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Interactions of microtubule-associated protein-2 with microtubules and neurofilaments

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Title:
Interactions of microtubule-associated protein-2 with microtubules and neurofilaments
Creator:
Joly, John Charles, 1963-
Publication Date:
Language:
English
Physical Description:
x, 125 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Amino acids ( jstor )
Cattle ( jstor )
Digestion ( jstor )
Gels ( jstor )
In vitro fertilization ( jstor )
Microtubule associated proteins ( jstor )
Microtubules ( jstor )
Polymerization ( jstor )
Proteins ( jstor )
Purification ( jstor )
Cytoskeleton -- physiology ( mesh )
Department of Biochemistry and Molecular Biology thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Biochemistry and Molecular Biology -- UF ( mesh )
Microtubule Associated Proteins -- physiology ( mesh )
Microtubules -- physiology ( mesh )
Neurofilament Proteins ( mesh )
Research ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1990.
Bibliography:
Bibliography: leaves 115-124.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by John Charles Joly.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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030487642 ( ALEPH )
22503298 ( OCLC )
AGZ6060 ( NOTIS )

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Full Text











INTERACTIONS OF MICROTUBULE-ASSOCIATED PROTEIN-2
WITH MICROTUBULES AND NEUROFILAMENTS
















BY

JOHN CHARLES JOLY


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


1990




INTERACTIONS OF MICROTUBULE-ASSOCIATED PROTEIN-2
WITH MICROTUBULES AND NEUROFILAMENTS
BY
JOHN CHARLES JOLY
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
1990


ACKNOWLE DGEMENTS
This work was made possible due to the love and support
of my parents. Their support through my undergraduate and
graduate studies enabled me to complete this long journey.
I must thank members of the Purich lab; especially Greg
Flynn for giving me my start in the lab and collaborating on
the work in Chapter 2, and Jim Angelastro for constantly
patient help with the flipping HPLC. Also Alexandra
Ainzstein for carrying out one of the digestion experiments
in Chapter 3. I am also greatly indebted to Dan Purich for
support through these years, for tolerating my antics and
for his gracious help in aiding my postdoctoral hunt.
Without his help I am sure I would not have enjoyed this
last year as much as I did.
ii


TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS ii
LIST OF TABLES V
LIST OF FIGURES vi
ABBREVIATIONS viii
ABSTRACT ix
CHAPTERS
1 INTRODUCTION 1
An Overview of the Cytoskeleton 1
Microtubules 2
Neurofilaments 6
Microtubule-associated Proteins 12
Proposal 26
2 INTERACTIONS OF MAP-2 WITH TUBULIN AND NF-L 29
Introduction 29
Materials and Methods 30
Results 33
Discussion 42
3 THE MICROTUBULE-BINDING FRAGMENT OF MAP-2:
LOCATION OF THE PROTEASE-ACCESSIBLE SITE 46
Introduction 46
Materials and Methods 47
Results 51
Discussion 62
4 THE MICROTUBULE-BINDING FRAGMENT OF MAP-2:
IDENTIFICATION OF AN ASSEMBLY-PROMOTING PEPTIDE
AND DISPLACEMENT OF HIGH-MOLECULAR-WEIGHT MAPS .. 66
Introduction 66
Materials and Methods 68
Results 74
Discussion 97
iii


5 CONCLUSIONS AND FUTURE DIRECTIONS
103
Interactions with Neurofilaments 103
Structure of MAP-2 107
MAP-2 Sequence Interactions with Microtubules .. 108
REFERENCES 114
BIOGRAPHICAL SKETCH 124
iv


LIST OF TABLES
Table page
1-1 Major classes of microtubule-associated
proteins 13
3-1 Amino acid composition of the 28 kDa
MAP-2 fragment 55
3-2 Amino terminal sequence analysis of the
28 kDa fragment 57
v


LIST OF FIGURES
Figure page
1-1 Comparison of the carboxyl termini of
both a- and B-tubulin from chick brain .... 7
1-2 Structural organization of the neuro
filament triplet protein 10
1-3 Comparison of the carboxyl termini of
murine MAP-2 and murine tau protein 25
1-4 Summary of MAP-2 structure in relation to
a microtubule 27
2-1 Thrombin digestion of radiolabeled MAP-2 .. 35
2-2 Binding of MAP-2 or MAP-2 fragments to
neurofilaments or tubulin 37
2-3 Autoradiogram of MAP-2 and MAP-2 fragments
binding to cytoskeletal protein 38
2-4 MAP-2 binding to purified neurofilament
triplet protein or the L subunit of
neurofilaments 40
2-5 Autoradiogram of MAP-2 and MAP-2 fragment
binding to neurofilament triplet protein
and L subunit 41
2-6 Determination of the isoelectric point of
the 28 kDa fragment of MAP-2 42
3-1 Purification of heat-stable microtubule
binding fragment and tau 52
3-2 HPLC purification of the microtubule-binding
fragment of MAP-2 digested with microtubules
present 54
3-3 Comparison of proteolytic fragmentation
patterns and the amino terminal sequences of
the microtubule binding fragments of MAP-2
and tau 58
vi


3-4 HPLC purification of the microtubule-binding
fragment of MAP-2 digested initially without
microtubules 62
4-1 Stimulation of microtubule assembly with
synthetic peptides 76
4-2 Time course of peptide induced assembly .... 77
4-3 Electron micrograph of peptide induced
assembly 79
4-4 Immunofluorescence of microtubules polymer
ized without and with m2 peptide 80
4-5 Critical concentration plot of peptide
induced tubulin polymerization 81
4-6 Seeded assembly of tubulin with synthetic
peptides 83
4-7 Effects of MAP-2 peptides on MAP binding to
microtubules 84
4-8 Densitometry of the coomassie blue stained
gel 86
4-9 Comparison of the stimulation of tubulin
polymerized by peptides m2 and m2' 87
4-10 Effects of increasing the m2' concentration
on high-molecular-weight MAP binding to
microtubules 89
4-11 32P-MAP-2 binding to taxol-stabilized
microtubules 91
4-12 Displacement of trace phosphorylated MAP-2
from taxol-stabilized microtubules by
unlabeled MAP-2 92
4-13 Displacement of trace phosphorylated MAP-2
from taxol-stabilized microtubules by
peptide m2' 94
4-14 Radiolabeled MAP-2 binding to taxol-
stabilized microtubules in the presence and
absence of 1.5 mM m2' peptide 95
4-15 Double reciprocal plot of MAP-2 binding .... 96
5-1 The octadecapeptide repeats of murine MAP-2,
murine tau, bovine tau, and a corresponding
sequence of the 190 kDa adrenal gland MAP .. 112
vii


ABBREVIATIONS
ATP, adenosine triphosphate
cAMP, cyclic adenosine monophosphate
cDNA, complementary DNA
DEAE, diethylaminoethyl
GTP, guanosine triphosphate
HPLC, high pressure liquid chromatography
MAP, microtubule-associated protein
mRNA, messenger RNA
NF, neurofilament
pi, isoelectric point
PMSF, phenylmethanesulfonyl fluoride
SDS, sodium dodecyl sulfate
viii


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
INTERACTIONS OF MICROTUBULE-ASSOCIATED PROTEIN-2
WITH MICROTUBULES AND NEUROFILAMENTS
by
John Charles Joly
May, 1990
Chairman: Dr. D. L. Purich
Major Department: Biochemistry and Molecular Biology
Bovine brain microtubule-associated protein-2 (MAP-2)
is a 280 kDa protein that binds to microtubules and
neurofilaments. MAP-2 was fragmented by thrombin into a 240
kDa projection domain and a 28 kDa microtubule-binding
domain. The 28 kDa cleavage fragment possessed a
neurofilament binding site for the L subunit of
neurofilaments. The thrombin cleavage site of MAP-2 was
very similar to the chymotryptic cleavage site of the
microtubule-associated protein tau. The 28 kDa microtubule
binding fragment was derived from the carboxyl terminus of
the intact protein and the 240 kDa projection domain was
from the amino terminus. The microtubule-binding fragment
was very rich in lysine and arginine residues and its
isoelectric point was approximately 10.0. The microtubule
binding fragment contained three octadecapeptide imperfect
repeats located fifty residues from the thrombin cleavage
site. These sequences were chemically synthesized to assay
ix


for promotion of tubulin polymerization. Only the second
promoted tubulin polymerization in vitro, yielding
microtubules of normal morphology. The time course of
peptide-induced microtubule assembly was similar to
microtubule-protein in vitro. This octadecapeptide
displaced MAP-lb from MAP-containing microtubules. The
addition of the next three amino acids in the MAP-2 sequence
to the carboxyl terminus of the peptide increased its
ability to promote tubulin polymerization at lower
concentrations and displaced all high-molecular-weight MAPs,
MAP-la,b and MAP-2a,b. This extended peptide displayed
competitive binding with radiolabeled MAP-2 to taxol-
stabilized MAP-free microtubules, suggesting the peptide
bound to the same site on microtubules as MAP-2. The
dissociation constant for MAP-2 binding was 3.4 /iM in the
absence of the extended peptide and 14 nK in the presence of
1.5 mM peptide. The estimated inhibition constant for the
extended peptide is 0.5 mM, about 100 times lower than for
the K,,, of MAP-2. These observations suggested that the
second repeated sequence of MAP-2 represents an important
recognition site for MAP-2 binding to microtubules and that
other structural features within MAP-2 may reinforce the
strength of MAP-microtubule interactions.
x


