka et al. 1997). SOCS proteins are
also negative regulators of signaling by other cytokines, growth factors, and hormones
(Reviewed in Alexander et al. 2002; Larsen and Ropke 2002; Alexander and Hilton 2004). There
are currently eight identified members of the SOCS family, SOCS-1 to SOCS-7 and cytokine-
induced SH2 domain containing protein (CIS). SOCS-1 is of particular interest, because it is a
negative regulator of the JAK kinases, as well as several cytokines, and hormone receptor
systems including epidermal growth factor receptor (EGFR) (Reviewed in Calo et al. 2003).
Our laboratory recently designed and synthesized a tyrosine kinase inhibitor peptide, Tkip
(WLVFFVIFYFFR) (Flowers et al. 2004). The characteristics of the Tkip 12-mer are
summarized in Chapter 2. In this study we show that Tkip inhibits IFNy-induced macrophage
activation and define Tkip cellular targets in antigen-induced immune responses. We also
describe a family of Tkip-related peptides, DTkip (WLVFFVIFYFFR) and DRTkip
(RFFYFIVFFVLW). DTkip and DRTkip, which like Tkip, bind to the JAK2
autophosphorylation site peptide, inhibit JAK2 autophosphorylation, and JAK2 phosphorylation
of IFNy receptor subunit, IFNGR-1.
It has been suggested that the binding of SOCS-1 to JAK2 requires the SOCS-1 SH2-
domain and that the kinase inhibitory region (KIR), while not required for the binding, is
essential for the inhibitory action of SOCS-1 (Yasukawa et al. 1999). We show here that a
peptide corresponding to SOCS-1 KIR region, SOCS 1-KIR, specifically binds to a peptide
representing the JAK2 autophosphorylation site and inhibits STATla activation. Further, we
show that SOCS1-KIR, as well as Tkip, inhibit IFNy-induced macrophage activation. We also
present data on a novel SOCS-1 antagonist peptide, pJAK2(1001-1013), which corresponds to
the JAK2 autophosphorylation site. pJAK2(1001-1013) enhances suboptimal IFNy-induced
antiviral activity, enhances IFNy-activated sequences (GAS) promoter activity, and inhibits
SOCS-1 suppression of STAT3 activation of LNCaP prostate cancer cells, thus functioning as a
SOCS-1 antagonist peptide.
SOCS proteins play an important role in the regulation of JAK/STAT signaling. Disruption
of the normal SOCS function may contribute to disease onset, progression or death. It has been
shown that deletion of SOCS-1-'- or SOCS-3- genes in mice results in death of the mice either as
neonates (SOCS-1-'-) or embryos (SOCS-3- -) (Naka et al. 1998; Starr et al. 1998). SOCS proteins
regulate immune response by inhibiting JAK kinases. Uregulated JAK kinases signaling may
result in inflammation and cancers.
Several cancers are characterized by constitutive activation of the JAK/STAT signaling
pathway. SOCS proteins may play a role in inhibiting malignant transformation of cells by
regulating JAK/STAT signaling, thereby preventing cancer onset and progression. This has been
shown in a number of cancers including leukemia (T cell acute lymphoblastic leukemia (ALL),
Pre-B ALL, and atypical chronic myelogenous leukemia (CML)), and hepatocellular carcinoma
(Alexander and Hilton 2004). These examples raise the interesting possibility of the role that
SOCS proteins and/or SOCS mimetics may play in the management of these cancers.
Dysregulation of JAK/STAT signaling also plays important roles in the pathogenesis of
some inflammatory diseases including rheumatoid arthritis (Suzuki et al. 2001), inflammatory
diseases of the gastrointestinal tract (SOCS2), (Suzuki et al. 2001; Lovato et al. 2003; Shouda et
al. 2001) as well as inflammations of the central nervous system for example in experimental
allergic encephalomyelitis (EAE), an animal model for multiple sclerosis in humans (Maier et al.
2002). Hence, dysregulation of JAK/STAT signaling plays an important role in the pathology of
some inflammatory diseases and therefore raises the possibility that SOCS proteins and/or SOCS
mimetics that negatively regulate JAK/STAT signaling may be possible therapeutics for the
control and treatment of the inflammatory disease.
There are several tyrosine inhibitors that are currently undergoing clinical trials for
treatment of various cancers, especially cancers resistant to chemotherapy- and radiation.
Herceptin (Trastuzumab) is a humanized monoclonal antibody specific for a member of the
epidermal growth factor receptor (EGFR) family called HER2, which binds to the extracellular
binding domain of HER2/neu on tumor cells inducing receptor internalization and inhibiting cell
cycle progression (Reviewed in Shawyer et al. 2002). The discovery of Herceptin resulted in the
treatment of aggressive forms of breast cancer, in which cancer cells overexpressed HER2.
These cancers are usually less responsive to chemotherapy (Shawyer et al. 2002). A second drug,
GleevacTM, iS a small molecule inhibitor of the oncogenes BCR-Abl, Abl, PDGFR, and c-kit.
GleevacTM blocks ATP binding to the kinase, thereby preventing phosphorylation events that are
required for signal transduction. GleevacTM has been shown to increase the effectiveness of
interferon therapy on chronic myelogenous leukemia patients (Shawyer et al. 2002). Other small
molecule tyrosine kinases targeted therapies include Erbitux (EGFR) for treatment of colorectal
cancer, Tarceva (EGFR) for treatment of pancreatic cancer, and Iressa (EGFR) for treatment of
non-small-cell lung cancer (Shawyer et al. 2002; Vincentini et al. 2003). This provides additional
evidence that tyrosine kinases targeted approaches may have potential as anti-cancer therapy.
Hence, it is possible that the SOCS-1 mimetic peptides developed in our laboratory, which
are tyrosine kinase inhibitors of STAT transcription factors such as the STAT3 oncogene, may
have potential as anti-cancer and anti-inflammatory disease agents. The mimetics would likely
augment the effect of endogenous SOCS-1. We will discuss these mimetic peptides and present
data on their effect on the JAK/STAT signaling pathway, IFNy-signaling, and cell proliferation.
SOCS-1 regulates signaling by a variety of cytokines including IFNy, IFNa, and several
interleukins. Put differently, SOCS-1 reduces the immune response mechanisms initiated by
these cytokines. In an immune competent individual, this is desirable because it prevents
excessive signaling, which would likely result in inflammation. However, in an
immunocompromised individual, there may be a need to enhance cytokine-induced immune
response to infection. One way of enhancing immune response to pathogen infection may be by
inhibition of SOCS-1 activity. It has recently been shown that silencing of SOCS-1 in dendritic
cells promoted cell activation, which led to enhancement of effective antigen-specific anti-tumor
immunity (Shen et al. 2004). This implies that reagents that negatively regulate SOCS-1 may
have potential in control and/or treatment of diseases arising from inadequate immune response.
Hence, we designed a SOCS-1 inhibitor peptide, pJAK2(1001-1013), that is derived from the
JAK2 autophosphorylation site. I will present data showing that pJAK2(1001-1013) is a SOCS-1
In this dissertation Tkip and other SOCS-1 mimetic peptides, which I hypothesized, would
function similar to Tkip and therefore similar to SOCS-1 are described. The first obj ective of this
study therefore was to determine whether these peptides; DTkip and DRTkip have Tkip-related
characteristics. Specifically, whether the peptides bind to JAK2 autophosphorylation site, inhibit
JAK2 autophosphorylation, and inhibit activated JAK2 activity.
It has previously been stated that the SOCS-1 domains that bind directly to JAK2
autophosphorylation site are the SH2 domain and the extended SH2 region (ESS), and that the
kinase inhibitory region (KIR) is not essential in the initial binding (Yasukawa et al. 1999). The
second objective of this study was therefore to determine whether a peptide corresponding to
SOCS-1 KIR, SOCS1-KIR, binds directly to JAK2 autophosphorylation site, inhibits JAK2
autophosphorylation, and inhibits activated JAK2 activity.
Since Tkip has been shown to be a SOCS-1 mimetic with possible therapeutic potential,
the third objective of this study was to define Tkip cellular targets. Specifically, to determine
whether Tkip targeted B cells, CD4+ T cells, CD8+ T cells, and antigen presenting cells. This
would provide insights on the direct effect of Tkip on specific cells of the immune system, which
is important if Tkip proves be a potential therapeutic agent.
The fourth objective of this study was to define a novel way of addressing inadequate
cytokine-induced immune response mechanism. Here, a novel SOCS-1 antagonist peptide,
pJAK2(1001-1013), is described and preliminary data showing that this peptide reverses SOCS-1
inhibition is presented.
Janus tyrosine kinases (JAKs) are a small but indispensable enzyme family of nonreceptor
tyrosine kinases that mediates the biological effects of cytokines, hormones, and growth factors
by tyrosine phosphorylation of signal transducers and activators of transcription, STAT,
(Reviewed in Ihle 1995; Parganas et al. 1998). The JAK family consists of four members (JAKl,
JAK2, JAK3, and TYK2) that are differentially activated in response to various cytokines (Ihle
1995). JAK proteins are approximately 120 to 130 kDa cytosolic proteins expressed in many
types of tissue with the exception of JAK3 whose expression is restricted to cells of the
hematopoietic system (Ihle 1995, Reviewed in Thompson 2005).
Members of the JAK protein family contain highly conserved structural domains
designated JAK homology domains (JH). There are currently seven known JH domains (JH1-
JH7) of which JH1 is the functional catalytic kinase. For JAK2, this domain possesses a critical
activation loop that becomes phosphorylated in order to activate the kinase. The phosphorylation
of tyrosine residue (Y1007) in the activation loop of JAK2 is essential for activation and
downstream signaling events (Feng et al. 1997). It has recently been suggested that the
phosphorylation of other tyrosines may also be necessary for JAK2 activation (Kurzer et al.
2004). Phosphorylation of Y1007 results in a conformational change in the activation loop,
which allows substrate access to specific binding sites in the catalytic groove (Yasukawa et al.
JH2 is a non-functional catalytic kinase domain but it bears sequence homology to typical
tyrosine kinase domains, hence it is referred to as a pseudokinase domain. The functional role of
the JH2 domain is unclear, however studies have suggested that it may have a kinase inhibitory
function. Both the JH1 and JH2 domains are located near the carboxyl terminus and comprise the
maj or portion of the JAK molecule (Reviewed in Leonard 2001; Kisseleva et al. 2002). The JH3,
JH4, and JH5 domains are poorly understood and require additional work to elucidate their
functions. JH6 and JH7 amino terminal domains have been implicated in the association between
the JAK molecule and the specific cytokine receptor (Leonard 2001; Kisseleva et al. 2002).
Figure 2-1 shows a diagrammatic representation of the domain structure of the JAK kinases.
Comparisons of the JAK2 autophosphorylation site amino acid sequences
(LPQDKEYYKVKEP) revealedl00% sequence homology among different mammalian species
including of the human JAK2 (Homo sapiens, genebank accession number NM_004972) mouse
(M~us musculus, AAH54807), rat (Rattus norvegicus, NP_113702), and pig (Sus scrofa,
BAA21662) as determined using the basic local alignment search tool (BLAST search,
http://www.ncbi .nlm.nih.gov/blast/). In addition, the amino acid sequence of the human JAK 1
(IETDKEYYTVKDD, accession number NP_002218) was 100% homologous to the mouse
JAK1 (NP_666257), as was human TYK2 (VPEGHEYYRVRED, accession number
NP_003322) and mouse TYK2 (NP_061263). Human JAK3 (LPLDKDYYVVREP,
NP 000206) and mouse JAK3 (LPLGKDYYVVREP, NP 034719 are nearly identical with a
one amino acid substitution, underlined. It is worth noting that in all four JAK kinases, the
sequences are similar, but not identical. The conservation in this region indicates the importance
of the autophosphorylation site in JAK function.
Signal Transducers and Activators of Transcription (STAT) Proteins
STAT proteins are a family of cytoplasmic transcription factors that participate in a variety
of cellular events, including differentiation, proliferation, cell survival, apoptosis, and
angiogenesis involving cytokines, growth factors, oncogenes, and hormones. Some of the
cytokines, growth factors, oncogenes, and hormones that utilize STAT proteins are shown in
There are currently seven known STAT family members: STAT1, STAT2, STAT3,
STAT4, STAT5A, STAT5B, and STAT6 (Darnell 1997). We are particularly interested in
STAT 1 and STAT3, both of which are negatively regulated by SOCS-1. It is worth noting that
other SOCS proteins may also negatively regulate STAT1 and STAT3.
The STAT proteins are comprised of six domains. These are oligomerization, coiled-coil,
DNA binding, linker, SH2, and transcription activation domains. The binding of STAT to the
receptors occurs through interaction of the SH2 to the receptor-docking site. The critical tyrosine
residues required for SH-phosphotyrosine interaction are STAT1-Y701; STAT2-Y690; STAT3-
Y705; STAT4-Y693; STAT5-Y694, and STAT6-Y641. These phosphotyrosines are located near
the SH2 domain (Calo et al. 2003).
The linker domain, which is alpha helical is the bridge between the DNA binding and the
SH2 domains. The transcription activation domain, located on the carboxyl terminus is involved
in communication with transcription complexes. The domain has a conserved serine residue
(except in STAT2 and STAT6) that when phosphorylated regulates STAT transcription activity
(Reviewed in Imada and Leonard 2000). The amino terminus region of STAT proteins is highly
conserved and provides protein to protein interaction, such as dimer interaction of STAT
molecules, which contributes to the stability of STAT-DNA binding, thereby increasing
transcription activity (Imada and Leonard 2000). The coiled-coil domain may be involved in
regulatory function and may be responsible for nuclear export of STAT.
Although some constitutively activated STATs have been observed in some human cancer
cell lines and primary tumors, STAT proteins are generally activated by tyrosine phosphorylation
(Cal6 et al. 2003). The STAT proteins become activated by a variety of receptors, such as
cytokine, growth factor, and hormone receptors, which may activate STATs directly or indirectly
through JAK kinases. In JAK/STAT signaling, the binding of a cytokine to its receptor results in
the associated JAK kinase becoming phosphorylated, and hence activated. The activated JAK
kinase phophorylates the receptor's cytoplasmic domain, at specific tyrosine residues, and opens
docking sites for STAT proteins. The STAT proteins docked on the receptor, are phosphorylated,
and thus become activated. Some models of JAK/STAT signaling indicate that the activated
STATs dissociate from the receptor, dimerize, and translocate to the nucleus where they activate
the transcription of specific genes (Bromberg and Darnell 2000). However, it has been shown
that in the case of IFNy signaling, IFNy, IFNGR-1, and the bound phospho-STAT1 dimer are
translocated to the nucleus as a complex (Subramaniam et al. 2001, Ahmed et al. 2003, Ahmed
and Johnson 2006), which implies that IFNGR-1 likely plays additional roles in signal
Interferon Signaling Through the JAK/STAT Signaling Pathway
There are two classes of interferon, type I and type II, both of which utilize the JAK/STAT
signaling pathway. Although type I and type II IFNs signaling pathways are similar, distinct
receptors, JAK kinases, and STAT proteins are utilized. IFNy, the type II IFN, signaling utilizes
IFNGR, which is a heterodimeric complex comprised of two subunits, oc (IFNGR-1) and P
(IFNGR-2), both of which are essential for biological activity of IFNy (Kotenko and Pestka
IFNGR-1 is associated with JAKl, while the IFNGR-2 is associated with JAK2.