CHAPTER 1
INTRODUCTION
An Overview of the Cytoskeleton
The cytoskeleton of eukaryotic cells is a dynamic
organelle responsible for maintaining cell shape and
rigidity, cell motility, and intracellular vesicle transport
and trafficking. The cytoskeleton consists of three main
types of filaments: microfilaments, intermediate filaments,
and microtubules. Microfilaments are seven nanometers in
diameter and are composed primarily of actin but also
contain actin-binding proteins. Intermediate filaments are
ten nanometers in diameter and vary in composition depending
on the particular cell type. In brain tissue, the neuronal
cell intermediate filaments are made of neurofilament
proteins while in glial cells, the intermediate filaments
are composed of glial fibrillary acidic protein.
Microtubules are 24 nanometer diameter structures composed
mainly of the heterodimer tubulin. In addition to tubulin,
there are cell specific microtubule-associated proteins
(MAPs) that bind to microtubules.
When specific cytoskeletal filaments are mixed in
vitro, they interact with each other. If bovine spinal cord
neurofilaments are mixed with bovine brain microtubules, the
viscosity of the resulting solution increases greatly and a
gel is produced (Runge et al., 1981). This is thought to be
1


2
a relevant and physiological interaction based on
microscopical examination of neuronal axoplasm which shows
neurofilaments and microtubules running parallel to each
other in neurite processes. The work of Hirokawa and his
coworkers over the years has demonstrated through microscopy
techniques that projections exist between the filamentous
structures (Hirokawa et al., 1985; Hirokawa 1982). These
projections could be cross-bridges connecting the two types
of filaments. Possible candidates for the cross-bridges are
MAPs. Both microtubule-associated protein-2 (MAP-2) and tau
proteins are MAPs and can bind to neurofilaments as well as
microtubules (Letterier et al., 1982; Heimann et al., 1985;
Miyata et al., 1986). It is the role of MAPs, specifically
MAP-2 and its interactions with microtubules and
neurofilaments, that is the focus of this thesis.
Microtubules
Microtubule Structure and Function
Microtubules are the most dynamic component of the
cytoskeleton and exist in all eukaryotic cells except
enucleated erythrocytes. The microtubule core is formed
from alpha and beta tubulin heterodimers arranged into
parallel rows, extending the length of the tubule, termed
protofilaments. Isolated microtubules and those observed in
sectioned cell specimens contain thirteen protofilaments,
while most microtubules assembled in vitro possess fourteen
protofilaments (McEwen and Edelstein, 1980). The tubulin
subunits arrange themselves in a head-to-tail fashion along
the protofilaments yielding the distinct polarity in


3
microtubules. This polarity was first observed by Rosenbaum
and Child (1976) and Witman (1975), who demonstrated biased
addition to microtubules in vitro. One end of the
microtubule polymer displays an increased rate of addition
compared to the other end. This characteristic polarity of
microtubules can be determined by interactions with
microtubule-binding proteins. One method is based on the
interaction of dynein with assembled microtubules (Haimo et
al., 1979; Haimo, 1982). Dynein is a large protein found in
flagella of Tetrahymena that binds to microtubules and
hydrolyzes adenosine triphosphate to produce the whip-like
motion during movement. The main globular head of the
dynein molecule tilts at an angle of 55 in the direction of
the end of preferred growth of the microtubule. Another
method to distinguish microtubule polarity takes advantage
of special in vitro solution conditions that favor formation
of microtubule walls decorated with additional
protofilaments. The extra protofilaments align themselves
into hook-shaped sheets and curve in one specific direction
depending on the polarity; either clockwise or
counterclockwise (Burton and Himes, 1978).
This intrinsic polarity of microtubules enables
microtubules to perform vectorial functions. When
intracellular vesicles travel along a microtubule in an
axon, the vesicle generally moves in one direction. Rarely
will a vesicle change its direction after starting its
journey, and most vesicles move toward the cell body in a
neuron (Hollenbeck and Bray, 1987). The polarity of a


4
microtubule is clearly a vital property for interactions
with other cytoskeletal components as well as for their
proper function. In a non-neuronal cell, microtubules are
capped at their minus end near the centrioles and extend the
plus end to the cell margin, yielding an overall radial
polarity in the cell.
Some other interesting properties of microtubules are
that they self-assemble in vitro in the presence of GTP at
warm temperatures and that they depolymerize at cold
temperatures or upon the addition of calcium ions. Each
alpha/beta dimer binds two moles of GTP per mole tubulin,
but each heterodimer has two types of nucleotide binding
sites (Weisenberg et al., 1968; Berry and Shelanski, 1972).
The beta subunit has an exchangeable nucleotide binding site
that readily exchanges GDP for GTP. The alpha subunit has a
nonexchangeable binding site that exchanges GDP for GTP very
slowly. Guanosine triphosphate nucleotides are hydrolyzed
only upon or after heterodimer incorporation into the
polymeric tubule.
Both in vitro and in vivo microtubule dynamics have
been monitored through the use of tubulin modified with a
fluorescent tag. This tag can be a direct covalent
attachment of a fluorochrome or by using an anti-tubulin
antibody with a secondary antibody labeled with a
fluorescent probe. In most cases examined the microtubules
have been shown to be highly dynamic structures rapidly
polymerizing and depolymerizing depending on the surrounding
conditions (Mitchison and Kirschner, 1984; Kristofferson et


5
al., 1986; Sammak et al., 1987). In vitro, the addition of
MAPs reduces microtubule dynamics (P.S. Yamauchi, personal
communication).
Microtubules are essential for proper mitotic function.
When the mitotic spindle poison colchicine is added to
cells, mitotic arrest is seen; particularly at metaphase
prior to microtubule depolymerization in anaphase which
achieves chromosome movement toward the spindle poles. The
chromosomes are connected to the microtubules through
kinetochores which cap the microtubules at one end, while
the opposite end is capped by the spindle pole or centriole.
During interphase the centrioles seem to act as microtubule
organizing centers and are located just outside the nucleus.
Some other important functions of microtubules are the
maintenance of cell anisometry and promotion of cell shape
changes. Nerve axons and retinal rod cells rely on the
microtubule network to support their distinctive cell
morphology, especially the neurite processes (Heidemann et
al., 1986). Conversely, the activation of platelets and the
action of polymorphonuclear leukocytes depend on changes in
the microtubule-cytoskeleton (Malawista, 1986).
Microtubules also provide the basic framework for the cell
motility machinery as they are essential components of
flagella and cilia.
Tubulin
The main component of microtubules is tubulin. This
protein exists as a heterodimer of 100 kDa with each subunit
possessing a molecular mass of approximately 50 kDa. There


6
is considerable homology between the a and 6 subunits
suggesting a common ancestral origin (Valenzuela et al.,
1981). Both a and B tubulin are very conserved across
species lines suggesting that there are stringent
requirements on the structure of tubulins over a reasonably
great phylogenetic range (Cleveland and Sullivan, 1985).
Both subunits contain a glutamate-rich carboxyl terminus
that is thought to be responsible for MAP binding to
microtubules (see Fig. 1-1). When the carboxyl termini of
both subunits are proteolytically removed with subtilisin,
the tubulin can self-assemble without MAP binding (Serrano
et al., 1984). In fact, when these tubules are sedimented
and the pellet fraction analyzed, the MAPs are found only in
the supernatant fraction. The presence of high
concentrations of salt (0.4 M -0.6 M) disrupts MAP binding
to microtubules indicating the interaction of MAPs with
microtubules is dependent on the ionic interactions. Thus
the glutamate rich carboxyl termini are thought to be the
binding sites for MAPs which in turn contain positively
charged residues (Littauer et al., 1986).
Neurofilaments
Neurofilament Structure
Neurofilaments are one member of the intermediate
filament protein family of which there are five main
components: (1) acid keratins, (2) basic keratins, (3) glial
fibrillary acidic protein, vimentin, peripherin, and
desmin, (4) neurofilaments and alpha-internexin, and (5)
lamins. Keratins are found in epithelial cells and their


Carboxyl Termini of a- and 6-tubulin
6 Asp-Glu-Gln-Gly-Glu-Phe-Glu-Glu-Glu-Gly-Glu-Glu-Asp-Glu-Ala
a Glu Gly-Glu-Gly-Gly-Glu-Glu-Gly-Glu-Glu Tyr
Fig. 1-1 Comparison of the carboxyl termini of both a- and 6-tubulin from chick brain
(Valenzuela et al., 1981). Note the high glutamate content of both sequences.


8
derivatives such as skin and nails (Steinert and Roop,
1988). Desmin filaments are found mostly in muscle cells
while vimentin filaments are located in mesenchymal cells
(Steinert and Roop, 1988). Glial fibrillary acidic protein
is the basic building block of glial filaments which are
found in glial cells (Steinert and Roop, 1988). Peripherin
is located in the neurons of the peripheral nervous system
(Portier et al., 1984) while neurofilament proteins are
found in most neuronal cells (Steinert and Roop, 1988).
These filaments are composed of three subunits. The main
core of the filament is made of the L subunit, a 70 kDa
protein. The other two subunits are the M subunit and H
subunit which are 150 kDa and 210 kDa respectively. Both of
these subunits contain multiple phosphates on serine and
threonine residues.
The neurofilament proteins have some common structural
features with each other and all other intermediate filament
proteins. Each has a 40 kDa conserved rod domain believed
to be derived from a common ancestral gene (Weber et al.,
1983). This common rod-shaped region is rich in alpha
helical content and possesses a very conserved epitope that
reacts with a mouse monoclonal antibody that recognizes all
intermediate filament proteins (Pruss et al., 1981). This
40 kDa region has been conserved and is essential for
intermediate filament assembly (Steinert et al., 1981;
Geisler and Weber, 1981). Each intermediate filament
protein contains hypervariable regions that flank the
central rod region. These hypervariable regions form the


9
amino and carboxyl termini of the proteins also known as the
head and tail regions respectively. It is the variation in
these regions that distinguishes each individual
intermediate filament protein. The difference between
neurofilament proteins and other intermediate filament
proteins like desmin and vimentin is the tail region.
Normally the tail region is only approximately 5 kDa in mass
but is over 55 kDa in the H subunit of neurofilaments and 50
kDa in the M subunit. It is the tail regions of the M and H
subunits (see Fig. 1-2) that contain many of the
phosphorylation sites found in these proteins (Julien and
Mushynski, 1983). The tail region of the L subunit is
considerably shorter than the M or H subunits and is rich in
glutamate content which may be important in MAP binding
similar to the glutamate rich carboxyl termini of alpha and
beta tubulin. The tail regions are considerably less
conserved across species lines.
Neurofilament Function
The neurofilament proteins are found mostly in axons as
opposed to the cell body and dendrites. Once assembled into
polymers, neurofilaments do not easily dissociate (Giesler
and Weber, 1981; Moon et al., 1981). In fact, they are
insoluble in aqueous buffers and their purification relies
on solubilization with high concentrations of urea
(Tokutake, 1984). Upon removal of the urea by dialysis, the
filaments reassemble. Filament formation after dialysis
from urea is not restricted to the neurofilament triplet
(i.e. neurofilaments composed solely L, M, and H subunits),


10
NF-L
Coilia Coil 1b Coil 2 Tail a E segment
NF-m ! k
Colli Coil 2 Talla El KSP1 E2 KSP2 KE including SP segments
Coil 1 Coil 2 Talla E and KSP agmant* KEP segment
Fig. 1-2 Structural organization of the neurofilament
triplet proteins. All three proteins contain coil-coil
regions common to all intermediate filament proteins.
Differences are seen in the amino terminal heads and
carboxyl terminal tails. The phosphorylation sites in NF-M
and NF-L are in the KSP-rich sequences.