Interaction of IFNy, primarily with IFNGR-1, causes the receptor subunits to dimerize and brings
the associated JAK1 and JAK2 into close proximity. The JAK kinases undergo
autophosphorylation at specific tyrosine residues (1007 for JAK2) and become activated
(Kotenko and Pestka 2000). The activated JAKs phosphorylate and activate the IFNGR subunits,
which results in a cascade of events including the phosphorylation of STATla. Two
phosphorylated STAT1 monomers dimerize and are phosphorylated at specific serine residues to
form an activated STAT1 transcription factor. According to some models of IFNy signaling, the
STAT 1 dimers dissociate from the IFNGR-1, translocate to the nucleus, bind to IFNy activation
sites (GAS), and induce expression of target genes (Bromberg and Darnell 2000). It has however
been shown that IFNGR-1 accumulates in the nucleus and colocalizes with STATla in a time
and dose dependent manner, implying that IFNGR-1 is likely also translocated into the nucleus
(Subramanian et al. 2001, Ahmed et al. 2003, Ahmed and Johnson 2006). Thus, IFNGR-1 may
play an active role in signal transduction events subsequent to binding of the receptor complex.
Studies have indicated that IFNGR-2 likely is not translocated to the nucleus (Ahmed and
Type I interferons, IFNa for example, utilize the interferon alpha-receptor (IFNAR) for
signal transduction. The IFNAR is comprised of two subunits, IFNAR-1 (associated with TYK2)
and IFNAR-2 (associated with JAKl) (Kotenko and Pestka 2000). The signal transduction
pathway is initiated when IFNa binds to the receptor, which results in IFNAR-1 and IFNAR-2
forming a heterodimer with subsequent autophosphorylation and activation of both JAK1 and
TYK2. The activated JAK kinases phosphorylate IFNAR-2, providing docking sites for STAT2,
which binds to the receptor and becomes phosphorylated at tyrosine 690. The phosphorylation
favors the binding of STAT1 to the phosphorylated STAT2 (Durbin et al. 1996). STAT1 is
phosphorylated (tyrosine 701) by the JAK kinases and the STAT2/STAT1 heterodimer is
released from the receptor and translocates to the nucleus. The STAT2/STAT1 heterodimer
associates with p48 nuclear factor to form the IFN-stimulated gene factor (ISGF3) complex
(Horvath et al. 1996), which stimulates activation of target genes within the IFN-stimulated
response elements (IRSE) (Li et al. 1998).
Regulation of the JAK/STAT Signaling Pathway
The JAK/STAT signaling pathway is carefully regulated. Unregulated JAK/STAT
signaling may result in excessive cytokine-induced immune response, which would likely result
in inflammation and be harmful to cells, and ultimately to the organism. Three classes of
regulators of the JAK/STAT pathway are currently known. These are the protein inhibitors of
activated STATs (PIAS), tyrosine phosphatases that include the Src-homology 2 (SH2)-
containing protein tyrosine phosphatases (SHPs), and the suppressors of cytokine signaling
protein family (SOCS) (Reviewed in Kile et al. 2001; Kisseleva 2002; Larken and Roipke, 2002,
Alexander and Hilton 2004). PIAS proteins regulate transcription through several mechanisms,
including blocking the DNA-binding activity of transcription factors, recruiting transcriptional
co-repressors and promoting protein sumoylation (Shuai 2006). SHPs regulate JAK/STAT
pathway by dephosphorylating activated phosphotyrosine (Reviewed in Rico-Bautista et al.
2006). SOCS can block cytokine signaling by acting as (i) kinase inhibitors of JAK proteins
(SOCS1 and SOCS3), (ii) binding competitors against STATs (SOCS3 and CIS) and (iii) by
acting as ubiquitin ligases, thereby promoting the degradation of their partners (SOCS1, SOCS3,
and CIS) (Rico-Bautista et al. 2006). I will discuss the SOCS proteins, in detail, as an example of
negative regulators of JAK/STAT signaling, because SOCS proteins are essential for the
regulation of JAK/STAT signaling and other signaling pathways.
Suppressors of Cytokine Signaling (SOCS)
SOCS proteins are a family of cytoplasmic proteins that negatively regulate signal
transduction of cytokines, hormones, and growth factors that utilize the JAK/STAT signaling
pathway (Naka et al. 1997, Starr et al. 1997, Endo et al. 1997). Loss or insufficient expression of
SOCS proteins may result in diseases including several immune disorders, inflammatory
diseases, and cancers (Reviewed in Alexander and Hilton 2004; Tan and Rabkin 2005). Mouse
studies have shown that deletion of the SOCS-1 gene results in neonatal death (Naka et al. 1998;
Starr et al. 1998), while deletion of the SOCS-3 gene results in embryonic death (Croker et al.
2003). In the case of human disease, it has been shown that a number of hematological
malignancies are characterized by constitutive activation of JAK/STAT signaling pathway.
These malignancies include T cell acute leukemia and atypical chronic myelogenous leukemia
(Alexander and Hilton 2004). Further, SOCS-1 is likely a tumor suppressor since aberrant DNA
methylation of SOCS-1 gene, resulting in transcriptional silencing, has been observed in human
hapatocellular carcinomas and hepatoblastomas. The restoration of SOCS-1 expression in cells in
which SOCS-1 gene had been silenced led to reduction in the transformed phenotype (Alexander
and Hilton 2004), providing direct evidence that lack of SOCS-1 expression may have played a
role in the development of the malignancies. In addition, constitutive activation of STAT3 and
deregulation of SOCS3 expression have been observed in a variety of inflammatory diseases
(Alexander and Hilton 2004). Thus, SOCS proteins play a fundamental role in maintaining
There are eight members of the SOCS family. These are the cytokine-induced SH2 domain
containing protein (CIS), SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, SOCS-6, and SOCS-7
(Reviewed in Alexander 2002; Larsen and Roipke, 2002; Alexander and Hilton, 2004). The
SOCS proteins are also known as JAK-binding protein (JAB), STAT-induced STAT inhibitor
(SSI), and cytokine-inducible SH2 containing (CIS) proteins (Larsen and Roipke, 2002). All
SOCS proteins have three shared domains, which are an N-terminal domain of varying length
and sequences, a central SH2 domain, which is essential for binding to the JH1 region of JAK
kinases (Yasukawa et al. 1999), and a C-terminal SOCS box. The SOCS box couples substrate-
specific interactions of the SH2 domain to the ubiqutination machinery, resulting in proteosomal
degradation of associated JAK (Alexander 2002). SOCS-1 and SOCS-3 also have the kinase
inhibitory region, KIR, which is hypothesized to be involved in catalytic activity of SOCS-1, but
not in the actual binding to JAK2 autophosphorylation site (Yasukawa et al. 1999, Giordanetto
and Kroemer 2003). In this manuscript, data will be provided indicating that the KIR region is
likely involved in direct binding to JAK2 autophosphorylation site. Figure 2-2 contains a
schematic representation of the domain structure of SOCS-1.
The physiological roles of four SOCS family members (CIS, SOCS-1, SOCS-2, and
SOCS-3) are well defined. I will briefly discuss the physiological role of SOCS-1 as an example
of the important role that SOCS proteins play in maintaining homeostasis.
Physiological Role of SOCS-1 Protein
SOCS-1 plays a vital role in negative regulation of IFNy signaling as shown by both in
vivo studies and in vitro assays. SOCS-1 double knockout mice (SOCS-1- -) die as neonates,
displaying low body weight and liver damage including necrosis. Diseased livers are
characterized by the presence of aggregates of granulocytes, eosinophils, and macrophages. The
SOCS-1--~ mice also exhibit monocytic invasion of the pancreas, the lung, and the heart (Starr et
al. 1998; Naka et al. 1998) and have a marked reduction in blood and spleen lymphocytes, as
well as severe deficiencies in both mature B- and T lymphocytes. In addition, mice thymuses are
reduced in size and show increased numbers of apoptotic cells both in the spleen and the thymus
when compared to normal mice. These symptoms indicate severe deficiencies in the immune
system (Naka et al. 1998; Starr et al. 1998) and show that SOCS-1 is indispensable for normal
The pathologies observed in SOCS-1-'- gene knock out mice were similar to those observed
in wild-type mice administered excess IFNy, which led to the hypothesis that the disease
observed in the SOCS-1-'- gene knock out mice was likely due to excessive response to IFNy.
Direct evidence that IFNy was required for the development of the lethal disease observed in
SOCS-1- gene knock out mice was obtained when the mice were treated from birth with IFNy
neutralizing antibodies. After three weeks of IFNy treatment the anti-IFNy treated mice remained
healthy, while all the untreated mice had succumbed to disease (Alexander et al. 1999). In
addition, the SOCS-1- -/IFNy- double knockout mice did not exhibit the lethal phenotype
observed in SOCS-1-'- knockout mice (Alexander et al. 1999).Thus, SOCS-1 is a key
physiological regulator of IFNy signaling. It is worth noting that SOCS-1- -/IFNy- double
knockout mice eventually died 6 months after birth with inflammation and polycystic kidneys
(Metcalf et al. 2002), which suggested that SOCS-1 regulation was not specific for IFNy.
The prediction that SOCS-1 regulates IFNy signaling was confirmed using biochemical
studies. Upon inj section of IFNy into mice, STAT1 phosphorylation was evident in the livers of
the SOCS-1+/ mice within 15 min but declined after 2 h. However, in the SOCS-1-'- gene knock
out mice, phosphorylated STAT1 remained and was detectable 8 h after IFNy administration
(Brysha et al. 2001), indicating continued IFNy signaling. These observations, taken together,
indicated that SOCS-1 is a physiological negative regulator of IFNy signaling (Alexander and
Hilton 2004) and that unregulated IFNy activity contributed to the pathology observed in the
SOCS-1- gene knock out mice. Thus, SOCS-1 plays a fundamental role in regulating IFNy
signal transduction. It has also been shown that SOCS-1 plays a fundamental role in the
regulation of IFNa and IFNP (Fenner et al. 2006) and other cytokines signaling through the
SOCS-1 Mimetic Peptides
Our laboratory has designed a family of SOCS-1 mimetic peptides, which are being tested
for SOCS-1-like activity. The first of these peptides was tyrosine kinase inhibitor peptide (Tkip,
WLVFFVIFYFFR), which was designed using a complementary peptide approach for
complementarity to the JAK2 autophosphorylation site (Flowers et al 2004). Tkip binds to the
JAK2 autophosphorylation site and inhibits JAK2 autophosphorylation and JAK2 mediated
phosphorylation of the IFNGR-1 (Flowers et al. 2004). Tkip also inhibits the
autophosphorylation of the epidermal growth factor receptor (EGFR), consistent with the fact
that EGFR is regulated by SOCS-1 and SOCS-3. In contrast, Tkip does not bind or inhibit
tyrosine phosphorylation of the vascular endothelial growth factor receptor (VEGFR) or the
substrate peptide of the protooncogene, c-Src (Flowers et al. 2004), both of which are not
regulated by SOCS-1 suggesting specifieity of Tkip-mediated inhibition. Although Tkip binds to
unphosphorylated JAK2 autophosphorylation site peptide, JAK2(1001-1013), it binds
significantly better to phosphorylated JAK2 autophosphorylation site peptide, pJAK2(1001-
1013). It has been suggested that SOCS-1 recognizes and binds only to phosphorylated JAK2,
therefore Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1, but not in
precisely the same way. Consistent with inhibition of JAK2, Tkip also inhibits the ability of
IFNy to induce an antiviral state as well as upregulation of IVHC class I molecules, and blocks
the phosphorylation of both STAT1 and STAT3 (Flowers et al. 2004). Tkip also inhibits the
proliferation of the prostate cancer cell lines DUl45 and LNCaP, in a dose dependent manner
(Flowers et al. 2005). In addition, Tkip has been shown to protect mice from EAE, an animal
model for the human inflammatory disease, multiple sclerosis (Mujtaba et al. 2005) via blockage
activation of inflammatory cytokines. Hence, Tkip appears to have both anti-inflammatory and
Tkip is synthesized from L-amino acids, which are the physiological form of amino acids
in nature and therefore are potentially readily degraded by proteases. Hence, if Tkip were to be
used as a therapeutic agent, there is potential that it may be readily degraded. This would likely
limit Tkip usefulness as a therapeutic agent. To address this limitation, we synthesized other
Tkip-related mimetic peptides. These were DTkip (WLVFFVIFYFFR) and DRTkip
(RFFYFIVFFVLW), which were synthesized from D-amino acids, which from a medicinal
chemistry view, were likely to be more resistant to proteolytic digestion and therefore likely to
be better therapeutics. DTkip has the same amino acid sequence as Tkip, while in DRTkip the
sequence is reversed, in other words DRTkip is a retro-inversion of Tkip. DTkip and DRTkip
were tested for SOCS-1-like activity such as the ability to bind to JAK2, inhibit JAK2
autophosphorylation, and inhibit JAK2-mediated phosphorylation of the substrates of the
JAK/STAT pathway. In addition, the peptides were also tested for effects on cell proliferation,
IFNy-induced antiviral activity and upregulation of IVHC class I molecules. In addition some
Tkip cellular targets were identified. I will present data describing a family of SOCS-1 mimetic
peptides and present a proof-of-concept for the therapeutic potential of Tkip.
Inhibition of SOCS-1 Activity and Immunological Relevance
As stated earlier, SOCS-1 is a negative regulator of immune factors including IFNs,
interleukins -2, -3, -4, -6, -7, and -12, tumor necrosis factor (TNFa) as well as a variety of
hormones such as growth hormone (Tan and Rabkin 2005). This raises the possibility that one
way of enhancing cytokine-mediated immune response to pathogen infection may be by
inhibition of SOCS-1 activity. It has recently been shown that silencing of SOCS-1 in dendritic
cells promoted cell activation, which led to enhancement of effective antigen-specific anti-tumor
immunity (Shen et al. 2004). This implies that reagents that regulate SOCS-1 may enhance
immune responses and therefore have potential in control and/or treatment of diseases arising
from inadequate immune responses. A SOCS-1 inhibitor peptide, pJAK2(1001-1013), that is
derived from the JAK2 autophosphorylation site was designed and synthesized. I will present
data showing that pJAK2(1001-1013) is a SOCS-1 antagonist.
JH7 JH6 JH5
JH4 JH3 JH2
N-l 65CI 125a Ra 65C 150 aa CI 6 I 245 aa _I 215 aa
Receptor binding Pseudo-kinase Kinase domain,
domain domain includes Y1007
Figure 2-1. A schematic representation of the domain structure of the JAK kinase family. The
JAK tyrosine kinase protein family contains four members: JAKl, JAK2, JAK3, and
TYK2. Each JAK molecule contains seven distinct regions: JH1-JH7. The JH1
domain is the catalytic domain, which includes the activation loop, in which
autophosphorylation site (Y1007) is located. The JH2 domain is the pseudo-kinase
domain and is believed to play a role in the autoregulatory activities of JAK2. The
JH6-JH7 domains mediate binding of JAK molecules to cytokine receptor proteins.
Adapted with modifications from Imada and Leonard 2000.
KIR ESS SH~2 domain
Figure 2-2. A schematic diagram showing the domain structure of SOCS-1 protein. All SOCS
proteins have an N-terminal region of varying length and sequence, a central SH2
domain, and a C-terminal SOCS box. SOCS-1 and SOCS-3 have a kinase inhibitory
domain (KIR), which lies between the N-terminal and the SH2-domain. In SOCS-1,
the 12-amino acids N-terminal and contiguous to the SH2 domain form the extended
SH2 (ESS) region, and the 12-amino acid residues N-terminal and contiguous with
the ESS form the kinase inhibitory region (KIR). The SOCS1-KIR peptide is derived
from the KIR, while the SOCS1-ESS peptide is derived from the ESS region.
Adapted with modification from Subramaniam et al. 2001
Table 2-1. A table showing the JAK kinases and STAT proteins utilized by some cytokines,
growth factors, hormones, and oncogenes.
Cytokines/hormones/oncogenes/ JAK STAT
JAK1 and JAK 2
JAK1 and TYK-2
JAK 1 and JAK 3
JAK 1 and JAK 3
JAK 1 and TYK-2
JAK 2 and TYK-2
STAT 1, 2, 3, 4, 5A, 6
STAT 1, 3, 5A/5B
STAT 1, 3, 5A/B, 6
STAT 3, 5A/B
STAT 1, 3
STAT 1, 3, 4, 5
STAT 1, 2, 3, 5
STAT 1, 3, 5A/B
STAT3, STAT 5B
JAK1 and JAK2
STAT 1, 3 and 5
MATERIALS AND METHODS
All the cell lines, except Sf9 insect cells, were obtained from the American Type Culture
Collection (Manassas VA). The human prostate cancer cells, LNCaP, and the murine
macrophage cells, Raw 264.7, were maintained in RPMI (JRH Biosciences, Lenexa, KS)
supplemented with 10% FBS (Hyclone, Logan, CT), 100 U/mL penicillin, 100 U/mL
streptomycin (complete media). Murine fibroblast cells, L929, were maintained in DMEM (JRH
Biosciences), supplemented with FBS, penicillin, streptomycin, non-essential amino acids,
sodium bicarbonate, and sodium pyruvate. The murine monocyte cell line, U937, was maintained
in RPMI complete media supplemented with 10 mM HEPES (Sigma-Aldrich, St. Louis, MO),
and 1 mM sodium pyruvate (Sigma-Aldrich). All the cell types were cultured at 37oC and 5 %
carbon dioxide humidified incubator. The Sf9 cells obtained from Invitrogen (Invitrogen
Corporation, Carlsbald, CA) were maintained at 27oC as adhesion cultures in complete TNM-FH
media (Grace Insect Medium, Supplemented, containing 10% FBS, 100 U/mL penicillin, and
100 U/mL streptomycin) or as suspension cultures in Sf-900 SFM media containing 5% FBS,
100 U/mL penicillin, and 100 U/mL streptomycin.
The peptides used in this study are listed in Table 3-1, and were synthesized by Mr.
Mohammed Haider, in our laboratory on an Applied Biosystems 9050 automated peptide
synthesizer (Foster City, CA) using conventional fluorenylmethyoxycarbonyl (Fmoc) chemistry
as previously described (Szente et al. 1996). A lipophilic group (palmitoyl-lysine) was added to
the N-terminus of peptides, to facilitate entry into cells, as a last step using a semi-automated
protocol (Thiam et al. 1999). Peptides were characterized by mass spectrometry and where
possible, HPLC purified. Peptides were dissolved in water or in DMSO (Sigma-Aldrich, St.
Louis, MO). The control peptides used in this proj ect did not show significant biological activity
in the systems tested.
Binding assays were performed as previously described (Flowers et al. 2004) with minor
modifications. Tkip, SOCS1-KIR, and control peptide, MulFNy(95-106) at 3 Gig/well, were
bound to 96-well plates, in binding buffer (0.1 M sodium carbonate and sodium bicarbonate, pH
9.6). The wells were washed three times in wash buffer (0.9% NaCl and 0.05% Tween-20 in
PBS) and blocked in blocking buffer (2% gelatin and 0.05% Tween-20 in PB S) for 1 h at room
temperature. Wells were then washed three times and incubated with various concentrations of
biotinylated JAK2(1001-1013) or biotinylated pJAK2(1001-1013), in blocking buffer, for 1 h at
room temperature. Following incubation, wells were washed five times and bound biotinylated
peptides were detected using HRP-conjugated neutravidin (Molecular Probes) and color detected
using o-phenylenediamine in stable peroxidase buffer (Pierce, Rockford, 1L). The chromogenic
reaction was stopped by the addition of 2 M H2SO4 (50 CIL) to each well. Absorbance was
measured at 490 nm using a 450-microplate reader (Bio-Rad Laboratories, Hercules, CA).
Peptide competition assays were carried out as described above except following peptide
immobilization, washing, and blocking, biotinylated JAK2(1001-1013) or biotinylated
pJAK2(1001-1013), which had been pre-incubated with various concentrations of soluble
peptide competitors (Tkip, SOCS1-KIR or control peptide) was added. Detection of bound
biotinylated peptide was conducted as described above. Data obtained from binding assays was
plotted using Graph Pad Prism 4.0 software (Graph Pad Software, San Diego, CA).
In2 vitro Kinase Assays
Autophosphorylation activity of JAK2 was measured in a reaction mixture containing
GST-JAK2 kinase fusion protein (Cell Signaling Technology, Danvers, MA), ATP (20 CIM, Cell
Signaling) and the appropriate peptide in kinase buffer (10 mM HEPES, pH 7.4, 50 mM sodium
chloride, 0.1 mM sodium orthovanadate, 5 mM magnesium chloride, and 5 mM manganese
chloride). It had previously been determined that soluble IFNGR-1 enhanced JAK2 activity
(Flowers et al. 2004), therefore IFNGR-1 (2 Cpg/ reaction) was added and the reaction mix
incubated at 250C for 30 min with intermittent agitation. The assays were carried out according
to a JAK2 kinase protocol obtained from Cell Signaling (Cell Signaling Technology, Danvers,
MA), but with modifications, derived in part from Flowers et al. (2004). The reactions were
terminated by addition of appropriate volume of 6 X SDS-PAGE loading buffer (0.5 M Tris-HCI
(pH 6.8), 36% glycerol, 10% SDS, 9.3% DTT, 0.012% bromophenol blue), and heating at 950C
for 5 min. The proteins were separated on a 12% SDS-polyacrylamide gel (Bio-Rad
Laboratories), transferred onto nitrocellulose membrane (Amersham Biosciences, Piscataway,
NJ), and probed with anti-pJAK2 antibodies (Santa Cruz Biotechnology, San Diego, CA).
Membranes were then stripped and re-probed with anti-JAK2 antibody (Santa Cruz
Biotechnology). Detection of proteins was accomplished using ECL protein detection reagents
Autophosphorylation activity of epidermal growth factor receptor (EGFR, Upstate) was
measured in a 50 Cl1 reaction containing kinase buffer, 0.2 Cpg EGFR (Upstate Biotechnology),
0.1 Cpg EGF (Upstate Biotechnology), 20 CLM ATP (Cell Signaling), and the appropriate peptide
(50 CLM) as described in Flowers et al. (2004). The reaction mix was incubated for 30 min at
250C, resolved by 12% SDS-PAGE, transferred onto nitrocellulose membrane, and
immunoblotted with anti-pEGFR antibody. The membranes were then stripped and reprobed
with anti-EGFR antibody. Detection of proteins was accomplished using ECL protein detection
reagents (Amersham Biosciences).
U937 murine fibroblast cells were plated on 6-well plates at a cell density of 1 x 106 CellS/
well and after an overnight incubation at 370C and 5% CO2, the cells were cultured in complete
media containing varying concentrations of lipophilic Tkip, lipophilic SOCS1-KIR, or lipophilic
control peptide for 18 h at 370C in a 5% CO2 incubator. To activate the JAK/STAT signaling
pathway, U937 cells were then incubated in the presence or absence of 1000 U/mL IFNy (PBL
Biochemical Laboratories, Piscataway, NJ) for an additional 30 min. The media was aspirated
out and the cells washed twice with cold PBS to remove media and cell debris. Cell lysates were
prepared by adding 250 CIL of cold lysis buffer (50 mM Tris-HCI (pH 7.4), 250 mM NaC1, 50
mM NaF, 5 mM EDTA, and 0. 1% NP40) containing protease inhibitor cocktail (Amersham
Bioscience) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Lysis was allowed to
proceed for 1 h at 40C (rocking) to ensure complete cell lysis. Lysates were then centrifuged and
supernatant transferred into fresh microcentrifuge tubes, protein concentrations determined, and
protein lysates resolved by SDS-PAGE on a 12% polyacrylamide gel (Bio-Rad Laboratories).
Proteins were transferred onto nitrocellulose membranes (Amersham Biosciences), placed in
blocking buffer (5% nonfat dry milk and 0.1% Tween-20 in TBS), and washed in 0.1% Tween-
20 in TB S. To detect phosphorylated STAT lo, membranes were incubated with pY701-STAT1 a
antibody (1:400 dilution; Santa Cruz) in blocking buffer for 2 h at room temperature. After four
washes, the membranes were incubated in HRP-conjugated goat anti-rabbit IgG secondary
antibody (1:2000 dilution; Santa Cruz) in blocking buffer for 1 h at room temperature, washed
four times, incubated for 1 min with ECL detection reagents (Amersham Biosciences) and
exposed to photographic film (Amersham Biosciences) to visualize protein bands.
Murine macrophage cells, Raw 264.7, were seeded on 24-well plates at a concentration of
3 x 105 cells/ well and allowed to adhere. Varying concentrations of the lipophilic peptides, Tkip,
SOCS1-KIR or MulFNy(95-106), were then added to the wells and the cells incubated for 2 h at
370C in a 5% CO2 incubator. Recombinant IFNy, in varying concentrations, was then added and
the cells incubated for an additional 72 h at 370C in a 5% CO2 incubator, after which
supernatants were transferred into fresh tubes and assayed for nitrite levels as a measure of nitric
oxide production using Gniess reagent, according to manufacturer' s instructions (Alexis
Biochemicals, San Diego, CA). To test for synergy between Tkip and SOCS1-KIR, the cells
were incubated in the presence of IFNy and varying concentrations of peptides as described
above and also in the presence of both lipophilic Tkip and lipophilic SOCS1-KIR or lipophilic
Tkip and lipophilic MulFNy(95-106). Supernatants were collected after 48 h and tested for nitric
oxide production as described above.
Tkip Cellular Targets
SJL/J mice were immunized with bovine myelin basic protein (MBP) as previously
described (Mujtaba et al. 2005). Briefly, 6 to 8 week old female SJL/J mice were immunized
subcutaneously at two sites on the base of the tail, with MBP (300 Cpg/mouse) in complete
Freund' s adjuvant. At the time of MBP immunization and 48 h later, pertusis toxin (400
ng/mouse) was administered (i.p). This protocol was approved by IACUC at the University of
Florida. The mice were observed daily for signs of EAE, and severity of disease was graded
using the following scale: 1) loss of tail tone; 2) hind limb weakness; 3) paraparesis; 4)
paraplegia; and 5) moribund/death. The first physical signs of disease were generally observed
beginning on day 18 to 21 after MBP immunization. Spleens were extracted after disease onset
and homogenized into a single cell suspension. Splenocytes (1 x 105 cells/well) were incubated
with medium, MBP (50 Clg/mL), lipo-Tkip, lipo-SOCS1-KIR or control peptide lipo-
MulFNGR(253-287) for 48 h at 370C in 5% CO2. To test the effect of the peptides on cell
proliferation, the cultures were pulsed with [3H]-thymidine (1.0 CICi/well; Amersham
Biosciences) for 18 h before harvesting onto filter paper discs using a cell harvester. Cell
associated radioactivity was quantified using a beta scintillation counter and data are reported as
counts per minute (cpm).
To test for the effect of Tkip on specific cellular targets, splenocytes obtained as described
above were enriched for the desired cell type using negative isolation kits purchased from Dynal
Biotech (Dynal Biotech, Oslo, Norway). The enrichments were carried out according to the
manufacturer' s instructions. Mouse B cells negative isolation kit, mouse CD4+ T cells negative
isolation kit, or mouse CD8' T cells negative isolation kit was used for B cells, CD4' T cells, or
CD8+ T cells, respectively. The use of the isolation kits results in cells enriched for the specific
cell type, but to confirm the purity level, the cells may be stained with cell-type specific
antibodies and FACS (fluorescence activated cell sorter) analysis carried out. This would have
given a better indication of the actual purity level, and would provide an indication what
contaminants (if any) were present.
Enriched cells were incubated with varying concentrations of appropriate lipophilic
peptides in the presence or absence of antigen presenting cells (APCs), and in the presence or
absence of antigen (MBP). The APCs were derived from splenocytes obtained from naive SJL/J
mice, which were incubated with MBP for 48 h, fixed with 2% paraformaldehyde for 30 min,
and washed extensively to remove residual paraformaldehyde. The cells were then transferred
into mouse IFNy-ELISPOT plates (Mabtech Inc, USA) and incubated for 48 h, after which the
wells were washed, incubated with secondary antibody, washed, and spots developed according
to manufacturer' s instructions. The plates were then blotted dry, spots counted, and the data
plotted using Graph Pad Prism 4.0 software (Graph Pad Software, San Diego, CA).
To test for the effect of Tkip on antibody production, B cells (5 x 105 cells/well) from
MBP sensitized mice obtained two months after disease remission were incubated with varying
concentrations of lipophilic peptides in the presence of MBP (50 Cpg/mL) and APCs and
incubated for 48 h. Culture supernatants were then harvested and tested for MBP-specific
antibodies by enzyme-linked immunoabsorbent assay (ELISA).