11
but for each individual subunit when purified from the other
two subunits can also be assembled by this method although
the filaments are not as structurally intact for the M and H
subunits (Tokutake et al., 1984). Unlike the case of facile
microtubule assembly/disassembly, intermediate filament
proteins form long-lived structures that provide the only
known function of these proteins, namely stabilization and
maintenance of the axon's structural integrity and caliber.
Neurofilaments increase the size of an axon by adding to the
volume occupied by the cytoskeleton (Lasek et al., 1983).
The contribution to axonal diameter or caliber, has a direct
effect on axonal function because the larger the axonal
diameter, the faster the action potential travels down the
axon. Also, this increase in size permits specialization of
nerve cells because the largest axons have the ability to
excite several target nerve cells at one time (Zucker,
1972). This can synchronize an entire population of cells
to act coordinately as with muscle cells to produce
movement. Axon size is very important to organisms which
must coordinate large muscles to produce rapid and essential
processes necessary to organismal survival. With a minimum
number of junctions, axon size is very important and
expression of the neurofilament genes can be crucial for
proper organismal behavior and survival (Bullock and
Horridge, 1965). Interestingly, some axons contain little
neurofilament proteins but still function properly
apparently as a result of increased microtubule content that


12
can replace neurofilaments in increasing the size of the
axon (Morris and Lasek, 1984).
While neurofilaments are required architecturally for
proper axon function, they are not required for such axonal
dynamics as axonal transport which is dependent on
microtubules. When an axon is disrupted by the neurotoxin
B,B'-iminodipropionitrile, the axonal inner structure is
rearranged with neurofilaments segregating to the outer
regions of the axon diameter and microtubules and organelles
segregating toward the center (Papasozomenos et al., 1981).
This model system has been used to examine interactions with
microtubules and neurofilaments where it has been shown that
cross-links exist between microtubules and neurofilaments as
well as within neurofilament networks themselves. An
antibody specific for MAP-2 has been found to co-localize
with both groups of filaments in iminodipropionitrile-
treated neurons implicating MAP-2 as a cross-linker within
and between these cytoskeletal systems (Papasozomenos et
al., 1985).
Microtubule-Associated Proteins
Introduction
MAPs are a varied class of proteins that have been
classified on the basis of their binding or modification of
microtubules. When one considers the diversity of
microtubule functions, it is not surprising that there
exists a great number of proteins that regulate temporal,
spatial, and metabolic controls of microtubule processes
(see Table 1-1). The first attempt to characterize proteins


Table 1-1
Major classes of microtubule-associated proteins
Protein
Subspecies
Subunit mass
(kDa)8
Primary
source
Properties
MAP 1
1A, IB
350
brain
thermolabile;projec-
tion on microtubule
Light chains
28,30
brain
associated with MAP-1
MAP 1C

350
brain
cytoplasmic dynein
MAP 2
2A,2B
270
brain
thermostable;proj ec-
tion on microtubule;
separable into pro
jection (235 kDa)
and binding (35 kDa)
domains phosphory-
lated;binds calmodu
lin
Type II CAMP- 53,39
dependent protein
kinase
Tau 3-5 55-62 brain
associated with MAP-2
projection domain
thermostable;number of
peptides depends on
age and species;
phosphorylated;binds
calmodulin
MAP 3
180
brain


Table 1-1 Continued
Protein
Subspecies Subunit mass
Primary
Properties
(kDa)a
source
MAP 4;210-kDa
HeLa MAP; 205-kDa
Drosophila MAP
3-4
200-240 depending
on species
cultured mammalian
cells; mouse tissues
(MAP 4); Drosophila
(205-kDa)
thermostable
125-kDa MAP

125
cultured mammalian cells
Chartins
69,72,80
cultured mammalian
cells;primary neurons
subspecies
thermolabile;phosphor-
ylated
STOPS
microtubules
140,72,56
brain
stable
associated with cold-
Sea urchin
MAPS

37,78,80
150,200,235
sea urchin eggs;
sea urchin spindles
spindle localization
Kinesin
""
110
134
squid axoplasm
sea urchin eggs
moves particles on
microtubles
Dynamin
100
calf brain
microtubule-activated
ATPase producing
movement of micro
tubules, bundles
microtubules
denatured mass of major polypeptides in each class as determined by SDS-PAGE.


15
associated with microtubule-networks was by Gibbons (1965)
who demonstrated that dynein could be selectively released
and then rebound to axonemes of Tetrahymena. Later,
Weisenberg (1972) observed that microtubules can be
assembled in vitro from crude brain extracts, and this
observation led to the identification of proteins associated
with polymerized microtubules. Through the use of
repetitive cycles of temperature-dependent assembly and
disassembly (Shelanski et al., 1973), quantitative MAPs or
MAPs that bind to microtubules in a defined molar ratio were
identified. The two most obvious classes of MAPs in this
category were the high-molecular-weight MAPs, MAP-1 and MAP-
2, and a family of polypeptides known as tau which has a
molecular mass of 55 to 68 kDa.
Unfortunately, the use of temperature-dependent cycles
has obscured the discovery of many less abundant, but
potentially very significant MAPs. During the course of
several temperature-dependent cycles much protein is lost,
both tubulin and MAPs. Only appoximately fifteen percent of
the microtubule-protein is yielded from bovine brain tissue
after three cycles. Also the use of nucleotides, both
adenosine- and guanosine triphosphate, increases the yield
of microtubule-protein by increasing the amount of tubulin
that polymerizes, but also influences MAP associations with
microtubules. For instance, the inclusion of adenosine
triphosphate in the first warming of the brain tissue
extract releases MAP-1C, a cytoplasmic retrograde dynein,
from the microtubule-lattice (Paschal et al., 1987b). This


16
protein was not characterized as a microtubule-associated
protein for years because it was routinely discarded during
the cycling of microtubule-protein.
One of the high-molecular-weight MAPs already mentioned
is MAP-1 which is actually a family of polypeptides at
appoximately 350 kDa. There are two closely spaced bands on
denaturing polyacrylamide gels that are MAP-la and MAP-lb.
Even though they are similar in molecular weight they
exhibit different monoclonal antibody reactivities and
produce different peptide digest patterns. These proteins
exhibit no preferential localization in brain tissue but
their purification from white matter as a family takes
advantage of the fact that other high-molecular-weight
contaminants (i.e. MAP-2) are enriched in gray matter
(Vallee, 1986). There is no available purification to date
for separating the subspecies of this family. MAP-1C on the
other hand has been purified to homogeneity and found to be
a microtubule-activated ATPase that can translocate
microtubules on glass slides and can translocate vesicles
along microtubules in an ATP-dependent fashion (Shpetner et
al., 1988). This very large protein has a native molecular
mass of approximately 450 kDa and scanning transmission
electron microscopy revealed that MAP-1C has a morphology
and mass of a two-headed dynein (Vallee et al., 1988).
Additionally, ultraviolet irradiation in the presence of
vanadate cleaved the protein into two fragments of about the
same size as those produced from flagellar dynein (Paschal
et al., 1987a).


17
Another microtubule-activated ATPase that can
translocate microtubules as well as intracellular vesicles
is kinesin. Kinesin was first discovered during studies of
organelle transport in the giant sguid axon, where
microtubules serve as tracks for the movements of organelles
(Vale et al., 1985a). Kinesin isolated from sguid axoplasm
can induce movement of carboxylated latex beads along
purified microtubules or gliding of microtubules on glass in
the presence of ATP (Vale et al., 1985b). The direction of
movement along the microtubules was anterograde or towards
the nerve terminal in an axon. This is exactly the opposite
direction of movement as MAP-1C (Paschal and Vallee, 1987).
Kinesin has since been found in a variety of organisms and
cell types from mammalian brain tissue (Brady, 1985) to
Drosophila melanoqaster (Saxton et al., 1988). This wide
distribution of kinesin suggests it may be involved in a
variety of microtubule-based motility systems in different
cell types. Immunolocalization studies in sea urchin eggs
and some mammalian cultured cells have demonstrated kinesin
is located in the mitotic spindle suggesting a role for
kinesin in mitosis (Scholey et al., 1985).
Kinesin is composed of two heavy chains of 120-124 kDa
and two light chains of 62-64 kDa and is a highly elongated
molecule with an axial ratio of appoximately 20:1 (Bloom et
al., 1988). Rotary shadowing of kinesin shows it is a rod
shaped molecule approximately 80 nanometers long. One end
of each kinesin molecule contains a pair of globular domains
while the opposite end is fan shaped (Hirokawa et al.,


18
1989). Monoclonal antibodies against the heavy chains stain
the globular structures while antibodies versus the light
chains stain the fan-shaped end (Hirokawa et al., 1989). A
60 kDa amino terminal section of the heavy chain corresponds
to the globular head region and contains the nucleotide-
dependent microtubule binding activitity and is thought to
the motor domain (Yang et al., 1989).
Among the lesser known MAPs is the MAP-4 class which
consists of a series of MAPs with molecular masses around
200-240 kDa depending on the species. It is found in almost
all cultured mammalian cells, in mouse tissues, and in
Drosophila melanogaster. MAP-4 shares the property of
thermostablity with MAP-2, a feature that is rare in
proteins of their size. The overall structure and binding
to microtubules of a few MAP-4 types are just starting to be
analyzed. Another group of not very well characterized MAPs
are STOPs which confer cold-stability on microtubules
(Margolis and Rauch, 1981). Normally, microtubules
depolymerize when exposed to cold temperatures; in the
presence of STOPs, the microtubules are stable. These
tubules can be depolymerized by calmodulin and low calcium
concentrations, and it has been shown that these proteins
are all retained on a calmodulin affinity resin (Job et al.,
1982) .
Tau Proteins
Among the best characterized MAPs to date are the tau
proteins, a family of closely related polypeptides. On
denaturing polyacrylamide gels the proteins exhibit


19
molecular masses of 55 to 70 kDa with usually four or five
distinct bands appearing. Tau is a phosphoprotein with
serines and threonines primarily phosphorylated. It is
located in neuronal tissues and restricted to axons of
neurons. Tau has been found in paired helical filaments and
in plaques from Alzheimer brain patients (Goedert et al.,
1988). It has the remarkable properties of being soluble in
2.5% (v/v) perchloric acid and being insoluble in 25% (v/v)
glycerol (Lindwall and Cole, 1984). Tau also is heat-
stable, and its purification takes advantage of this
property.
The source of heterogeneity in tau was unclear until
recently when it has been demonstrated that the different
tau polypeptides are the result of alternative splicing from
one mRNA transcript (Himmler, 1989). By the selective
splicing of specific exons from the transcript, a specific
tau translation product is synthesized. This work was
performed with bovine brain mRNA. The first tau sequence to
be identified was the murine system by Lee et al. (1988) who
showed two tau transcripts were made in vitro with differing
carboxyl termini. An interesting structural feature of the
transcripts noticed in the murine tau investigation was the
presence of three imperfect octadecapeptide repeats in the
center of the molecule. Each repeated sequence ended with a
proline followed by three glycines. All the repeats
contained serines and threonines, and they were rich in the
basic amino acids arginine and lysine. These investigators


20
hypothesized the repeats were important for tau binding to
microtubules.
Another group of investigators showed a proteolytic
fragment of tau could bind to microtubules (Aizawa et al.,
1988) A 14 JcDa chymotryptic fragment present in each
bovine tau polypeptide was found to bind to microtubules.
The amino terminus of this fragment was determined by Edman
sequencing which localized where the fragment was derived
from after comparison with the murine cDNA predicted
sequence. This proteolytic fragment contained two of the
three repeats believed to be involved in microtubule
binding. This was the first indication that not all the
repeats were necessary for tau binding to microtubules. An
interesting feature of bovine tau protein not elucidated by
the protein chemistry work of Aizawa et al. (1988) was shown
by the cDNA sequencing work of Himmler et al. (1989). They
determined that all the bovine forms of the tau polypeptides
contain four repeats rather than three as seen in the murine
forms. It is unclear what the significance of having four
repeats versus three repeats is although tighter or better
binding could be the result.
Some interesting work with the repeats of tau has been
done in vitro. Ennulat et al. (1989) has shown that
synthetic peptides corresponding to the first and second
repeats of murine tau protein can promote the polymerization
of tubulin. The third repeat has also been tested but
failed to promote tubulin polymerization into microtubules.
Himmler et al. (1989) also has demonstrated that a


21
polypeptide consisting of the four repeats can cosediment
with taxol-stabilized microtubules and he demonstrated that
just two repeats fused together also possessed this ability.
The importance of the cosedimentation data cannot be
overlooked, but the work of Ennulat et al., (1989) is more
significant because this was the first demonstration of a
synthetic peptide of a microtubule-associated protein
performing the same function as a MAP.
Interestingly, Aizawa et al. (1989) found a sequence
that is similar to one of the repeats of tau in the 190 kDa
adrenal gland-specific microtubule-associated protein. This
sequence could also promote the polymerization of tubulin in
vitro. An emerging theme in microtubule cytoskeletal
research is that a group of similar sequences in some MAPs
are responsible for promoting MAP-tubule interactions.
Microtubule-Associated Protein-2
Microtubule-associated protein-2 (MAP-2) is a very
large protein specific for neuronal tissue and restricted to
dendrites. Its molecular mass on denaturing gels is
approximately 280 kDa but the predicted mass from its cDNA
is only about 200 kDa (Lewis et al., 1988). MAP-2 is very
similar to tau in that it is heat-stable, a phosphoprotein,
and the murine form contains a trio of imperfect
octadecapeptide repeats. MAP-2 also shows heterogeneity on
denaturing gels splitting into two high-molecular-weight
forms, MAP-2a and MAP-2b. It is unknown what the cause of
this heterogeneity is. The only known function of MAP-2 is
to polymerize tubulin into microtubules.


22
MAP-2 can be phosphorylated by a variety of kinases.
The cAMP-dependent protein kinase, calmodulin dependent
protein kinase, calcium/phospholipid-dependent protein
kinase, and protein kinase C have all been shown to use MAP-
2 as a substrate (Goldenring et al., 1985; Tsuyama et al.,
1986; Akiyama et al., 1986). MAP-2 can also be
phosphorylated by non-neuronal specific kinases such as the
insulin receptor kinase and an epidermal growth factor
stimulated kinase (Kadowaki et al., 1985; Hoshi et al.,
1988). When MAP-2 is isolated by standard cycling
procedures from brains it contains about 10 moles phosphate
per mole of MAP-2 (Tsuyama et al., 1986). About ten more
phosphates can be added with exogenous cAMP-dependent
protein kinase. Rat brain MAP-2, isolated immediately
following rapid in vivo heat-treatment, contains
approximately 46 moles of phosphate per mole of MAP-2
(Tsuyama et al., 1987). Such microwave treatment reduces
the activity of phosphoprotein phosphatases. The
phosphorylation state of MAP-2 has a direct influence on its
function. The more phosphorylated the protein is, the less
its affinity for tubulin is and the ability to promote
polymerization is reduced (Murthy and Flavin, 1983).
MAP-2 has very little organized secondary structure.
Circular dichroic measurements of MAP-2 revealed it
contained very little alpha-helix or beta-sheet (Hernandez
et al., 1986). This same study determined that MAP-2
possessed a highly elongated structure as analyzed by gel
filtration chromatography and analytical


23
ultracentrifugation. In addition, the predicted structure
from the cDNA studies of Lewis et al., (1988) also revealed
little organized secondary structure.
Surprisingly, when MAP-2 is digested with various
endoproteases, only two major fragments are usually
produced: the first, a small fragment approximately 28-36
kDa in mass and the second, a large 240 kDa fragment. The
small fragment contains the microtubule-binding site of MAP-
2 (Vallee, 1980; Flynn et al., 1987), and the large fragment
is known as the projection domain because it is seen in
electron micrographs before protease treatment protruding
from the microtubule wall but is absent after protease
digestion (Vallee and Borisy, 1978). This limited digest
pattern with several proteases suggests some higher order of
structure. A completely random structure would give greater
heterogeniety in protease digests. If trypsin or
chymotrypsin are employed, the digest patterns are more
complex but the microtubule-binding products are 34-36 kDa.
The projection domain contains a majority of the
phosphorylation sites. The significance of these
phosphorylation sites in the projection domain remains
unclear. One known function of the projection domain is
that it contains a binding site for the regulatory subunit
of cAMP-dependent protein kinase (Vallee, 1986). This
suggests that MAP-2 may be associated with a protein kinase.
This kinase could phosphorylate the microtubule-binding
domain thereby modulating the affinity of MAP-2 for
microtubules.


24
The entire cDNA structure of murine MAP-2 recently was
reported by Lewis et al. (1988), and it showed that MAP-2
possessed three imperfect octadecapeptide repeats similar to
murine tau. Secondary structure predictions from the cDNA
confirmed the findings of Hernandez et al. (1986) showing
MAP-2 contained little alpha-helical content or beta-pleated
sheet content. A synthetic polypeptide of 100 amino acids
of MAP-2 sequence containing the first and second repeats
was shown to bind to MAP-stabilized microtubules and could
cycle with these microtubules (Lewis et al., 1988). This
report revealed extensive homology of MAP-2 with tau
especially in the carboxyl termini of both proteins as
depicted in Fig. 1-3. It will be interesting to know the
bovine MAP-2 sequence to see if it contains three
octadecapeptide repeats like murine MAP-2 or four repeats as
in the bovine tau proteins.
Recently a third form of MAP-2 has been found in
addition to MAP-2a and b (Garner et al., 1988). This
protein is a 70 kDa heat-stable MAP that cross-reacts with
MAP-2 antibodies but is only expressed in neonatal and
juvenile rats. Northern blots of different developmental
stages in rat show a 6 kilobase mRNA in neonatal brain
tissue and a 9 kilobase mRNA in adult brain tissue when
probed with a MAP-2 specific cDNA (Garner and Matus, 1988).
When this 70 kDa protein, now termed MAP-2c, was sequenced,
the sequence was the same as MAP-2a and b except for a 1,372
amino acid deletion corresponding to the central section of
the adult form (Papandrikopoulou et al., 1989). This was


1610
PPSYSSRTPGTPGTPSYPRT
* * * *
123
GERSGYSSPGSPGTPGSRSR
1670
DLKNVKSKIGSTDNIKYOPK
***** ****** * **
183
DLKNVRSKIGSTENLKHOPG
1730
KLDFKEKAOAKVGSLDNAHH
***** * ***** *
243
KLDFKDRVOSKIGSLDNITH
1790
AS PRRLSNVSSSGSINLLES
*** ********** *
303
TS PRHLSNVSSTGSIDMVDS
1630
PGTPKSGILVPSEKKVAIIR
**** *
143
TPSLPTPPTREP-KKVAWR
1690
GGQVQIVTKKIDLSHVTSKC
** **** *** *****
203
GGKVOIVYKPVDLSKVTSKC
1750
VPGGGNVKIDSQKLNFREHA
****** *** ** *** *
263
VPGGGNKKIDTHKLTFRENA
1810
PQLATLAEDVTAALAKQGL
******* * ******
323
PQLATLADEVSASLAKQGL
1650
TPPKSPATPKQLRLINQPLP
******* * *
163
TPPKSPASKSRLQTAPVPMP
1710
GSLKNIRHRPGGGRVKIESV
*** ** **** *
223
GS LGNIHHKPGGGOVEVKSE
1770
KARVDHGAEIITQSPSRSSV
** ****** ** *
283
KAKT DHGAEIV Y KS PW S GD
Fig. 1-3 Comparison of the carboxyl termini of murine MAP-2 (upper sequence) and murine
tau protein (lower sequence). The asterisks indicate an exact match and the underlined
sequences refer to the imperfect octadecapeptide repeats. The hyphen at position 155 of
tau represents a space created to allow for better alignment of the sequences.