Antiviral Assays for SOCS-1 Antagonist Function
Antiviral activity was determined using the standard viral cytopathogenic effect assay
described previously with minor modifications (Langford et al. 1981). Briefly, human fibroblast
WISH cells at 70-80% confluency, were incubated in media alone or 0.4 U/mL human IFNy
(PBL Biomedical Laboratories) or both 0.4 U/mL human IFNy and lipo-pJAK2(1001-1013) or
lipo-JAK2(1001-1013) for 22 h in DMEM containing 2% FBS (maintenance media). Following
incubation, WISH cells were washed once with maintenance media and infected with
encephalomyocarditi s virus (EMCV) (200 pfu/well) for 1 h at 3 70C. The WISH cells were then
washed once to remove unbound viral particles and incubated in fresh maintenance media for an
additional 24 h at 370C in a 5% CO2 incubator. Plates were subsequently blotted dry and stained
with 0.1% crystal violet solution for 5 min. Unbound crystal violet was aspirated and the plates
thoroughly rinsed with deionized water, blotted, and air-dried. Viral plaques were counted using
a dissecting microscope and antiviral activity was determined by comparing experimental
treatment groups with the virus only control group.
Transfections of LNCaP Cells with SOCS-1 DNA
Transfections were carried out to introduce SOCS-1 DNA into mammalian cells and test
the ability of pJAK2(1 00 1-1 013) peptide to reverse SOC S- 1 inhibition of STAT3
phosphorylation. Human prostate cancer cells, LNCaP, were plated in a 6-well plate and allowed
to grow to 60% confluency. SOCS-1 plasmid DNA (1.6 Gig/well) (pEF-FLAG-I/mSOCS1), a gift
from Dr. David Hilton (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia)
or an empty vector, was transfected into the LNCaP cells using lipofectamine (Invitrogen
Corporation, Carlsbad, CA) according to manufacturers instructions, but with modifications. The
cells were incubated for 4 h, after which the transfection media was aspirated, fresh complete
media (DMEM supplemented with 10% FBS and 100 U/mL streptomycin and 100 U/mL
penicillin) added, and the cells incubated for an additional 72 h at 370C. The complete media was
then aspirated out, fresh media containing lipo-pJAK2(1001-1013) (20 CIM) or control peptide,
lipo-MulFNy(95-125), added to the transfected cells, and the JAK/STAT signaling pathway
activated by adding IL-6 (50 ng/mL). Cells were incubated for 30 min prior to harvesting. Cell
extracts were resolved by SDS-PAGE on a 12% polyacrylamide gel, transferred onto
nitrocellulose membrane (Amersham Biosciences), and probed with phosphorylated (pY705)
STAT3 antibody (Santa Cruz Biotechnology). The membranes were stripped and reprobed with
unphosphorylated STAT3 antibody (Santa Cruz Biotechnology). Detection of proteins was
accomplished using ECL detection reagents (Amersham Biosciences).
For immunoprecipitations, LNCaP cells growing on 60 mm plates and at 50% confluency
were transfected with either SOCS-1 plasmid DNA (8 Cpg/plate) or empty vector (8 Cpg/plate) in
lipofectamine and incubated for 4 h after which 5 mL complete media was added and cells
allowed to grow for 72 h. The cells were harvested in lysis buffer as described for western blot
analysis, and the cell lysates centrifuged at 10000 x g to remove cellular debris and nuclei.
Supernatants were transferred into fresh tubes and incubated with 2 Cpg/mL anti-Flag antibody
(Sigma-Aldrich) for 2 h at 4oC while rotating. Protein G PLUS-agarose beads (Santa Cruz
Biotechnology) were added to the supernatants and allowed to incubate for 2 h at 4oC while
rotating, followed by centrifugation to pellet protein G immune complexes. Supernatants were
discarded and the immune complexes washed three times with lysis buffer and once with PBS.
The immune complexes were then heated (95oC/5 min) in 50 CLL of 1 X SDS sample buffer,
resolved on a 12% polyacrylamide gel, transferred onto nitrocellulose, and immunoblotted with
anti-SOCS-1 antibody (Santa Cruz Biotechnology). Detection of proteins was accomplished
using ECL detection reagents (Amersham Biosciences).
GAS Promoter Activity
A plasmid, pGAS-Luc, that contains the promoter for IFNy-activated sequence (GAS)
linked to firefly luciferase gene was obtained from Statagene (La Jolla, CA). A constitutively
expressed thymidine kinase promoter-driven Renilla luciferase gene (pRL-TK) (Promega
Corporation, Madison, WI) was used as internal control in reporter gene transfections. WISH
cells (1 x 105 cells/well) were seeded in a 12-well plate and incubated overnight at 37oC,
following which 3 Clg GAS promoter-driven firefly luciferase expressing plasmid DNA and 10
ng pRL-TK were cotransfected into the WISH cells, using Fugene 6 (Roche Diagnostics
Corporation, Indianapolis, IN). Two days later, the cell lysates were used to assay for firefly
luciferase and Renilla luciferase, using a dual luciferase assay kit (Promega Corporation).
Luciferase activity, in relative luciferase units, was calculated by dividing firefly luciferase
activity by Renilla luciferase activity in each sample.
Primer Design and PCR Amplification of Murine SOCS-1 DNA
Primers were designed to amply full-length SOCS-1 from the mouse SOCS-1 intracellular
expression vector, pEF-FLAG-I/mSOCS-1 previously described. Primers were designed for
compatibility with the multiple cloning site (MCS) of pBlueBac4.5/V5-His TOPO TA vector
(Invitrogen). The primers were purchased from Integrated DNA Technology Inc and the primer
sequences are shown below.
Forward primer 1
5'- AGG ATG GTA GCA CGC AAC CAG GT 3'
5'- GAT CTG GAA GGG GAA GGA AC 3'
PCR reactions were carried out in 50 CLL reactions containing dNTPs (0.2 mM each),
MgCl2 (1.5 mM), primers (0.2 CLM each), template DNA (1 Cpg), platinum Taq DNA polymerase
(1 unit), platinum Pfxc polymerase (1 unit), 10 X PCR buffer (5 pIL) and DNAse free water. The
thermocycler was programmed for 30 cycles at 94oC for 30 sec, 50oC for 30 sec, 72oC for 1 min,
with initial denaturation at 94oC for 4 min and final extension at 72oC for 7 min. The PCR
products were resolved on a 1% TAE agarose gel, stained with ethidium bromide, and
Cloning SOCS-1 into pBlueBac4.5/V5-His TOPO TA Expression Vector
The PCR products of correct size (650 bp) were excised out of the gel, purified using
Wizard PCR prep DNA purification system (Promega, Madison WI) and cloned into the
pBlueBac4.5/V5-His TOPO TA expression vector according to the manufacturer's instructions
(Invitrogen). Following cloning, the recombinant vector was used to transform TOP10
chemically competent cells according to manufacturer' s instructions (Invitrogen), and the
transformed cells grown on LB-ampicilin nutrient agar plates overnight at 370C. Positive and
negative control cloning reactions were carried out similar to those for SOCS-1 DNA, but in the
absence of SOCS-1 DNA. Positive clones were identified by PCR, and plasmid DNA isolated
from the PCR positive clones. Restriction enzyme digestion and DNA sequencing were used to
confirm the presence of SOCS-1 DNA.
Expression of SOCS-1 in Sf9 Cells
The recombinant pBlueBac4.5/His-V5 TOPO vector carrying the murine SOCS-1 gene
(pBlueBac4.5/muSOCS-1) was transfected into the expression vector Bac-N-Blue (Invitrogen)
according to the manufacturer' s instructions. Briefly, the Bac-N-Blue vector contains a triple-cut,
linearized AcMNPV (Autographa californica multiple nuclear polyhedrosis virus). The
linearized virus lacks sequences essential for efficient propagation, specifically sequences in the
ORFl1629. Hence, for successful propagation and isolation of viable virus, the essential
ORFl1629 sequences need to be supplied by a transfer vector. The pBlueBac4.5/His-V5 TOPO
vector contains these essential sequences. The pBlueBac/muSOCS-1 and Bac-N-Blue were co-
transfected into Sf9 (Spodoptera Jfrugiperda)) insect cells growing at 50% confluency (Invitrogen)
in the presence of cellfectin reagent (Invitrogen) in unsupplemented Grace Insect Medium
(Invitrogen). The transfection was carried out at room temperature and the transfection
complexes incubated with the cells for 6 h following which complete TNM-FH medium was
added and the cells allowed to grow for an additional 72 h. Half the volume of the culture media
was harvested and used for plaque assay. The same volume of fresh TNM-FH media was added
to the cells, and the cells allowed to grow for an additional 72 h at which point significant cell
lysis was observed. The virus (Pl stock) was then collected by centrifugation and tested for the
presence of murine SOCS-1 DNA by PCR using the Baculovirus primers.
Forward primer 5'- TTTACTGTTTTCGTAACAGTTTTG 3'
Reverse primer 5'- CAACAACGCACAGAATCTAGC 3'
In order to generate high titer recombinant virus, PCR positive clones were propagated
further and used to infect fresh Sf9 cells growing in suspension culture. The cells were allowed
to grow and harvested at different time points to determine the best time for harvesting cells
expressing SOCS-1 protein. It was determined that 96 h infection provided the highest SOCS-1
protein yield, and subsequently Sf9 cells were harvested 96 h post infection. Cell lysates were
harvested using native conditions as described in the ProBond purification manual (Invitrogen)
and the expression of murine SOCS-1 confirmed by immunoblot analysis using anti-SOCS-1
antibody (Santa Cruz).
The data were not normally distributed therefore nonparametric statistical analyses tests,
the Mann-Whitney and Wilcoxon tests were used. The Mann-Whitney signed rank sum test
compares two groups and performs calculations on the rank of the values, rather than the actual
data (Motulsky 1995). It is considered to be similar to the t-test, except the data are not normally
distributed. The Wilcoxon signed rank sum test compares two paired groups of nonparametric
data (Motulsky 1995). It is similar to the paired t-test, but the data are not normally distributed.
All calculations were performed using the GraphPad Prism statistical package (GraphPad
Software Inc, San Diego, CA).
Table 3-1. List of peptides used in this study. DTkip and DRTkip were synthesized from D-
amino acids. All the other peptides were synthesized from L-amino acids. The Y1007
in pJAK2(1001-1013), in italics, is phosphorylated.
MulFNGR1 (253 -287) 2 53 TKKN SFKRK SIMLPK SLL SVVK SATLETKPE SKY S
MulFNy(95-106), MulFNy(95-125), and MulFNR1(253-287) were used as control peptides.
These peptides do not show significant biological activity in the assays for which they have
been used as control peptides.
Tkip Family Members Bind to JAK2 Autophosphorylation Site
It has previously been shown that Tkip binds to the autophosphorylation site of JAK2 and
inhibits JAK2 autophosphorylation and phosphorylation of IFNGR-1 (Flowers et al. 2004). Here
I show that DTkip and DRTkip but not the control peptide, MulFNy(95-106), bound to the
JAK2(1001-1013) peptide in a dose-dependent manner (Figure 4-la). Thus, these SOCS-1
mimetic peptides bind JAK2 autophosphorylation site peptide in a dose-dependent manner.
Current literature suggests that phosphorylation of JAK2 tyrosine 1007 is important in
SOCS-1 mediated JAK2 ubiquitin-proteosome-dependent degradation (Ungureanu et al. 2002).
Hence, I wanted to determine whether these peptides, like SOCS-1, bind to phosphorylated
JAK2, pJAK2(1001-1013). Dose response solid-phase ELISA were carried out with biotinylated
pJAK2(1001-1013) peptide in place of biotinylated JAK2(1001-1013) peptide. DTkip and
DRTkip bound with two to threefold greater affinity to pJAK2(1001-1013) than to JAK2(1001-
1013) as shown in Figure 4-1b. This is consistent with previous data showing that Tkip binds
with a greater affinity to pJAK2(1001-1013) than to JAK2(1001-1013) (Flowers et al. 2004).
The binding observed was dose dependent, implying specificity in binding. Hence, the SOCS-1
mimetic peptides recognize JAK2 autophosphorylation site similar to Tkip, implication of which
is that they recognize JAK2 autophosphorylation similar to SOCS-1.
In order to determine whether Tkip and DRTkip bind to the same site on JAK2
autophosphorylation site, competition for binding assays were carried out. Soluble Tkip and
soluble DRTkip, but not the control peptide (MulFNy95-106) inhibited binding of biotinylated
JAK2(1001-1013) to immobilized Tkip in a dose dependent manner (Figure 4-2a). Next, the
competition for binding of Tkip with DRTkip to pJAK2(1001-1013) was determined. As shown
in Figure 4-2b, Tkip and DRTkip inhibited the binding of pJAK2(1001-1013) to immobilized
Tkip. Hence, Tkip and DRTkip likely bind to the same site on JAK2 autophosphorylation site,
the implication of which is that DRTkip may have similar binding characteristics as Tkip to
JAK2. The D amino acid isomer thus, binds similar to the L isomer, suggesting a stable form of
Tkip for functional studies.
Tkip Family Members Inhibit JAK2 Kinase Activity
SOCS-1 regulates JAK2 activity by interacting with the autophosphorylation site and
inhibiting JAK2 kinase activity. Therefore, DRTkip was tested for its ability to inhibit JAK2
autophosphorylation and JAK2 phosphorylation of substrate (IFNGR-1). DRTkip but not the
control peptide, MulFNy(95-106) inhibited JAK2 autophosphorylation and JAK2 induced
phosphorylation of IFNGR-1 (Figure 4-3). These results are similar to what has been previously
shown with Tkip (Flowers et al. 2004). The data suggest that DRTkip may inhibit both JAK2
activation and JAK2-mediated phosphorylation of IFNGR-1, the implication of which is that the
SOCS-1 mimetic peptides, like SOCS-1, may potentially regulate IFNy signaling. It is worth
noting that like Tkip, DRTkip did not inhibit tyrosine phosphorylation of c-Src kinase (data not
shown), which is consistent with the fact that c-Src kinase is not inhibited by SOCS-1.
Tkip Inhibits Superantigen-induced Proliferation of Mouse Splenocytes
The staphylococcus superantigens are potent T cell mitogens that exert their effects by
forming complexes with 1VHC class II molecules on antigen presenting cells, and binding to the
T cell receptor (TCR) via the Vp-element of the (TCR), resulting in activation of the T cells
(Reviewed in Torres et al. 2001). Our laboratory has shown that superantigens such as
staphylococcus entrotoxin A and B (SEA and SEB) can exacerbate immunological disease and
induce relapses in the mouse model for multiple sclerosis, experimental allergic
encephalomyelitis, EAE, (Torres et al. 2001). It has previously shown that Tkip inhibits antigen-
induced proliferation of splenocytes (Mujtaba et al. 2005). Here the ability of Tkip and DRTkip
to inhibit superantigen-induced proliferation of primary cells was tested. Mouse splenocytes
were stimulated with SEB (500 ng/mL) in the presence of lipophilic Tkip, lipophilic DRTkip, or
lipophilic control peptide, MulFNy(95-106) (a lipophilic group, lysyl-palmitate, is added to the
N-terminal ends of the peptides to facilitate entry into the cells) and incubated for 72 h prior to
pulsing with 3-[H]-thymidine. As shown in Figure 4-4a, Tkip but not DRTkip inhibited SEB
induced proliferation of the splenocytes. Next I tested the ability of Tkip and DRTkip to inhibit
SEA-induced splenocytes proliferation. Tkip, but not DRTkip inhibited SEA-induced
splenocytes proliferation (Figure 4-4b). Next the ability of Tkip and DRTkip to inhibit STATla
phosphorylation in murine fibroblast cells (U937) was tested. Tkip, but not DRTkip inhibited
STAT la phosphorylation (Data not shown). The lysyl-palmitate group is an L-lysine and may be
affected by proteases in such a way as to affect the efficiency of DRTkip uptake by cells. It is
also possible that the D-isomer amino acids may result in a peptide whose conformation is
different enough from Tkip to have slightly different cellular function. However, since only a
limited number of cellular function assays were carried out, it can not be conclusively be
determined whether DRTkip has or does not have intracellular SOCS-1 like function. Thus,
factors currently unknown may affect DRTkip intracellular function.