26
due to alternative splicing of the mRNA from one gene of
MAP-2. The protein still contained the carboxyl terminus
ofadult MAP-2 with the triad of imperfect repeats. It is
currently unclear what the significance of the embryonic and
adult forms is, although a reduced degree of cytoskeletal
cross-linking during axonal and dendritic growth with the
embryonic form is a possibility (Papandrikopoulou et al.,
1989) .
Altogether, the findings on the structural organization
of MAP-2 can be depicted as shown in Fig. 1-4. This figure
shows the section spliced out of mature MAP-2 creating the
MAP-2c form. The binding site for the regulatory subunit of
cAMP-dependent protein kinase is located at the amino
terminus and the proposed microtubule-bundling domain is
located at the extreme carboxyl terminus of the molecule.
When this bundling sequence is removed, MAP-2 loses its
ability to cause microtubule bundling (Lewis et al., 1989).
This sequence contains homology to leucine zipper proteins
and can be functionally replaced by these leucine zippers
such as the one contained in GCN4. It has been hypothesized
that the bundling domain at the carboxyl terminus of MAP-2
interacts with another MAP-2 carboxyl terminus causing the
microtubules to bundle.
Proposal
The aim of this study was to determine how MAP-2
interacted with two different cytoskeletal components,
neurofilaments and microtubules. This was to be
accomplished through the use of proteolytic digests to


27
MAP-2 Structure:
Microtubule
Protein Kinase
R2C2
Binding Site
Hinge Region
rv
M,
M2
1 -
1 1
Absent in MAP-2C
\
Microtubule x Projection
Binding
Fragment
Bundling Domain
Arm
M1 =VKSKIGSTDNIKYQPKGG
M 2 = VTSKCGSLKNIRHRPGGG
M3=AQAKVGSLDNAHHVPGGG
Fig. 1-4 Summary of MAP-2 structure in relation to a
microtubule. The amino terminus of MAP-2 contains a binding
site for the regulatory (R) subunit of cAMP-dependent
protein kinase. The carboxyl terminus contains the
imperfect repeats as well as a proposed bundling sequence.
The hinge region contains a protease sensitive site.


28
determine which fragment(s) of MAP-2 could interact with the
different polymers. Once the fragment was determined, a
structural characterization of its biochemical properties
was to be carried out. This included a determination of its
isoelectric point, amino acid composition, and amino
terminal analysis. With the availability of the entire
seguence of murine MAP-2, internal sequences responsible for
promoting tubulin polymerization in vitro were to be
identified. In addition to tubulin polymerization, MAP-2
sequences responsible for displacement of high-molecular-
weight MAPs from microtubules were to be identified.


CHAPTER 2
INTERACTIONS OF MAP-2 WITH TUBULIN
AND NF-L
Introduction
Neuronal cytoplasm is highly organized, and both
microtubules and neurofilaments run parallel with respect to
the axon's longitudinal axis in a manner suggesting
microtubule-to-neurofilament cross-linking (Wuerker and
Palay, 1969; Ellisman and Porter, 1980; Hirokawa, 1982). In
vitro observations indicate that microtubules interact with
neurofilaments, and MAPs can enhance the attainment of high
solution viscosity and/or gelation (Runge et al., 1981;
Aamodt and Williams, 1984b; Minami and Sakai, 1983;
Letterier et al., 1982). Aamodt and Williams (1984a) used
falling-ball viscosmetry to demonstrate the occurrence of an
optimal MAP level in plots of viscosity/gelation versus MAP
concentration; they likened this MAP concentration profile
to that of bivalent antibody cross-linking in
immunoprecipitin formation. Previous studies have traced
this apparently optimal MAP profile to the presence of
endogenous GTPase activity, and the inhibition of cross
linking/gelation at high MAPs can be eliminated with a GTP-
regenerating system (Flynn and Purich, 1987). The
requirement of GTP is to maintain microtubule stability as
microtubules will disassemble after all the GTP is converted
to GDP. Nonetheless, high-molecular-weight MAPs do bind to
29


30
microtubules (Kim et al.f 1979; Vallee, 1982; Purich and
Kristofferson, 1984) and neurofilaments (Runge et al., 1981;
Aamodt and Williams, 1984b; Minami and Sakai, 1983;
Letterier et al., 1982), and some interactions of the 280
kDa neuronal MAP-2 have been explored by limited proteolytic
fragmentation. Tubule binding is restricted to a 34-36 kDa
tryptic or chymotryptic fragment of MAP-2, and the remaining
240 kDa component corresponds to the lateral projections
observed in electron micrographs of microtubules decorated
with MAP-2 (Kim et al., 1979; Vallee and Borisy, 1977).
During the course of studies on neurofilament-
microtubule-MAP-2 interactions I sought to localize the
site(s) of neurofilament binding with respect to the tubule
binding and -projection domains of MAP-2. A thrombin
cleavage technique was developed to obtain these MAP-2
fragments in higher yields than that obtained with trypsin
or chymotrypsin. Interestingly, a 28 kDa tubule-binding
domain was found to contain a neurofilament-binding site.
My studies also suggest that this binding interaction has
considerable ionic character, as suggested by isoelectric
point determinations of the MAP-2 fragment.
Materials and Methods
Materials
Bovine thrombin (catalog number, T 4648) and the
catalytic subunit of cAMP-dependent protein kinase were
purchased from Sigma. Ultrapure ammonium sulfate and urea
were purchased from Schwarz-Mann, and carboxymethyl Sephadex
from Calbiochem. [32P]ATP (specific activity > 7000


31
Curies/mmol) was an ICN product, and Ampholines were
obtained from LKB. Assembly buffer for preparation of
microtubule-protein contained 0.1 M piperazine-N, N'-bis[2-
ethanesulfonic acid], 1 mM ethyleneglycol-bis[B-aminoethyl
ether]-N,N,N1,N1-tetracetic acid, and 1 mM magnesium
sulfate.
Preparation of proteins
Bovine brain microtubule-protein was prepared by the
procedure of Shelanski et al., (1973). Neurofilaments were
prepared from fresh bovine spinal cord by the method of
Delacourte et al., (1980) as modified by Letterier et al.,
(1982). Neurofilament triplet protein was prepared as
described by Tokutake et al., (1983), and the NF-L subunit
of neurofilaments was purified according to the method of
Geisler and Weber (1981). Tubulin was separated from MAPs
by the phosphocellulose method of Weingarten et al., (1975).
MAP-2 preparation and phosphorylation
MAP-2 was purified by the method of Herzog and Weber
(1978), concentrated by ammonium sulfate precipitation, and
phosphorylated by the catalytic subunit of cAMP-dependent
protein kinase prior to gel filtration chromatography.
Typically 500 units of kinase was dissolved in 0.025 ml
dithiothreitol (50 mg/ml), incubated at room temperature for
10 minutes, and used immediately with 1.5 millicuries
[32P]ATP, 0.02 mM unlabeled ATP, for 30 minutes at 37 C in
the presence of approximately 40 mg heat-stable MAPs. The
MAPs were separated on a BioGel A-1.5M column with the MAP-
2 fractions pooled and concentrated in a dialysis bag


32
against dry carboxymethyl Sephadex at 4C. This purified
[32P]MAP-2 was clarified by centrifugation at 130,000 x g
for 25 minutes in a Beckman Airfuge prior to digestion and
incubations with cytoskeletal proteins.
Digestion of MAP-2 with thrombin
Purified and radiolabeled MAP-2 (50,000 CPM//xg) was
incubated at 0.4 mg/ml with 4 units/ml thrombin. To
determine the optimum time of digestion, aliquots were taken
at 5 minute intervals from 0 to 30 minutes. Once an optimum
time of 30 minutes was determined, all digestions of MAP-2
prior to incubation with cytoskeletal proteins were
conducted for 30 minutes and quenched by the addition of 1
mM phenylmethylsulfonyl fluoride and incubation on ice.
Sedimentation of polymerized protein
Thrombin-digested or undigested MAP-2 was incubated
with neurofilaments for 10 minutes at 4C, or microtubules
for 30 minutes at 37C. The incubations were then layered
over 20% (w/v) sucrose in assembly buffer and centrifuged at
130,000 x g for 20 minutes. Supernatant fractions were
discarded and the pellet fractions were washed with 1 mg/ml
bovine serum albumin and 0.1% (v/v) Triton X-100 and
resuspended in 8 M urea. Aliquots of both supernatant and
pellet fractions were analyzed for radioactivity by liquid
scintillation spectrophotometry, and an equal number of
counts were loaded on 7-17% (w/v) polyacrylamide gels.
Polyacrylamide gel electrophoresis
Sodium dodecyl sulfate gel electrophoresis was carried
out as described by Laemmli (1970), and nonequilibrium pH


33
gel electrophoresis (NEPHGE) was performed by the method of
Roberts et al., (1984). For the NEPHGE gels radiolabeled
MAP-2 was digested with thrombin and the digestion was
quenched by the addition of an equal volume of 9.3 M urea,
0.5% (w/v) dithiothreitol and 2% (v/v) Nonidet NP-40 in 5 mM
potassium carbonate. The digests were loaded onto "NEPHGE"
tube gels with 1% (v/v) Ampholine 9-11 and 2% (v/v)
Ampholine 3.5-10, and run for 2000 volt-hours. Gels were
stained with coomassie blue, destained, dried under vacuum,
and exposed to Kodak X-AR5 film at -80C.
Results
Binding to microtubules and neurofilaments
In view of the high molecular weight of MAP-2,
proteolytic fragmentation by trypsin or chymotrypsin has
proven to be useful in defining the MAP-2 domain(s)
interacting with other cytoskeletal components (Olmsted,
1986). Vallee (1980) first demonstrated that MAP-2 can be
fragmented into 35 and 240 kDa components by chymotrypsin or
trypsin. The smaller fragment contains the microtubule
binding domain, and the larger is designated as the
projection-arm domain. While these protease cleavage
products have been very useful in many investigations of
microtubule self-assembly, chymotryptic and tryptic cleavage
do not yield stable limit polypeptides, and the stability of
such proteolytic fragments is quite limited, leading to the
loss of the initially cleaved domains and the ability of
these fragments to stimulate microtubule assembly. In a
survey of the action of other proteases, I observed that


34
thrombin, an arginine-specific serine protease,
predominantly yielded MAP-2 fragments of 28 kDa and 240 kDa.
MAP-2 cleavage can be readily assessed by SDS gel
electrophoresis of [32P]MAP-2 because this protein is
extensively phosphorylated (Theurkauf and Vallee, 1983). As
seen in Fig. 2-1 this 28 kDa fragment is very stable and
very resistant to further digestion over the time points
shown. Indeed, phenylmethanesulfonyl fluoride at 1.0 mM
final concentration blocked any further degradation over a
five to seven day period at 4C.
This development has allowed for probing with much
greater ease the interactions of MAP-2 fragments with
neurofilaments and microtubules. The basic approach is to
determine which proteins or protein fragments cosediment
with assembled microtubules or neurofilaments using
ultracentrifugation and subsequent electrophoretic analysis.
First MAP-2 was enzymatically phosphorylated with the cAMP-
dependent protein kinase to a level of 1 mole of added
phosphate per mole of MAP-2 based on the conditions of
Letterier et al., (1982). After purification of the
radiolabeled protein by gel filtration chromatography, the
MAP-2 was concentrated, and aliquots were digested with
thrombin. Next, the thrombin-digested fragments (indicated
by the plus sign) or undigested MAP-2 (indicated by the
minus sign) were incubated with microtubules or
neurofilaments under the conditions listed in Fig. 2-2. The
polymerized and unpolymerized cytoskeletal proteins were
separated into pellet [p] and supernatant [s] fractions by


35
Fig. 2-1 Thrombin digestion of radiolabeled MAP-2.
[32P]MAP-2 was incubated at 37 C with 4 units/ml thrombin
for the indicated time in minutes. The digestion was
quenched by heating at 100C for 5 minutes in the presence
of sodium dodecyl sulfate and the products resolved on a 15%
(w/v) polyacrylamide gel. The gel was then dried under
vacuum and expose to Kodak X-AR 5 film.


36
ultracentrifugation. Because the projection and tubule
binding domains do not contain identical phosphorylation
sites, a constant total amount of radioactivity was applied
for each electrophoretic sample. Lanes 1-4 of the coomassie
stained gel in Fig. 2-2 and the corresponding lanes of the
autoradiogram in Fig. 2-3 demonstrate that only the 28 kDa
thrombin-produced fragment of MAP-2 binds to neurofilaments.
The next four lanes in both Fig. 2-2 and Fig. 2-3
demonstrate that this thrombin fragment behaves as the so-
called microtubule-binding domain of MAP-2 as it binds to 1
mg/ml of taxol-stabilized microtubules composed solely of
tubulin. In the absence of neurofilaments or microtubules
the 28 kDa fragment remained in the supernatant fraction
even after ultracentrifugation as seen in lanes 9-12 of Fig.
2-3. Indeed, the entire pellet fraction was used for the
electrophoretic analysis in lanes 10 and 12 of Fig. 2-3, and
virtually no high-molecular-weight or fragmented MAP-2
cosedimented without neurofilaments present. These
observations verified that the fragment is only sedimentable
as a result of interactions with either neurofilaments or
microtubules.
Next, I sought to determine the neurofilament
protein(s) interacting with MAP-2 or the 28 kDa fragment.
Earlier work by Miyata et al., (1986) and Heimann et al.,
(1985) demonstrated MAP binding to the low-molecular-weight
subunit of neurofilaments. A second series of binding
assays were conducted and Fig. 2-4 shows the coomassie blue
staining pattern and Fig. 2-5 shows the corresponding


37
NF NoNF
Protein Tubulin or Tb
M /++ \/+ +V+ + M
\S p s pAs p S paS P s P/
12 34 56 78 9 10 II 12
Fig. 2-2 Binding of MAP-2 or MAP-2 fragments to
neurofilaments or tubulin. A coomassie blue stained
gradient 7-17% (w/v) polyacrylamide gel is shown.
Radiolabeled MAP-2 was incubated with (+) or without (-)
thrombin as described in Fig. 2-1, quenched with 1 mM
phenylmethylsulfonyl fluoride and incubated with 2 mg/ml
neurofilament protein or 1 mg/ml taxol-stabilized tubulin
with 1 mM guanosine triphosphate. Samples were then handled
as described in "Methods. M refers to molecular weight
markers, s to supernatant fraction, and p to pellet
fraction.


38
NF No NF
Protein Tubulin or Tb
4 + -A4 + _A/h+--\
r rr ii | -otk
116K
-97.4 K
-66.2K
-42.7 K
4
\S p S P/\S p S P/\S p S P/
Fig. 2-3 Autoradiogram of MAP-2 and MAP-2 fragment binding
to cytoskeletal protein. The coomassie blue stained
polyacrylamide gel shown in Fig. 2-2 was soaked in 25% (v/v)
glycerol for 30 minutes after destaining and dried under
vacuum. The dried gel was exposed to x-ray film for 4 hours
without intensifying screens.


39
autoradiogram. Lanes 3, 4 and 7, 8 of Fig. 2-5 verified
that uncleaved MAP-2 binds to the neurofilament triplet and
to filaments composed solely of L subunit. The data shown
in lanes 1, 2 and 5, 6 of Fig. 2-5 extend the earlier
observations by clearly demonstrating the binding of the 28
kDa fragment to the neurofilament triplet and L component of
neurofilaments. It should be noted that only trace levels
of tubulin are evident in the 50-55 kDa molecular weight
range in the gels shown in Fig. 2-2. Much higher levels of
tubulin are required for binding of MAP-2 to assembled
tubules. This suggests that binding depends on presence of
neurofilaments and does not require tubulin or assembled
microtubules for binding.
Determination of the isoelectric point of the 28 kDa
fragment
Tubulin and neurofilament proteins contain negatively
charged regions that may be important in MAP binding
(Olmsted, 1986). The data presented in the preceding
section demonstrate that the 28 kDa fragment of MAP-2 binds
to both cytoskeletal organelles. Accordingly, I tried to
use conventional isoelectric focusing to determine the
isoelectric point of the radiolabeled fragment. This
consistently failed at all ranges of ampholytes, and the
fragment never migrated into the first dimension of a two
dimensional gel. This is indicative of very basic proteins.
In order to estimate the isoelectric point of very basic
neurofilament subunit proteins the Nonequilibrium pH gel
electrophoresis technique is used (Roberts et al., 1984).
When this technique was tried, the fragment readily migrated


40
NF NF
Triplet Low NoNF
M /++ \/++ \4+__\ M
\s P s PAS p S P7\S p s P /
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 2-4 MAP-2 binding to purified neurofilament triplet
protein or the L subunit of neurofilaments. A coomassie
blue stained gradient 7-17% (w/v) polyacrylamide gel is
shown. Radiolabeled MAP-2 was incubated with thrombin as
described in Fig. 2-2 and in "Methods", and incubated with
either 2.2 mg/ml neurofilament triplet or 1.1 mg/ml L
subunit at 4C for 10 minutes. Nomenclature is the
in Fia. 2-2.
same as


41
NF NF No
Triplet Low NF
4T^/FT^- r i:ri ?' -2ook
116K
97.4 K
66.2K
-42.7 K
\s p s P/\s p s p/\s psp/
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 2-5 Autoradiogram of MAP-2 and MAP-2 fragment binding
to Neurofilament triplet protein and L subunit. The
coomassie blue gel shown in Fig. 2-4 was soaked in 25% (v/v)
glycerol for 30 minutes after destaining and dried under
vacuum. The dried gel was exposed to x-ray film for 4 hours
without intensifying screens.