The observations that DRTkip, unlike Tkip, did not seem to significantly affect biological
activity suggested that the use of the Tkip retro-inversion, DRTkip, may have changed the
orientation of the amino acids enough to affect function. Hence, this research focused on Tkip
and SOCS1-KIR and not the other SOCS-1 mimetics. There is however, continued interest in
why DRTkip is not the same as Tkip in cell functional comparisons.
SOCS-1 Kinase Inhibitory Region (SOCS1-KIR) Binds to JAK2 Autophosphorylation Site
Yasukawa et al. (1999) suggested that the binding of SOCS-1 to JAK2 requires the SOCS-
1 SH2-domain and the extended SH2 domain (ESS) and that the kinase inhibitory region (KIR),
while not required for the initial binding is essential for the inhibitory action of SOCS-1. Here, I
present results showing that a peptide corresponding to SOCS-1 KIR region, SOCS1-KIR,
specifically binds to a peptide representing the JAK2 autophosphorylation site and inhibits
STAT la activation. Further, I show that SOCS1-KIR, as well as Tkip, inhibit IFNy-induced
macrophage activation. In addition, I show that a peptide corresponding to the ESS region,
SOCS 1-ES S. Does not bind to the JAK2 autophosphorylation site, the implication of which is
that the SH2, ESS, and KIR regions may all play role in the binding of SOCS-1 to JAK2.
Tkip and SOCS1-KIR Bind to JAK2 Autophosphorylation Site
First I determined whether SOCS1-KIR, like the SOCS-1 mimetic Tkip, binds to the JAK2
autophosphorylation site by carrying out dose-response solid-phase binding assays with JAK2
autophosphorylation site peptide, JAK2(1001-1013). Tkip, SOCS1-KIR, or a control peptide,
MulFNy(95-106), were immobilized on 96-well microtiter plates and incubated with biotinylated
JAK2(1001-1013) at various concentrations. Tkip and SOCS1-KIR, but not the control peptide,
bound to the JAK2(1001-1013) peptide in a dose-dependent manner (Figure 4-5a). Thus, both
Tkip and SOCS-1-KIR specifically bind to the JAK2 autophosphorylation site peptide. While
this is consistent with the SOCS-1 mimetic character of Tkip, it also provides direct evidence
that the KIR region of SOCS-1 can interact directly with JAK2 autophosphorylation site,
suggesting that Tkip and KIR recognized a similar site on JAK2.
Since phosphorylation of Y1007 is required for high catalytic activity of JAK2, it is logical
that SOCS-1 would bind with higher affinity to Y1007 phosphorylated JAK2. Consistent with
this, it has previously been shown that Tkip binds with greater affinity to Y1007 phosphorylated
JAK2(1001-1013) peptide, pJAK2(1001-1013), than to unphosphorylated JAK2 peptide
(Flowers et al. 2004). I therefore determined whether SOCS1-KIR bound to pJAK2(1001-1013)
with greater affinity than to JAK2(1001-1013). Both SOCS1-KIR and Tkip bound to
pJAK2(1001-1013) with two to three-fold greater affinity than to JAK2(1001-1013) as shown in
Figure 4-5b. In addition, SOCS1-KIR bound to pJAK2(1001-1013) with higher affinity than
Tkip. Thus, SOCS1-KIR recognizes JAK2 autophosphorylation similar to Tkip, the implication
of which is that Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1.
To determine whether Tkip and SOCS1-KIR bind to the same site on the JAK2
autophosphorylation site, binding competition assays were carried out. Tkip or SOCS1-KIR was
immobilized on a 96-well plate and biotinylated JAK2(1001-1013) or biotinylated pJAK2(1001-
1013), which had been pre-incubated with Tkip, SOCS1-KIR or a control peptide, was allowed
to bind to the immobilized peptides. As shown in Figure 4-6a, soluble Tkip and soluble SOCS1-
KIR, but not soluble control peptide, inhibited the binding of biotinylated JAK2(1001-1013) to
immobilized Tkip. A similar pattern of inhibition was observed with biotinylated JAK2(1001-
1013) binding to immobilized SOCS1-KIR (Figure 4-6b). Homologous inhibition was slightly
better for both Tkip and SOCS1-KIR, which suggests slight differences in recognition of JAK2
Next, the binding competition of Tkip and SOCS1-KIR to pJAK2(1001-1013) was
determined. The competition for binding to pJAK2(1001-1013) was similar to that observed in
competition for binding to unphosphorylated JAK2 peptide (Figure 4-6c and 4-6d). Again,
homologous competition was slightly better, which again suggests slight differences in
recognition of the JAK2 autophosphorylation site. These data provide direct evidence that the
mimetic effect of Tkip is applicable to the KIR region of SOCS-1 and that Tkip and KIR
recognize JAK2 autophosphorylation site similar but not exactly the same.
JAK2 Kinase Activity and STATla Activation
SOCS-1 regulates JAK2 activity at least at two levels. One involves interaction with the
autophosphorylation site, which affects JAK2 phosphorylation of substrates such as STAT 1 and
STAT3 (Yasukawa et al. 1999). The other level involves induction of proteosomal degradation
of both JAK2 and SOCS-1, requiring the SOCS box domain of SOCS-1 (Zhang et al. 2001).
Obviously, neither Tkip nor SOCS1-KIR has a SOCS box, so the two peptides were compared
for their relative ability to inhibit JAK2 autophosphorylation as well as phosphorylation of the
transcription factor, STAT1. As shown in Figure 4-7a, Tkip but not SOCS1-KIR inhibited JAK2
autophosphorylation. This would suggest that the similar but slight differences in recognition of
JAK2 resulted in significant differences in regulation of JAK2 autophosphorylation.
Next the two peptides were compared for their relative ability to inhibit IFNy activation of
STATla in murine U937 cells. In contrast to inhibition of JAK2 autophosphorylation, both Tkip
and SOCS1-KIR inhibited JAK2 mediated phosphorylation of STAT la (Figure 4-7b). Thus,
Tkip inhibits JAK2 autophosphorylation as well as JAK2 mediated phosphorylation of STATla
in murine U937 cells, while SOCS1-KIR does not inhibit JAK2 autophosphorylation, but does
inhibit JAK2 mediated phosphorylation of STAT la transcription factor. SOCS1-KIR thus shows
the same regulatory pattern as SOCS-1, in that JAK2 autophosphorylation is not inhibited, while
STATla substrate phosphorylation by activated JAK2 is inhibited. It has previously showed that
Tkip, like SOCS-1, also inhibited EGFR autophosphorylation (Flowers et al. 2004). Thus
SOCS1-KIR was tested for ability to inhibit EGFR phosphorylation. As shown in Figure 4-7c,
both peptides inhibited EGFR phosphorylation with Tkip being the more effective inhibitor.
SOCS1-KIR is thus similar to SOCS-1 in its kinase inhibitory function.
Tkip and SOCS1-KIR Inhibit IFNy-induced Activation of Macrophages
IFNy plays an important role in activation of macrophages for innate host defense against
intracellular pathogens as well as serving to bridge the link between innate and adaptive immune
responses (Reviewed in Boehn et al. 1997). Thus, Tkip and SOCS1-KIR were examined for their
ability to block IFNy activation of the murine macrophage cell line Raw 264.7 as determined by
inhibition of nitric oxide (NO) production using Griess reagent (Alexis Biochemicals).
Lipophilic (lipo) versions of the peptides were synthesized with palmitic acid for penetration of
the cell membrane (Thiam et al. 1999). Both Tkip and SOCS-KIR, compared to control peptide,
MulFNy(95-106), inhibited induction of NO by various concentrations of IFNy as shown in
Figure 4-8a. Dose-response with varying concentrations of the peptides against IFNy (6 U/mL)
resulted in increased inhibition of NO production by Tkip and SOCS1-KIR with Tkip being the
more effective of the inhibitors as shown in Figure 4-8b. The control peptide, MulFNGR1(253-
287) was relatively ineffective at inhibition, providing evidence for the specificity of Tkip and
SOCS1-KIR inhibition. Tkip and SOCS1-KIR in combination (33 CIM each) were the most
effective in inhibition of IFNy induction of NO in macrophages. This synergy may reflect
differences in recognition of the autophosphorylation site of JAK2 by the two peptides. Thus,
Tkip and SOCS 1-KIR both inhibited IFNy induction of NO in macrophages with Tkip being the
more effective inhibitor.
Tkip and SOCS1-KIR Inhibit Antigen-specific Lymphocyte Proliferation
Our laboratory has previously shown that Tkip inhibits antigen-specific proliferation of
mouse splenocytes in vitro (Mujtaba et al. 2005). Specifically, Tkip inhibited proliferation of
splenocytes from mice immunized with bovine myelin basic protein (MBP). Here, I compared
Tkip and SOCS1-KIR for their relative ability to inhibit proliferation of MBP-specific
splenocytes in cell culture. Splenocytes (3 x 105 cells/well) were incubated with MBP (50
Clg/mL) in the presence of lipo-Tkip, lipo-SOCS 1-KIR, or lipo-control peptide for 48 h and
proliferation assessed by testing for [3H]-thymidine incorporation. As shown in Figure 4-9, both
Tkip and SOCS1-KIR inhibited MBP-induced proliferation of splenocytes, while the control
peptide had a negligible effect on proliferation. Similar to inhibition of NO production by
macrophages, Tkip was more effective than SOC S1-KIR in inhibition of MBP induced
splenocyte proliferation with 84, 88, and 97% inhibition at 1.2, 3.7, and 11 CIM, respectively,
compared to 61, 67, and 72% for SOCS1-KIR. Thus, both Tkip and SOCS1-KIR inhibited
antigen-induced splenocyte proliferation, which is consistent with SOCS-1 protein inhibition of
antigen-specific lymphocyte activity (Cornish et al. 2003).
An Extended SH2 Sequence (SOCS1-ESS) Peptide does not Bind to pJAK2 (1001-1013)
Based in part on binding experiments with truncations of SOCS-1 protein, it has been
proposed that the SH2 domain plus ESS bind to JAK2 at the activation site represented by
peptide pJAK2(1001-1013), while SOCS1-KIR binds primarily to the catalytic site of JAK2
(Yasukawa et al. 1999). Therefore the SOCS1-ESS peptide, 68ITRASALLDACG, was
synthesized and compared with SOCS1-KIR for binding to biotinylated pJAK2(1001-1013).
As shown in Figure 4-10, SOCS1-KIR as well as Tkip bound biotinylated pJAK2(1001-
1013) in a dose-response manner, while SOCS1-ESS failed to bind. Hence, the SOCS1-KIR
peptide (residues 56-68), but not the SOCS1-ESS peptide (68-79) binds to the JAK2
autophosphorylation site. The SOCS1-KIR peptide, except for residues 53-55 is contained in the
SOCS-1 ESS-SH2 construct, dN56, (Yasukawa et al. 1999) that bound to JAK2
autophosphorylation site. dN56 is a construct in which sequences N-terminal to amino acid
residue 56 have been truncated, therefore it contains KIR, ESS, SH2 domain, SOCS box, and the
C-terminal sequences. Thus, SOCS1-KIR, which is N-terminal and contiguous with SOCS1-
ESS, probably shares overlapping functional sites with SOCS1-ESS. Clearly, SOCS1-KIR is
preferentially recognized by JAK2(1001-1013) compared to the 12-mer SOCS1-ESS. The
specific role of the various residues in the SH2 domain of SOCS-1 in JAK2 and pJAK2(1001-
1013) binding remains to be determined.
Tkip Cellular Targets
Studies carried out in our laboratory have shown that Tkip affects the growth of cells
growing in culture, as well as the progression of EAE. Specifically, Tkip inhibits LNCaP and
DUl45 prostate cancer cells proliferation and inhibits antigen-specific cell proliferation Tkip at
63 Cpg/mouse, given every other day prevented development of acute form of EAE, and induced
stable remission in the chronic relapsing/remitting form of EAE (Flowers et al. 2004, Flowers et
al. 2005, Mujtaba et al. 2005). Moreover, no toxicity was observed when Tkip at 200 Cpg/mouse,
given every other day for one week (Mujtaba et al. 2005). These data suggested that Tkip may
have direct effect on cells of the immune system. Hence, I attempted to define Tkip cellular
targets. Data are presented showing that Tkip specifically targets antigen presenting cells
(APCs), CD4' T, CD8' T, and B cells. In addition preliminary data on SOCS1-KIR peptide
cellular targets are also presented.
Effect of Tkip and SOCS1-KIR on CD4+ T Cells
In order to identify Tkip cellular targets, I first asked whether Tkip had any effect on
primary splenocytes derived from myelin basic protein (MBP) sensitized mice. Sensitization of
mice with MBP, in the presence of adjuvant, results in the development of experimental allergic
encephalomyelitis, EAE, a disease characterized by paralysis. As shown in Figure 4-9, Tkip and
SOCS1-KIR inhibit antigen-specific primary cell (splenocytes) proliferation in a dose-dependent
manner. Next I asked, which cell populations were targeted by Tkip. First the effect of Tkip and
SOCS1-KIR on CD4+ T cell subset was tested. CD4' T cells are important in generating effective
cell mediated immunity and in mediating humoral immune responses. CD4' T cells obtained
from MBP sensitized mice after disease onset were incubated with lipophilic peptides, in the
presence or absence of APCs and MBP. Both Tkip and SOCS1-KIR (33 CLM), but not the control
peptide, MulFNGR1(253-287) inhibited CD4+ T cell proliferation (Figure 4-11a). In addition,
Tkip and SOCS1-KIR (33 CLM), inhibited antigen-induced IFNy production by CD4' T as
determined by IFNy ELISPOT assays (Figure 4-11b). The presence of MBP enhanced cell
proliferation and the number of IFNy-producing cells, but the presence of APCs did not seem to
have such effects. The data presented for peptides were obtained in the presence of both MBP
and APCs. Hence, Tkip and SOCS1-KIR target CD4' T cells and inhibit antigen-specific CD4
T cell proliferation and IFNy production.
Effects of Tkip and SOCS1-KIR on CD8+ T Cells
CD8+ T cells play important roles in the immune response mechanism including cytotoxic
T cell activity and production of cytokines that serve as effectors for various other immune
responses. Since Tkip and SOCS1-KIR inhibited CD4+ T cell activity, I asked whether the
peptides could also target CD8' T cells. CD8' T cells derived from MBP-sensitized mice that
were showing signs of active disease were treated with lipophilic peptides in the presence or
absence of MBP and APCs. IFNy ELISPOT assays carried out. Both Tkip and SOCS1-KIR, but
not the control peptide, MulFNGR1(253-287) showed a general trend in inhibiting MBP-induced
IFNy-production by CD8' T cells (Figure 4-12). The presence of MBP lead to a slight increase in
the number of IFNy-producing cells, but the presence of APCs did not seem to have such an
effect. The data presented for peptides was obtained in the presence of both MBP and APCs.
Effect of Tkip and SOCS1-KIR on B Cells
B cells also play an important role in immune response mechanism. Activation of B cells
results in B cell proliferation and differentiation into antibody producing cells and memory cells.
B cells are also APCs. Tkip was tested for effect on B cells derived from IVBP-sensitized mice,
two months after disease remission. The B cells were treated with either lipophilic Tkip,
lipophilic SOCS1-KIR, or lipophilic control peptide, MulFNGR1(253-287), or no peptide, in the
presence APCs and in the presence, or absence of unprocessed antigen (1VBP). Tkip and SOCS1-
KIR, in the presence oflVIBP and APCs, inhibit antigen-specific B cell proliferation (Figure 4-
13a). In addition, both Tkip and SOCS1-KIR inhibit the secretion oflVIBP-specific antibodies in
IVBP-treated B cells (Figure 4-13b). The addition of APCs did not significantly affect the
experimental results. The data shows that Tkip and SOCS1-KIR target B cell and inhibit both B
cell proliferation and secretion of antibodies by plasma cells, the implication of which is that
Tkip and SOCS1-KIR may have an effect on B cell activity. This is of specific interest in the
study of EAE and multiple sclerosis, because the presence of 1VBP-specific antibodies and of
inflammatory cytokines such as IFNy has been shown to exacerbate disease. Hence, this may be
one of the mechanisms by which Tkip inhibits disease progression in IVBP-sensitized mice.
Effect of Tkip on Macrophages
I have described experiments showing that Tkip and SOCS1-KIR inhibit IFNy-induced
macrophage activation (Figure 4-8). I also tested the ability of Tkip to inhibit LPS-induced
macrophage activity. Both Tkip and SOCS1-KIR inhibited LPS-induced macrophage activity
(Figure 4-14). LPS signaling utilizes the Toll Like Receptor 4 (TLR4), a maj or pathway used in
immune response to gram-negative bacteria. Hence, these results have possible implication on
the effect that Tkip on TLR4 signaling and therefore in inhibition of excessive signaling through
the TLR4. Such excessive signaling has been implicated in the development of inflammation.
Additional research is being carried out to determine additional effects that Tkip may have on
TLR4 signaling. Since macrophages are APCs, these data are preliminary evidence that Tkip and
SOCS1-KIR target APCs.
I have therefore shown that Tkip and SOCS1-KIR target and affect the activity of immune
cells including CD4+ T cells, CD8+ T cells, B cells, and APCs, the implication of which Tkip and
SOCS1-KIR may have effects on antigen-induce immune responses.
SOCS-1 Antagonist Activity of pJAK2 (1001-1013) Peptide
The demonstration that the KIR region of SOCS-1 could bind to the autophosphorylated
JAK2 peptide raised the possibility that the phosphorylated peptide, pJAK2 (1001-1013), may
inhibit the function of endogenous SOCS-1 and thus enhance IFNy and 1L-6 activities that are
mediated by JAK2. As shown in Figure 4-15a, the antiviral activity of a suboptimal dose of IFNy
(0.4 U/mL) was enhanced against encephalomyocarditi s virus (EMCV) in WISH cells by
pJAK2(1001-1013). Specifically, unphosphorylated JAK2(1001-1013) at 11 CIM final
concentration reduced EMCV plaques relative to IFNy alone by 42%, while the same
concentration of pJAK2(1001-1013) reduced plaques by 59%. This is consistent with better
binding of pJAK2(1001-1013) by SOCS1-KIR as shown above and by previous studies showing
that SOCS-1 is active against JAK2 phosphorylated at tyrosine 1007 (Yasukawa et al. 1999). The
peptide alone had no effect on EMCV, similar to media. Thus, pJAK2(1001-1013) boosts the
activity of a suboptimal concentration of IFNy, possibly interfering with endogenous SOCS-1
At the level of signal transduction, I examined the effects of pJAK2(1001-1013) on
activation of STAT3 transcription factor in the LNCaP prostrate cancer cell line. The cells were
treated with IL-6 to activate STAT3 signaling, which occurs through JAK2 kinase (Flowers et al.
2005). The SOCS-1 gene was overexpressed in these cells, which resulted in reduction of the
level of IL-6 induced activation of pSTAT3 as shown in Figure 4-15b. Treatment of the cells
with pJAK2(1001-1013) (20 CIM) resulted in approximately a two-fold increase in activated
STAT3 compared to IL-6 treated cells that were transfected with SOCS-1, as per densitometry
readings. Expression of SOCS-1 protein in LNCaP cells is shown in Figure 4-15b. Thus,
pJAK2(1001-1013) has an inhibitory effect on SOCS-1 at the level of signal transduction.
I next determined if pJAK2(1001-1013) could enhance GAS promoter activity of IFNy.
Accordingly, a plasmid with the GAS promoter element linked to the firefly luciferase reporter
gene was cotransfected along with Renilla luciferase reporter plasmid as a control, into human
WISH cells. As shown in Figure 4-16c, treatment of the WISH cells with IFNy (1 U/mL)
resulted in a four-fold relative increase in luciferase activity, which was increased to ten-fold and
five-fold by 5 and 1 CIM pJAK2(1001-1013), respectively. pJAK2(1001-1013) alone did not
activate reporter gene (data not shown) and control peptide did not enhance IFNy activation of
reporter gene. Thus, consistent with the anti-SOCS-1 effects of pJAK2(1001-1013), the peptide
also enhanced IFNy function at the level of gene activation. It has recently been shown that
suppression of SOCS-1 in dendritic cells by siRNA enhanced anti-tumor immunity (Shen et al.
2004). In order to determine the effect of pJAK2(1001-1013) on cell-mediated immune response,
C56BL/6 mice were treated with pJAK2(1001-1013), control peptide, or PBS, following
immunization with BSA. It was shown that pJAK2(1001-1013) enhanced BSA-induced
proliferation of splenocytes by four to five fold when compared to control peptide or PBS
(Waiboci et al. 2007). Further, I showed that supernatants containing SOCS-1 protein competed
with SOCS1-KIR for binding to pJAK2(1001-1013) (Figure 4-15d). The demonstration of
SOCS-1 competition for pJAK2(1001-1013) is consistent with pJAK2(1001-1013) antagonism
via sequestration of critical functional site(s) on SOCS-1. Hence, the data have shown that
pJAK2(1001-1013) antagonizes SOCS-1 activity at five different levels; IFNy antiviral function,
IL-6 signal transduction, IFNy activation of reporter gene via the GAS promoter, enhancement of
antigen-specific proliferation, and possible sequestration of binding sites on SOCS-1.
Expression of SOCS-1 Protein
SOCS-1 is a negative regulator of immune factors including IFNs, IL-2, IL-4, and IL-6.
SOCS-1 also modulates signaling by a variety of hormones. However, in spite of the presence of
SOCS-1 and other immune modulators, the host defense system can pathologically perpetuate
inflammation by overproducing immune mediators, such as inflammatory cytokines, that cause
damage to multiple organs, resulting in what are referred to as inflammatory diseases/disorders.
Further, it is estimated that approximately 20% of human cancers result from chronic
inflammation. In addition, silencing of the SOCS-1 gene, by methylation, has been found in
several human cancers (Hanada et al. 2006). Research carried our in our laboratory has shown
that Tkip protects IVBP sensitized mice from developing EAE, an inflammatory disease. Further,
it has shown that Tkip inhibits proliferation of prostate cancer cells. Since Tkip is a SOCS-1
mimetic, I reasoned that recombinant SOCS-1 protein may have the same effect as Tkip in
preventing inflammation and inhibiting proliferation of cancer cells. Therefore, recombinant
SOCS-1 protein was expressed with the aim of obtaining protein to first characterize SOCS-1
functional sites and second to determine functional relationship to Tkip. The recombinant SOCS-
1 protein was tested for binding to JAK2 autophosphorylation site.
First, primers were designed to amplify murine SOCS-1 (muSOCS-1) from an expression
library, pEF-FLAG-I/mSOCS-1, a gift from Dr. D. Hilton (Walter and Eliza Hall Institute of
Medical Research, Victoria, Australia). SOCS-1 DNA was amplified from the expression library,
gel purified, and cloned into pBlueBac4.5/V5-His TOPO TA vector (Invitrogen). Plasmid DNA
was isolated and the presence of muSOCS-1 confirmed by restriction enzyme digestion (Figure
4-16a) and DNA sequencing (Figure 4-16b). I refer to the cloned product as pBlueBac/muSOCS-
1. The constructs were co-transfected, with the baculovirus vector Bac-N-Blue (Invitrogen), into
Sf9 insect cells and propagated. Co-transfection of the pBlueBac4.5/His-V5 TOPO vector
carrying muSOCS-1, with Bac-N-Blue vector ensured that generally, only recombinant virus
would be viable and therefore, would grow. Following transfection and propagation, viral
plaques were picked and tested for presence of muSOCS-1 by PCR. As shown in Figure 4-17,
several plaques had viral DNA of the correct size (650 bp). Of these, two were purified and used
for protein expression. Western blot analysis of the expression of SOCS-1 protein in the Sf9 cells
is shown in Figure 4-15d. The SOCS-1 lysates and the SOCS1-KIR peptide were used for
competition for binding assays to test for ability to inhibit binding of pJAK2(1001-1013) peptide
to immobilized SOCS1-KIR. Figure 4-15d, shows that SOCS1-KIR and SOCS-1 lysate, similar
to Tkip and SOCS1-KIR competed for binding sites on pJAK2(1001-1013), the implications of
which is that SOCS1-lysate may have characteristics similar to Tkip. The goal of the laboratory
is to obtain sufficient purified SOCS-1 protein for detailed characterization of functional sites.
S0.75-1 a DTkip
o 0 DRTkip
I /1 0 Tkip
8 0.501 *, Control peptide
1 / r No peptide
0.0 0.5 1.0 1.5 2.0 2.5
Peptide concentration (mM)
o o DRTkip
0 .7 a DTkip
II ;d Tkip
S0.50-9 Control peptide
I~l r No peptide
0.0 0.5 1.0 1.5 2.0 2.5
Peptide concentration (mM)
Figure 4-1. JAK2 autophosphorylation site peptides JAK2(1001-1013) and pJAK2(1001-1013)
bind to SOCS-1 mimetic peptides. A) JAK2(1001-1013) peptide binds to Tkip,
DTkip, and DRTkip. Biotinylated JAK2(1001-1013), at the indicated concentrations,
was added in triplicate to 96-well plates coated with Tkip, DTkip, DRTkip, control
peptide (MulFNy(95-106)), or buffer alone. The assays were developed using
standard ELISA methods with neutravidin-HRP conjugate to detect bound
biotinylated JAK2(1001-1013). B) Biotinylated pJAK2(1001-1013) binds to Tkip,
DTkip and DRTkip. Biotinylated pJAK2(1001-1013) was added to wells coated with
the peptides or buffer and binding assays were carried out as described above. The
binding of JAK2(1001-1013) or pJAK2(1001-1013) to Tkip, DTkip or DRTkip, when
compared to control peptide was statistically significant as determined by Mann-
Whitney signed rank test (P < 0.02 and P < 0.004, respectively).
-- 60 DTkip
I 8 I M Mul FNy(95 106)
50 300 500
Competitior peptide concentration, CLM
S60-( D IDRTkip
50 III Mul FNy(95 -106)
50 300 500
Peptide competitor concentration, tLM
Figure 4-2. Both soluble DRTkip and soluble Tkip inhibit the binding of biotinylated
JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip. A)
DRTkip and Tkip inhibit the binding of JAK2(1001-1013) to immobilized Tkip. The
inhibition of binding by Tkip or DRTkip is statistically significant when compared to
control peptide (MulFNy 95-106) (P < 0.05 as determined by Mann-Whitney signed
rank test. B) DRTkip and Tkip inhibit the binding of pJAK2(1001-1013) to
immobilized Tkip. The differences in inhibition of binding by Tkip to pJAK2, when
compared to control peptide, is statistically significant (P < 0.05), but that of DRTkip
is not statistically significant (P > 0.05) as determined by Mann-Whitney signed rank
test. All experiments were carried out in triplicate and the data are representative of
three independent experiments.
+ + + + +
-+ + + +
Figure 4-3. DRTkip and Tkip but not the control peptide, MulFNy(95-106), inhibit JAK2
autophosphorylation. Kinase assays were carried out in the presence of JAK2 kinase,
soluble IFNGR-1, and radiolabelled ATP as described in Chapter 3. To show equal
protein loading, an immunoblot with JAK2 antibody of the reactions was carried out
as described above, but in the presence of unlabelled ATP.
rlM~ :"" ~111
4000*= HHMTkip + SEB
IIIIIl DRTkip + SEB
sooo. O Control peptide + SEB
1.2 3.7 11
Peptide concentration, CLM
30000* Medla + SEA
555Tki + SEA
E ,,,,,III I IIIII DR C;T kip+ SEA
a 20000* 1m Co iintrol peptide + SEA
Peptide concentration, CLM
Figure 4-4. Tkip, but not DRTkip inhibits superantigen-induced splenocyte proliferation. A)
Tkip, but not DRTkip inhibits SEB-induced splenocyte proliferation. Splenocytes (4 x
105 cells/mL) from naive NZW mice were incubated with varying concentrations of
lipophilic peptides Tkip, DRTkip or control peptide MulFNy(95-106) and SEB (0.5
Cpg/mL) for 72 h, followed by pulse labeling with 3-[H]-thymidine, and harvesting on
filter discs. Cell associated radioactivity was quantified using a p-scintillation counter
and is reported as counts per minute (cpm). Differences between Tkip and control
peptide were statistically significant (P < 0.05) as determined by Mann-Whitney
signed rank test. Differences between DRTkip and control peptide were not
statistically significant (P > 0.05). B) Tkip, but not DRTkip inhibits SEA-induced
splenocyte proliferation. The experiment was carried out as described for SEB, but in
the presence of SEA (0.5 Cpg/mL). The differences between Tkip and control peptide
were statistically significant (P < 0.05), but the differences between DRTkip and
control peptide are not statistically significant (P > 0.05) as determined by the Mann-
Whitney signed rank test. The experiments were carried out in triplicate and data are
representative of three independent experiments.