42
into the first dimension of the gel as seen in Fig. 2-6, and
it migrated between two very basic isoelectric point
markers, ribonuclease (pl=9.3) and lysozyme (pl=10.5-11.0).
The thick arrow denotes the position of the 28 kDa fragment
in the gel. From this migration pattern, the isoelectric
point of the 28 kDa fragment of MAP-2 was estimated to be
approximately 10. The migration of the trace-labeled
fragment in both dimensions also indicated that the
phosphorylation conditions do not lead to significant
heterogeneity in overall ionic charge or molecular weight.
Furthermore, the dephosphorylated form will necessarily
display an even higher isoelectric point. The large smear
at the top right side of the gel is the projection domain
and further breakdown products of this domain. These
fragments are acidic and do not readily migrate into the
gel.
Discussion
The findings presented in this chapter indicate that
neurofilaments, a specific class of intermediate filaments,
interact with MAP-2 in the region corresponding to the 28
kDa microtubule-binding domain. In earlier work with actin,
Sattilaro (1986) reached a similar conclusion about the
binding of chymotryptic fragments of MAP-2 to polymerized
actin. Likwise, this same fragment of MAP-2 constitutes the
microtubule-binding domain (Vallee, 1980). Thus, all three
major cytoskeletal classes (i.e. microfilaments,
intermediate filaments, and microtubules) interact with a
common structural region of MAP-2. In view of our estimated


43
NEPHGE
Fig. 2-6 Determination of the isoelectric point of the 28
kDa fragment of MAP-2. An autoradiogram of a two
dimensional gel is shown. The first dimension is
Nonequilibrium pH gel electrophoresis and the second
dimension is sodium dodecyl sulfate polyacrylamide gel
electrophoresis with a 7-17% (w/v) gradient of acrylamide.
Protein molecular weight/isoelectric point reference
standards were: 1) phosphorylase b; 2) bovine serum albumin;
3) carbonic anhydrase; 4) soybean trypsin inhibitor; 5)
ribonuclease and 6) lysozyme.


44
isoelectric point value of 10 for the 28 kDa component, all
of these MAP interactions must be strongly influenced by
electrostatic charge. Vallee (1982) has clearly
demonstrated that 0.35 M sodium chloride can remove MAPs
from taxol-stabilized microtubules, and this observation is
also in harmony with the notion of ionic interactions
between tubules and MAPs. Furthermore, it has been shown
that increased phosphorylation of MAP-2 reduces its ability
to promote tubulin polymerization (Murthy and Flavin, 1983).
My use of trace labeling of heat-stable MAPs with
[32P]ATP and protein kinase reinforces the general utility
of this method as first applied by Heimann et al., (1985).
The radiolabeled low-molecular-weight thrombin fragment of
MAP-2 is more stable than that obtained with trypsin and
chymotrypsin, and this stability may also facilitate studies
of the stoichiometry and dissociation constants for fragment
binding to microtubules, neurofilaments, or actin. Other
more approximate methods using intact MAPs and either
densitometry or pelleting of assembled cytoskeletal elements
(Miyata et al., 1986; Heimann et al., 1985) still require
refinement and/or verification.
Finally, the thrombin results underscore the facility
and generality of serine protease of MAP-2 into low- and
high-molecular-weight fragments. This behavior is
reminiscent of proteolytic action on myosin. Furthermore,
intact MAP-2 has an isoelectric point of 5.4 (Berkowitz et
al., 1977), whereas I found that the tubule/filament-binding
domain has a value of 10. This suggests that there must be


45
significant acidic and basic charge localization in the
high- and low-molecular-weight fragments, respectively, of
MAP-2. Nonetheless, the biological significance of this
protein design feature awaits further understanding of the
role of MAP-2 in the cytomatrix.


CHAPTER 3
THE MICROTUBULE-BINDING FRAGMENT OF MAP-2:
LOCATION OF THE PROTEASE-ACCESSIBLE SITE
Introduction
The thrombin protease digestion of MAP-2 shown in Fig.
2-1 revealed a very stable 28 kDa fragment. This fragment
possessed the sequences responsible for cosedimentation of
MAP-2 with microtubules as well as neurofilaments. Since
this fragment contained the "active site" of MAP-2, I wanted
to learn more about its biochemical properties. It was
already known the fragment had a very basic isoelectric
point and it was suspected that the basic residues were
involved in the binding to the anionic termini of both alpha
and beta tubulin. A second interesting point concerning the
structure of the fragment was the specificity and stability
of the protease digestion products. The autoradiogram of
Fig. 2-1 showed no heterogeneity in the 28 kDa microtubule
binding fragment and limited heterogeneity in the very
protease-sensitive 240 kDa domain. However, limited
heterogeneity in the larger fragment could be observed by
the low resolving power of the 15% (w/v) polyacrylamide gel
for large molecular weight proteins. Nevertheless, the
specificity of the digestion in producing the 28 kDa
fragment was impressive. This specificity implied some
higher order structure in the MAP-2 molecule yet a previous
46


47
report on the circular dichroism spectrum of MAP-2 revealed
little alpha-helical or beta-pleated sheet content. If
there was no higher order structure, I would expect to see a
far more complex pattern of digestion. In order to resove
this paradox a large scale purification of the microtubule
binding fragment of MAP-2 was undertaken.
In addition, the structure of MAP-2 was probed by
protease digests with and without the presence of
microtubules. The low-molecular-weight digestion products
were isolated and their amino terminal composition checked
by Edman sequencing.
Materials and Methods
Materials
[32P]ATP (7000 Ci/mmol) was purchased from ICN along
with ultrapure grades of ammonium sulfate, sodium dodecyl
sulfate (SDS), acrylamide, and bis-acrylamide. Immobilon
was obtained from Millipore Corporation and coomassie
brilliant blue R-250 was from Serva. DEAE-Sephadex A-50 was
purchased from Pharmacia; and bovine thrombin,
trifluoroacetic acid, Mes buffer, and phenylmethane-
sulfonylfluoride (PMSF) were from Sigma.
Preparation of proteins
Bovine brain microtubule protein was prepared by the
procedure of Shelanski et al. (1973). MAP-2 was purified by
the method of Herzog and Weber (1978) and radiolabeled as
previously described (Flynn et al. 1987).


48
Preparation of heat-stable microtubule-binding fragments
Heat-stable microtubule-binding fragments were prepared
according to Vallee (1986) with the following modifications.
Thrombin was used instead of chymotrypsin at 8 U/ml and 37C
for 30 minutes to digest thrice cycled bovine microtubule-
protein at a concentration of 5 mg/ml. PMSF was added to 1
mM at the end of the digestion to stop proteolysis. The
assembled tubules were sedimented at 100,000 x g for 75
minutes at 30C and the pellet was resuspended in 0.75 M
NaCl and 1 mM dithiothreitol. After homogenization and
incubation on ice for 30 minutes the protein was heated in a
boiling water bath for nine minutes followed by cooling on
ice for 20 minutes. The resulting slurry was centrifuged
for 30 minutes at 15,000 x g at 4C. The supernatant
fraction contained heat-stable microtubule-binding fragments
from tau and MAP-2. These heat-stable binding fragments
were concentrated by ammonium sulfate precipitation and then
dialyzed against microtubule assembly buffer (100 mM Mes, pH
6.8, 1 mM EGTA, and 1 mM magnesium sulfate) at 4C with 1 mM
PMSF, and passed over a 1 ml DEAE-Sephadex A-50 column
equilibrated in the same buffer. The breakthrough fractions
were pooled and precipitated with 60% (w/v) ammonium
sulfate. After sedimentation, the precipitate was
resuspended in assembly buffer and used for HPLC analysis or
Immobilon blotting.


49
Preparation of the 28 kDa fragment of MAP-2 without
microtubules present during digestion
Purified MAP-2 corresponding to 0.6 mg at a
concentration of 2.0 mg/ml was digested with 5 U/ml thrombin
for 30 minutes at 37C. Digestion was quenched with 2 mM
PMSF and cooling on ice for 10 minutes. The digestion
products were passed over a 1 ml DEAE-Sephadex A-50 column
and handled as described in the preceding section.
HPLC purification of the microtubule-binding fragment of
MAP-2 and its amino acid analysis
High performance liquid chromatography was carried out
on a Hewlett Packard Model 1090a chromatograph, equipped
with a diode array detector. The ammonium sulfate
concentrated, microtubule-binding fragments were clarified
by centrifugation at 3000 x g for 5 minutes prior to loading
on a Waters Associates C-18 column, equilibrated in 0.1%
(v/v) trifluoroacetic acid. The protein was eluted with a
linear gradient from 0-50% (v/v) acetonitrile with 0.1%
(v/v) trifluoroacetic acid at a flow rate of 0.5 ml/min and
1 ml fractions were collected. The elution profile was
monitored at a wavelength of 220 nm because the fragments
are very low in aromatic amino acid content. Initially,
polyacrylamide gel electrophoresis was used to check the
composition of material in each peak. Fractions containing
the MAP-2 microtubule-binding domain were pooled, dialyzed
against 100 mM ammonium bicarbonate, lyophilized, and then
hydrolyzed in 6 N HC1 for 24 hours at 110C. Samples were
analyzed with a Beckman Model 6300 Amino Acid Analyzer.


50
SDS electrophoresis and blotting
Immobilon was handled according to the manufacturer's
instructions prior to electroblotting. The
polyvinylidenedifluoride membrane was wetted in 100% (v/v)
methanol for 5 minutes followed by soaking in distilled
water for another 5 minutes and then it was allowed to dry.
Ultrapure grades of SDS, acrylamide, and bis-acrylamide were
used to avoid blocking the N-terminus. The electrophoretic
samples for sequencing were heated to 80C for 5 minutes
after adding Laemmli sample buffer which contained ultrapure
SDS but no bromophenol blue dye. As an indicator of when
the electrophoresis was finished molecular weight markers
were run in adjacent lanes with dye present. To scavenge
any radicals that could possibly react with the samples,
0.1% (v/v) thioglycolate was added to the top chamber
buffer. A 12% (w/v) acrylamide SDS-gel containing heat-
stable microtubule-binding fragments was electrophoretically
transferred to the membrane in 10 mM CAPs, pH 10.0, 10%
(v/v) methanol for 6 hours at 70 volts. The membrane was
stained with Coomassie Brilliant Blue R-250, destained in
50% (v/v) methanol-10% (v/v) acetic acid. The blot was air
dried and stored at -20C in the dark until the sequencer
was available. The band of interest was excised with a
razor blade and sequenced in a gas-phase protein sequencer
(Applied Biosystems 470A protein sequencer) with on-line
phenylthiohydantoin analyzer at the Protein Chemistry core
facility.