2 + 95-106
0.3 a buffer
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Biotinylated JAK2 (1001 1013)
E2.0-1 O SOCS1-KIR
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Biotinylated pJAK2(1001 1013), mM
Figure 4-5. JAK2 autophosphorylation site peptides bind to SOCS1-KIR. A) JAK2(1001-1013)
peptide binds to both SOCS1-KIR and Tkip. Biotinylated JAK2(1001-1013), at the
indicated concentrations, was added in triplicate to a 96-well plate coated with either
Tkip, SOCS1-KIR, control peptide (MulFNy(95-106)), or binding buffer and binding
assays carried out as described in Chapter 3. B) Biotinylated pJAK2(1001-1013)
binds to both SOCS1-KIR and Tkip. Biotinylated pJAK2(1001-1013) was added to
wells coated with either Tkip, SOCS1-KIR, MulFNy(95-106), or buffer and binding
assays were carried out as described in Chapter 3. The binding of JAK2(1001-1013)
and pJAK2(1001-1013) to Tkip or to SOCS1-KIR, when compared to control peptide
were statistically significant (P < 0.05) as determined by the Mann-Whitney signed
Figure 4-6. Both soluble SOCS1-KIR and soluble Tkip inhibit the binding of biotinylated
JAK2(1001-1013) and biotinylated pJAK2(1001-1013) to immobilized Tkip or
SOCS1-KIR. A) SOCS1-KIR and Tkip inhibit the binding of JAK2(1001-1013) to
immobilized Tkip. B) SOCS1-KIR and Tkip inhibit the binding of JAK2(1001-1013)
to immobilized SOCS1-KIR. C) SOCS1-KIR and Tkip inhibit the binding of
pJAK2(1001-1013) to immobilized Tkip. D) SOCS1-KIR and Tkip inhibit the
binding of pJAK2(1001-1013) to immobilized SOCS1-KIR. For all competition for
binding assays the differences in inhibition of binding by Tkip or SOCS1-KIR, when
compared to the control peptide, were statistically significant as Mann-Whitney
signed rank test (P < 0.05). All experiments were carried out in triplicate and the data
are representative of three independent experiments.
100 300 500
Soluble Peptide, pM
100 300 500
Soluble peptide, pM
100 300 500
Soluble Peptide, pM
r' m SOCS 1-KIR
mm~ 95 106
Soluble peptide, pLM
2 3 45
+ + + +
+ + ++
6 7 8 9 10
Peptide, 100 uM
Peptide, 50 uM
JAK2 so ~r C
+ + + +
1 2 7
- + -
Tkip (ll uM)
Tkip (33 uM)
SOCS l-KIR(ll uM)
SOCS l-KIR (33 uM)
253-287 (ll uM)
253-287 (33 uM)
.. anm em
Figure 4-7. Differences in the kinase inhibition patterns of SOCS1-KIR and Tkip in JAK2
autophosphorylation, STATla phosphorylation, and EGFR phosphorylation. A)
Tkip, but not SOCS1-KIR or the control peptide, MulFNy(95-106), inhibits JAK2
autophosphorylation. B) SOCS1-KIR and Tkip, but not the control peptide
MulFGR1(253-287) inhibit IFNy-induced STATla activation in murine U937 cells.
Immunoblots with phosphorylated (pY701) STATla and the corresponding
densitometry readings of band intensities are shown. The membrane was stripped and
reprobed with STATla antibody. C) Both Tkip and SOCS1-KIR inhibit EGFR
phosphorylation. In vitro kinase assays were carried out in which SOCS1-KIR, Tkip
or control peptide was incubated with EGF and EGFR and ATP for 30 min at 250C.
The kinase reaction mixtures were resolved on 12% SDS-PAGE, transferred onto a
nitrocellulose membrane and immunoblotted with anti-phosphorylated EGFR
antibody, with the densitometry readings of band intensities shown. The membrane
was stripped and reprobed with EGFR antibody.
Media 3.12 6.25 12.5 25
& Media+ IFRy
-@ 253-287 + IFRy
-# SOCS1-KIR + IFRy
SOCS1-KIR + Tkip + IFRy
8 11 22 33 44 55 66 77 88 99 110
Figure 4-8. SOCS1-KIR and Tkip inhibit IFNy-induced macrophage activation. A) SOCS1-KIR
and Tkip, but not the control peptide significantly inhibited IFNy-induced
macrophage activation as determined by testing for nitrite concentration using Greiss
reagent. The inhibition by Tkip, or SOCS1-KIR compared to control peptide was
statistically significant as determined by Mann-Whitney rank test (P < 0.05). B) Tkip,
and SOCS1-KIR, but not the control peptide show dose-dependent synergy in
inhibiting IFNy-induced induction of NO. Raw 264.7 cells were treated with IFNy, in
the presence of varying concentrations of Tkip and SOCS1-KIR and screened for NO
production as described above. The differences between Tkip or SOCS1-KIR,
compared to the control peptide were statistically significant as determined by
Wilcoxon matched pairs test (P < 0.05).
SMBP + Tkip
900. IIIIIIII MBP + SOCS1-KIR
SMBP + 253 287
0 4000*7 1
Peptide concentration, pCM
Figure 4-9. Both SOCS1-KIR and Tkip inhibit proliferation of murine splenocytes. Splenocytes
(1 x 105 cells/well) were obtained from MBP sensitized SJL/J mice that had
developed EAE and were in remission. The splenocytes were incubated with RPMI
1640 complete media containing MBP (50 Clg/mL) and varying concentrations of
lipophilic SOCS1-KIR, lipophilic Tkip or lipophilic control peptide,
MulFNGR1(253-287), for 48 h. Cultures were then incubated with [3H]-thymidine
for 18 h before harvesting. Radioactivity was counted on a p-scintillation counter and
data reported as cpm above background (media only). Both lipo-SOCS 1-KIR and
lipo-Tkip, but not the control peptide inhibited splenocyte proliferation in a dose-
dependent manner. The inhibition of proliferation by lipo-SOCS1-KIR or lipo-Tkip,
compared to the control peptide, was statistically significant as determined by Mann-
Whitney signed rank test (P < 0.05). The data are representative of two independent
experiments, each carried out in triplicate.
c O SOCS1-KIR
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Biotinylated pJAK2(1001-1013), mM
Figure 4-10. Biotinylated pJAK2(1001-1013) binds to SOCS1-KIR but not to SOCS1-ESS.
Biotinylated pJAK2(1001-1013) was added to wells coated with either Tkip, SOCS1-
KIR, SOCS 1-ES S, control peptide MulFNy(95-106), or buffer and binding assays
were carried out as described in Chapter 3. The binding of pJAK2(1001-1013) to
SOCS1-KIR when compared to control peptide was statistically significant as
determined by Mann-Whitney signed rank test (P < 0.01), while no significant
binding was observed between SOCS1-ESS and the control peptide (P > 0.05).
I C~ Media + MBP
looooo. 1 IC II MBP + SOCS1-KIR
E I MBP +253-287
Peptide concentration, pM
30 m Tkip
T I IIIIIIIIIII SOCS1-KIR
I I I TI 1 253-287
3.7 11 33 100
Peptide concentration, pM
Figure 4-11. Tkip and SOCS1-KIR inhibit antigen-specific CD4+ T cell proliferation and CD4+
T cell-induced IFNy production. A) Tkip and SOCS1-KIR inhibit CD4+ T cell
proliferation. Splenocytes obtained from MBP sensitized SJL/J mice, in remission,
were enriched for CD4+ T cells and incubated (5 x 105 cells/well) with varying
concentrations of lipophilic peptide in the presence or absence of MBP (50 Cpg/mL)
and APCs for 72 h. The cultures were pulsed with 3[H]-thymidine for 18 h before
harvesting. Radioactivity was counted and is reported as cpm. Differences in
inhibition of proliferation by Tkip or SOCS1-KIR, compared to control peptide,
MulFNGR1(253-287) were statistically significant (P < 0.05) as determined by
Mann-Whitney signed rank test. B) Tkip and SOCS1-KIR inhibit CD4+ T cell
induced IFNy production. CD4' T cells were incubated with lipophilic peptides in the
presence or absence of MBP and APCs. The cells were transferred onto IFNy-
ELISPOT plate, and incubated for 48 h. Spots, representing IFNy-producing cells,
were detected using HRP-conjugated secondary antibody. Differences in reduction of
the number of IFNy-producing cells by Tkip or SOCS1-KIR, compared to control
peptide, were not statistically significant as determined by Mann-Whitney signed
rank test (P < 0.05). The experiments were carried out in triplicate and the data are
representative of two independent experiments.
mmMBP + Tkip
1411 I mmIr MBP + SOCS1-KIR
12,1 1III MBP + 253 287
1.2 3.7 11 33
Peptide concentration, CIM
Figure 4-12. Tkip and SOCS1-KIR inhibit CD8' T cell-induced IFNy production. Splenocytes
were obtained from MBP sensitized SJL/J mice that a few days after signs of active
disease. The splenocytes were enriched for CD8+ T cells (5 x 105 cells/well) and
incubated with varying concentrations of lipophilic peptides in the presence or
absence of MBP (50 Cpg/mL) and APCs. The cells were transferred into IFNy-Elispot
plates and incubated for 48 h. The spots, representing IFNy-producing CD8' T cells,
were detected using HRP-conjugated secondary antibody. The differences in
inhibition of IFNy production by Tkip-treated or SOCS1-KIR treated cells, compared
to control peptide-treated cells ( 11 and 33 CLM) were statistically significant as
determined by Mann-Whitney signed rank test (P < 0.03). The experiment was
carried out in triplicate and the data are representative of two independent
z I Media
I I I Media + MBP
6000 IIIIIIIISOCS 1-KIR
E 5000,1 I I 253-287
3.7 11 33
Peptide concentration, CIM
E 0.7- -1 I 11 1 MBP
c z mmTkip
3 "~ ~II11 1~ I IIIII~ SOCS1-KIR
4 0.5, 253 -287
3.7 11 33
Peptide concentration, pM
Figure 4-13. Tkip and SOCS1-KIR inhibit antigen-induced B cell proliferation and antibody
production. A) Tkip and SOCS1-KIR inhibit B cell proliferation. B cells (5 x 105
cells/well) obtained from MBP sensitized SJL/J mice 2 months after last remission
were incubated with lipophilic peptides in the presence or absence of MBP and APCs.
The data presented for peptides was in the presence of MBP and APCs. The cultures
were pulse labeled with 3[H]-thymidine for 18 h and harvested. Radioactivity was
counted and is reported as cpm. The differences in inhibition of proliferation by Tkip
or SOCS1-KIR (3.7, 11, and 33 CLM), compared to the control peptide were
statistically significant as determined by Mann-Whitney test (P < 0.01). B) Tkip and
SOCS1-KIR inhibit the production of MBP-specific antibodies. B cells (5 x 105
cells/well) from MBP sensitized SJL/J mice were incubated with lipophilic peptides
in the presence of MBP (50 Cpg/mL) and APCs for 48 h. Culture supernatants were
then harvested and tested for MBP-specific antibodies by ELISA. The difference
recorded for Tkip or SOCS1-KIR (33 CLM), compared to control peptide were
statistically significant (P < 0.01) as determined by Wilcoxon matched pairs test. The
experiments were carried out in triplicate and data is representative of two
7.5.1 HMLF5 Tk~p
555LF' + S0C51-VIRh
0.1 0.5 1.0
LPS concentration, Clg/ml
Figure 4-14. Tkip inhibits LPS-induced macrophage activity. Murine macrophage cells, Raw
264.7, were incubated with varying concentrations of LPS alone or with either
lipophilic Tkip, lipophilic SOCS1-KIR or lipophilic control peptide, MulFNy(95-
106), each at 24 CLM final concentration, for 48 h. Culture supernatants were collected
and nitrite concentration determined using Greiss assay. The experiments were
carried out in triplicate and data are representative of two independent experiments.
There was statistically significant difference between Tkip and control peptide (P <
0.05), but none between SOCS1-KIR and control peptide (P > 0.05), at the
concentrations tested, as determined by Wilcoxon matched pairs test.
Figure 4-15. pJAK2(1001-1013) peptide has SOCS-1 antagonist properties. A) pJAK2(1001-
1013) enhances suboptimal IFNy-induced antiviral activity against EMC virus in
human fibroblast WISH cells as determined using a cytopathic assay. B)
pJAK2(1001-1013) reverses SOCS-1 inhibition of STAT3 phosphorylation in human
prostate cancer cells (LNCaP) transfected with a SOCS-1 plasmid DNA, pEF-FLAG-
I/mSOCS-1, provided by Dr. David Hilton. The results of an immunoblot assay with
phosphorylated STAT3 (pY705) and the corresponding densitometry readings of
band intensities are shown. Also shown are results of an immunoblot assay with
unphosphorylated STAT3 antibody as well as SOCS-1 expression in LNCaP cells.
The data are representative of two independent experiments. C) pJAK2(1001-1013)
enhances GAS promoter activity. WISH cells were transfected with a vector
expressing firefly luciferase, driven by a GAS promoter, along with a vector
expressing Renilla luciferase as a control vector. IFNy (1 U/mL) and lipo-
pJAK2(1001-1013) at 5 or 1 CIM final concentration, or a control peptide,
MulFNy(95-125) (5 CIM) were added to the cells. After 48 h incubation, the cell
extracts were assayed for relative luciferase activities using a luminometer. D)
Soluble SOCS-1 protein, similar to SOCS1-KIR inhibits the binding of biotinylated
pJAK2(1001-1013) to SOCS1-KIR. Biotinylated pJAK2(1001-1013) that had been
preincubated with varying concentrations of soluble SOCS1-KIR, SOCS-1 lysate or
control lysates, was added in triplicate to a 96 well plate coated with SOCS1-KIR.
Bound biotinylated pJAK2(1001-1013) was detected using neutravidin-HRP
conjugate as described in Chapter 2. Also shown is an immunoblot lysate showing
expression of SOCS-1 in Sf9 insect cells. Aliquots of the cell lysate were used for the
competition for binding assay.
Media IFNy IFNy +JAK2 IFNy+ pJAK2
I2 3 4 5 6 7 8 9
II- + +
1 1 1 1 1 '
Treatment None IFN IFN+ IFN+ IFN+
pJAK2 pJAK2 95-125
pIM 5 1 5
Control plasmid +
O Control lysate
Peptide (pM) 100
Protein (pg/ml) 1 2
Lane 1 2 3 4 5 6 7 8 9 1011121314
10 20 30
MVARNVA NAISPAAEPR RRSEPSSSSS
PGDTHFRTF~R SHSDYRRITIR TSALLD~ACGF
TFLVRDSRQR NCFFIALSVKM ASGPTSIRVH
LLEHYVAAPR RMLGAPLRQR RVRPLQELCR
Figure 4-16. SOCS-1 protein was expressed in baculovirus infected Sf9 insect cells. A) The
restriction enzyme digestion pattern of pBlueBac/muSOCS 1 plasmid DNA with Lane
2 showing the correct restriction enzyme digestion product. B) Amino acid sequence
of the cloned plasmid showing full-length SOCS-1 sequence, sequences derived from
the cloning vector are underlined.
Lane 1 2 3 4 5 6 7 89101112131415
Figure 4-17. Recombinant baculovirus containing muSOCS-1 DNA. PCR amplification of
recombinant plaques (blue) with Baculovirus forward and baculovirus reverse
primers. Lanes 1, 4, 5, 6, 9, and 10 are carrying inserts of the expected size.
Constructs represented by lane 4 and 6 were chosen for further analysis.
SOCS-1 is absolutely essential for survival of the individual. Although SOCS-1 knockout
mice appear to be normal at birth, they exhibit stunted growth and die neonately by three weeks
of age (Alexander et al. 1999). These mice exhibit a syndrome characterized by severe
lymphopenia, activation of T lymphocytes, fatty degradation and necrosis of the liver,
hematopoetic infiltration of multiple organs, and high levels of constitutive IFNy as well as
abnormal sensitivity to IFNy (Alexander et al. 1999, Reviewed in Alexander and Hilton 2004,
Yoshimura 2005). IFNy plays a central role in the pathology as SOCS-1 knockout mice that are
deficient in IFNy or IFNy receptor do not die as neonates. Similar pathology and lethality is
observed in normal neonates that are injected with IFNy. It is worth noting that SOCS-1- -, IFNy~I
double knockout mice die by 6 months of age of severe inflammatory disease (Metcalf et al.
2002) indicating that SOCS-1 regulation is not specific for IFNy.
The dynamics of induction of SOCS-1 by IFNy in cells and the activation of STATla is
illustrative of how SOCS-1 attenuates IFNy functions under physiological conditions. For
example, treatment of monocytes or astrocytes with IFNy was followed by activation of the
SOCS-1 gene at approximately 90 min (Dickensheets et al. 1999, Brysha et al. 2001). Low doses
of IFNy resulted in transient increases in SOCS-1 mRNA that returned to baseline after 4 h,
while high concentrations of IFNy resulted in increases of SOCS-1 mRNA up to 24 h. Thus, the
SOCS-1 response appears to be induced by the IFNy signal. Treatment of hepatocytes from
SOCS-1" mice with IFNy resulted in STATla activation within 15 min, which peaked by 2 h
before declining (Brysha et al. 2001). Although STATla is similarly activated in SOCS-1
deficient livers, it persists for 8 h. SOCS-1 thus appears to attenuate IFNy persistent activation of
STATla, which nonetheless allows the beneficial effects of IFNy-induced signaling.
Given the critical importance of SOCS-1 in modulating the activities of IFNy and other
inflammatory cytokines that use tyrosine kinases such as JAK2 in their signaling pathways, our
laboratory developed the small tyrosine kinase inhibitor peptide, Tkip, which is a mimetic of
SOCS-1 (Flowers et al. 2004). Tkip was designed to recognize the autophosphorylation site on
JAK2 involving residues 1001 to 1013 containing the critical tyrosine at 1007 (Yasukawa et al.
1999). Flowers et al (2004) showed that Tkip blocked JAK2 autophosphorylation as well as
tyrosine phosphorylation of substrates such as STATla and IFNy receptor chain, IFNGR-1. The
authors also showed that like SOCS-1, Tkip blocked EGFR autophosphorylation, while not
affecting the tyrosine kinase function of c-Src and vascular endothelial growth factor receptor.
Additional experiments showed that Tkip blocked IL-6 induced activation of the STAT3
oncogene in LNCaP prostate cancer cells, which involved inhibition of JAK2 activation (Flowers
et al. 2005). These studies presented a proof-of-concept demonstration of a peptide mimetic of
SOCS-1 that regulates JAK2 tyrosine kinase function.
Because of its potential for regulation of inflammatory conditions where tyrosine kinases
such as JAK2 play a role in the resultant pathology, Tkip was tested in a mouse model of
multiple sclerosis called experimental allergic encephalomyelitis (EAE) (Mujtaba et al. 2005).
SJL/J mice were immunized with myelin basic protein (MBP) for induction of the
relapsing/remitting form of EAE. Tkip, 63 Clg every other day, given intraperitoneally,
completely protected the mice against relapses when compared to control groups in which
greater that 70% of the mice relapsed after primary incidence of disease. Protection of mice
correlated with lower MBP antibody titers in Tkip-treated groups as well as suppression of MBP-
induced proliferation of splenocytes taken from EAE-afflicted mice. Consistent with its JAK2
inhibition function, Tkip also inhibited the activity of inflammatory cytokine TNF-u, which uses
the STATla transcription factor. Thus, Tkip, like SOCS-1, possesses anti-inflammatory activity
that protects mice against ongoing relapsing/remitting EAE.
The design of Tkip was independent of any knowledge of the structural and functional
domains of SOCS-1. Given that the design focused on Tkip binding to the autophosphorylation
site of JAK2, I compared Tkip with regions of SOCS-1 that have been proposed to be either
directly involved in such binding or to be involved in enhancement of SOCS-1 binding to the
autophosphorylation site. Yasukawa et al. (1999) identified three regions that were directly
involved in SOCS-1 binding to JAK2, the large SH2 domain, a N-terminal 12-amino acid
sequence called extended SH2 (ESS), and an additional N-terminal 12-amino acid region called
the kinase inhibitor region (KIR). The ESS and SH2 domains were felt to bind to the
autophosphorylation or activation site of JAK2, while KIR bound to the catalytic site in this
model. The 12-amino acid ESS sequence consists of residues 68-79, while KIR consists of
residues 56-67. The SOCS1-KIR peptide sequence compared to the KIR above consists of
residues 53-68, sharing just the 168 with the ESS and containing three additional residues in its
N-terminus. In comparative binding, the SOCS1-KIR peptide bound to pJAK2(1001-1013),
while SOCS1-ESS peptide failed to bind (Figure 4-10). Close examination of N-terminal
truncated SOCS-1 expressed proteins, designated dN51 (missing residues N-terminal to 51) and
dN68, in Yasukawa et al (1999) showed that removal of the KIR region resulted in loss of
binding to JAK2 in transfected cells. Further, in direct binding to pJAK2 autophosphorylation
site peptide by truncated dN56 SOCS-1 protein, the SOCS1-KIR peptide sequence except for
residues 53-55, was present along with ESS and SH2 (Yasukawa et al. 1999). Although the
region of SOCS-1 that binds to the autophosphorylation site of JAK2 as per Yasukawa et al.
(1999) is referred to as SOCS-1 SH2 plus ESS, the bindings that they refer to also contained our
SOCS1-KIR sequence. Additionally, I have shown that SOCS-1 protein competed with SOCS1-
KIR for pJAK2(1001-1013), suggesting that the two recognized the JAK2 autophosphorylation
site similarly. Thus, I feel that the binding data with our SOCS1-KIR are consistent with the
binding studies of these authors. It should be noted that the peptide binding approach used here
does not involve assessment of collaboration and/or synergism among the KIR, ESS, and the
SH2 domains of SOCS-1. Thus, based on the studies reported here, along with those by others
(Yasukawa et al. 1999), KIR, ESS and SH2 may all be involved in binding to JAK2
autophosphorylation site. It remains to be determined as to the extent of their relative roles.
I have shown in this study that SOCS1-KIR, independent of other domains of SOCS-1,
can bind directly to a peptide, JAK2(1001-1013), that corresponds to the autophosphorylation
site of JAK2. Further, I showed that SOCS1-KIR competed with Tkip for binding to JAK2(1001-
1013). The competitions suggest that the peptides recognized JAK2 similarly but not exactly the
same way. Phosphorylation of tyrosine 1007 on the JAK2 peptide enhanced binding of Tkip and
SOCS1-KIR. Tkip blocked JAK2 autophosphorylation as well as JAK2 phosphorylation of
STATla, while SOCS1-KIR did not block autophosphorylation but did block phosphorylation
of STATla, similar to the pattern or profile of SOCS-1 inhibition of phosphorylation (Alexander
and Hilton 2004). The peptides were also functionally similar in inhibiting IFNy activation of
macrophages to produce NO and inhibiting antigen-specific induction of proliferation of
splenocytes, with Tkip being the more effective inhibitor. Thus, I have shown here that the KIR
region of SOCS-1 can directly bind to the autophosphorylation site of JAK2. These data are
consistent with the observation made by Babon et al. (2006) on SOCS-3, which like SOCS-1 has
KIR. These authors showed that the SH2, ESS, and the C-terminal half of KIR directly contacted
the phosphotyrosine binding loop of IL-6 receptor, gpl30, and that the N-terminal half of KIR
likely bound the JAK kinase. Hence, SOCS-1 KIR likely binds directly to JAK2
Tkip is relatively hydrophobic, while SOCS1-KIR is hydrophilic. However, both peptides
have hydropathic profiles that are complementary to that of pJAK2(1001-1013). ESS however,
had a hydropathic profile different from Tkip. Tkip was designed to have a hydrophathic profile
complementary to that of pJAK2(1001-1013) (Flowers et al. 2004, Weathington and Blalock
2003). Thus, Tkip would recognize primarily hydrophobic residues or groups in the pJAK2
peptide, while SOCS1-KIR would recognize primarily hydrophilic residues or groups. The
binding competition could thus be due to a steric interference, which is consistent with
differential effects of Tkip and SOCS1-KIR on JAK2 kinase activity.
It has previously been shown that Tkip has potential anti-tumor (Flowers et al. 2005) and
anti-inflammatory (Mujtaba et al. 2005) properties. Hence, Tkip may have potential as a
therapeutic agent. Here, I have shown that Tkip and SOCS1-KIR directly affect the activity of
CD4' T cells, CD8 T cells, B cells, and macrophages, which provides additional insights of the
direct effect that these peptides have on the cells of the immune system. These data further show
the probable therapeutic potential of Tkip.
The fact that the KIR region of SOCS-1 can bind directly to pJAK2(1001-1013) raises
the possibility that pJAK2(1001-1013) can function as an antagonist of SOCS-1. It has thus been
shown here under four different types of experiments that pJAK2(1001-1013) possesses SOCS-1
antagonist properties. First, pJAK2(1001-1013) enhanced suboptimal IFNy activity. Second,
prostate cancer cells transfected for constitutive production of SOCS-1 protein had reduced
activation of STAT3 by IL-6 treatment. pJAK2(1001-1013) reversed the SOCS-1 effect. Third,
pJAK2(1001-1013) enhanced IFNy activation of the luciferase reporter gene via the GAS
promoter. Fourth, pJAK2(1001-1013) enhanced antigen-specific splenocyte proliferation. As
indicated above, treatment of cells with IFNy resulted in activation of the SOCS-1 gene in
approximately 90 min and it has been proposed that it is associated with the physiological
attenuation of the IFNy response by SOCS-1 (Dickensheets et al. 1999, Brysha et al. 2001)
Consistent with this, it has recently been reported that small-interfering RNA inhibition of
SOCS-1 expression in bone marrow dendritic cells resulted in enhanced CTL activity and IFNy
production by ELISPOT, culminating in enhancement of anti-tumor immunity (Shen et al. 2004).
I have thus shown here that SOCS1-KIR binds directly to the autophosphorylation site of
JAK2, similar to the binding of Tkip SOCS-1 mimetic, which results in inhibition of JAK2
phosphorylation of substrate. This directly identifies a region of SOCS-1 that possesses intrinsic
anti-kinase function. Related to this, the autophosphorylation site peptide, pJAK2(1001-1013),
functioned as an antagonist of SOCS-1. These findings with SOCS-1 mimetics and antagonists
have implications for novel therapeutic approaches to mimicking SOCS-1 for treatment of
inflammatory diseases and for suppressing SOCS-1 in order to enhance the immune response
against cancer and infectious diseases.
Future work will involve expressing and purifying large quantities of murine SOCS-1 and
using the purified protein to characterize the functional regions of SOCS-1 protein. I have also
designed additional experiments to determine specifically the role of the SOCS-1 KIR and ESS
regions. This would provide additional insight on the minimum domain essential for SOCS-1
Mouse studies are currently underway attempting to determine whether Tkip can be used
for treatment of ongoing relapsing/remitting form of EAE, with implications for treatment of
multiple sclerosis. Further, additional experiments are being designed to determine whether Tkip
binds to the other JAK kinases, JAKl, JAK3 and TYK2, implication of which Tkip would be
used to regulate signaling by these kinases.
For SOCS-1 antagonist studies, mouse studies are being carried out in which attempts are
being made to determine whether pJAK2(1001-1013) can enhance protection against ongoing
APPENDIX: VECTOR MAP OF THE TRANSFER (CLONING) VECTOR
~I c~4~y~i r=IF;~EI
Cosmments fo~r pBlueBac4.5NB-His
Polyhedrin promoter (pp,J bases 7-95 y
Multiple cloning site: bases 132-189
V5 epitope: bases 196-237
8xH is tag: bases 247-284
SV40 polya8denylation sequence: 289-416
F en d of Ac-PH: bases 420-521
S end of ORF1829: 1429-551 (complemenrtary)
Sequence homologous to ORF1829 sequence in BeeN-Blue DNA: bases 833-1431 (complemaentary)
Arupicill; n resistance gene: 1817-2674
pUC origin: 2822-3495
EE tecZ fragmenrt: 4725-381 3 (complementary)
Sequence homologous to incZ sequence in Bee-N-Blue DBNA: bases 4498-3613 (corplernentary)
Early to late promoter: bases 5027-4728 (corplemnentary)
Figure A-1. A map of the pBlueBac4.5/V5-His vector. Adapted from pBlueBac4.5/V5-His
TOPO Expression Kit manual (Invitrogen).
s~ ~ib I
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Lilian Wangechi Waiboci was born on October 5, 1971, in Nyeri, Kenya to Mr. Francis
Waiboci Matu and Mrs. Esther K. Waiboci. She grew up in Nyeri, graduating from Bishop
Gatimu Ngandu Girls High School in 1989. She earned her B.S. in Biochemistry and Zoology
and her M.S. in Biochemistry from the University of Nairobi, Kenya in 1995 and 2001,
Upon graduating in 1995, Lilian worked as a high school teacher, teaching Chemistry and
Biology. During her M.S. program, she was a research assistant at the International Livestock
Research Institute (ILRI) and upon completion of her degree requirements worked as a part-time
lecturer at Jomo Kenyatta University of Agriculture and Technology (JKUAT), and later for the
Walter Reed Army Medical Research Proj ect, HIV Laboratory in Kericho, Kenya as a
Laboratory Manager and Research Technician.
Lilian earned her Ph.D. from the Department of Microbiology and Cell Science,
University of Florida in May 2007. She will pursue a carrier that includes both aspects of
research and teaching in a University or a Research Institute. Lilian is married to Dr. George
Muhia Kariuki and they have a son, Victor Kariuki Muhia.