51
Results
Site of thrombin cleavage
In order to gain more information about this site of
facile thrombin cleavage, a high-yield isolation method was
developed for amino acid analysis and sequencing
experiments. I again employed thrombin, but digested
assembled three-cycle microtubule-protein (i. e.. tubulin and
MAPs), followed by heat-treatment of the resulting pellet to
remove all tubulin and heat-labile MAPs. Then, DEAE-
Sephadex ion-exchange chromatography was used to separate
the heat-stable microtubule-binding fragments from any high
molecular weight digestion products. This ion-exchange
chromatography step was a key part of the purification and
took advantage of the basicity of the microtubule-binding
fragments as they did not interact with the resin while any
incompletely digested MAPs and any contaminating projection
domain did interact with the resin very strongly. This is
shown in Fig. 3-1. The breakthrough fractions from the
DEAE-Sephadex chromatography contained the microtubule
binding fragments of the heat-stable proteins MAP-2 and tau
(see lane 5) resulting in greater heterogeneity than that
seen in Fig. 2-1 where purified MAP-2 was digested. I also
found that a tau monoclonal antibody recognized the upper
bands in lane 5, identifying them as putative digestion
products of tau.
At this point in the purification scheme, I observed
four closely spaced bands on a gel ranging from 28 to 36


52
M 1 2 3 4 5
Fig. 3-1 Purification of heat-stable microtubule-binding
fragment of MAP-2 and tau. Microtubule-protein was digested
with thrombin as described under "Methods". The coomassie
blue stain of a 12% (w/v) polyacrylamide gel shows the
purification during its various stages. Lane 1 is before
cleavage; 2, after cleavage; 3, supernatant after
centrifugation; 4, heat-stable protein; 5, after DEAE-
Sephadex chromatography; 6, a larger amount of sample 5. M
corresponds to molecular weight markers.


53
kDa. Many different ion-exchange resins were tried to
separate these bands such as strong and weak cation
exchangers, and hydroxyapatite chromatography, but all were
unsuccessful. A two-dimensional gel using the NEPHGE
technique in the first dimension was run and all four
fragments were found to be very basic but each fragment was
slightly less basic with increasing molecular weight. This
indicated that the use of ion-exchange resins to separate
the fragments was probably futile and another means of
separation was necessary. The microtubule-binding fragments
in lane 5 of Fig. 3-1 can be readily separated by reverse-
phase HPLC as shown in Fig. 3-2 where SDS gel
electrophoresis revealed that peak C corresponded to the 28
kDa component. This lane also revealed that there is only
minor contamination by a faster migrating component.
Fraction C was subjected to acid-catalyzed hydrolysis and
amino acid analysis, and these results are listed in Table
3-1. I had previously reported this fragment had an
unusually high isoelectric point in comparison with intact
MAP-2 (Flynn et al. 1987), and the amino acid analysis
confirmed this observation. The fragment is comprised of
nearly 14 mole percent in lysyl and arginyl residues.
Curiously it contains a higher than usual proline content.
The analysis also confirmed the fact that this fragment was
low in aromatic amino acids as it contained only two
tyrosine residues and three phenylalanine residues. This
low content of aromatic residues makes it very difficult to
monitor its purification at 280 nm as is usually done for


Absorbance, 220 nm
54
Time after Injection, min
Fig. 3-2 HPLC purification of the microtubule-binding
fragment of MAP-2 digested with microtubules present. The
MAP-fragments seen in lane 5 of Fig. 3-1 were separated by
reverse phase HPLC and the fractions resolved by a 12% (w/v)
polyacrylamide gel (see inset) which is coomassie stained.


55
TABLE 3-1
Amino acid composition of the 28 kDa MAP-2 fragment
Amino Acid
24 h Hydrolysate
(mol%)
Estimated
Residues/mol
Asx
in

CO
21
Thr
5.2
14
Ser
9.7
24
Glx
9.2
22
Pro
6.9
18
Gly
9.0
24
Ala
7.5
18
Val
5.5
15
Met
0.2
0
He
4.7
12
Leu
8.1
20
Tyr
0.8
2
Phe
1.4
3
Lys
9.6
25
His
2.9
7
Arg
4.3
11


56
proteins. Also the analysis was rich in its glutamate +
glutamine content as well as its aspartate + asparagine
content even though it has already been established the
fragment was very basic. This meant that most of these
amino acids were in the amide form rather than the
carboxylic acid form.
Amino-terminal sequence of the microtubule-binding fragment
Microsequencing techniques were employed with the 28
kDa fragment electroblotted from SDS-polyacrylamide gels to
a derivatized nylon screen (Immobilon). This allowed
further reduction in any contamination by other MAP
fragments such as that observed in the HPLC preparation.
The amino terminal sequence obtained was, Thr-Pro-His-Thr-
Pro-Gly-Thr-Pro-Lys-Ser-Ala-Ile-Leu-Val-Pro-Ser-Glu-Lys-
Lys, based on the results listed in Table 3-2. In the
absence of thrombin treatment, identical sequence
experiments with either electroblotted MAP-2, as well as
MAP-2 in solution, did not yield any phenylthiohydantoin
derivatized amino acids at detectable levels. Likewise,
experiments with the immobilized 240 kDa projection-arm
fragment yielded no sequence data. Protein samples failing
to yield detectable levels of amino acid derivatives were
subjected to acid-catalyzed hydrolysis and amino acid
analysis to assure that sufficient levels of protein for
sequencing had been employed. In all cases adequate levels
of protein were present above the detection limits for


57
TABLE 3-2
Amino-terminal sequence analysis of the 28 kDa
MAP-2 fragment
Cycle No.
Residue
(pmol)a
Cycle No.
Residue
(pmol)
1
Thr
74
11
Ala
103
2
Pro
131
12
He
74
3
His
15
13
Leu
144
4
Thr
42
14
Val
82
5
Pro
122
15
Pro
102
6
Gly
84
16
Ser
65
7
Thr
54
17
Glu
39
8
Pro
105
18
Lys
105
9
Lys
141
19
Lys
78
10
Ser
85
a Values reported correspond to the sum of the major,
leading, and trailing cycle yields for each amino acid.


Fig. 3-3 Comparison of proteolytic fragmentation patterns and the amino-terminal sequences
of the microtubule-binding fragments of MAP-2 and tau protein. The polypeptide chains and
cleavage patterns for MAP-2 and tau are represented as heavy lines. The closed circles
represent the blocked MAP-2 N-terminus, and the underlined amino acid residues represent
identical and/or conserved amino acid residues common to both MAP-2 and tau proteins. The
tau protein scheme is based on the data of Aizawa et al. (1988) and Lewis et al. (1988).
Arrowheads denote octadecapeptide imperfect repeats.


MAP-2:
240 kDa 28 kDa
: JT
r ''1
Thr-Pro-His-Thr-Pro-GIv-Thr-Pro-Lvs-Ser-Ala-lle-Leu-Val-Pro-Ser-Glu-Lvs-Lvs
Tau Proteins:
. J I i A A
Ser-Ser-Pro-Gly-Ser-Pro-GIv-Thf-Pro-GIv-Ser-Ara-Ser-Arq-Thr-Pro-Ser-Leu-Pro


60
sequencing. These findings suggested that the amino-
terminus of MAP-2 is blocked and that the 240 kDa fragment
is derived from the amino-terminus whereas the 28 kDa
fragment resided at the carboxyl end. These observations
are in accord with the findings by Kosik et al. (1988) who
reported that the N-terminus of MAP-2 appears to be blocked;
moreover, Lewis et al. (1988) reported the entire derived
amino acid sequence using murine MAP-2 cDNA clones. The
primary sequence data with bovine brain MAP-2 correspond to
the murine sequence spanning residues 1626 to 1644 with only
three exceptions. As shown in Fig. 3-3, there is a similar
protease-accessible sequence in the microtubule-binding
fragment of bovine tau protein. In that case, however,
fragments were generated by chymotryptic cleavage (Aizawa et
al., 1988). Both of these cleavage-site sequences reside
approximately thirty-to-forty residues toward the N-terminal
side of the first of three nonidentical octadecapeptide
repeats (indicated schematically by the arrowheads) found in
both MAP-2 and tau (Lewis et al., 1988; Lee et al., 1988).
Amino-terminal sequence of the microtubule-binding fragment
after digestion in the absence of microtubules
My initial experiments to determine if the microtubule
binding fragment of MAP-2 also bound to neurofilaments,
digested purified MAP-2 alone in solution. In the preceding
section the amino terminal sequence of the binding fragment
was determined but the fragment was generated by digestion
of microtubule-protein at 37C in the presence of 1 mM
guanosine triphosphate. In this regard, MAP-2 was digested
in the presence of microtubules, a condition that might have


61
influenced the cleavage point of thrombin were the preferred
site of digestion hindered by interaction with the
microtubule lattice. To determine whether the cleavage
point is the same for the microtubule-binding fragment under
the two different conditions, the amino terminal sequence
was checked once again but now in the absence of
microtubules during the digestion. Purified MAP-2 was
cleaved alone in solution, and passed over a DEAE ion-
exchange column to remove the projection domain. The
breakthrough fractions containing the microtubule-binding
domain were pooled, electrophoresed through a polyacrylamide
gel, transferred to a polyvinylidenedifluoride membrane, and
sequenced by automated Edman chemistry. Ten cycles were
performed yielding ten residues that were exactly the same
as the first ten residues in Table 3-2 and Fig. 3-3. This
indicated that thrombin cleaved MAP-2 at the same argininyl
residue in the presence and absence of microtubules.
Additionally, the breakthrough fractions from the DEAE-
Sephadex column were subjected to reverse phase HPLC similar
to the fragments generated in the presence of microtubules.
When loaded on a C-18 column and eluted with the same
gradient as mentioned in the preceding section, one major
peak corresponding to the 28 kDa fragment was seen (see Fig.
3-4) and one minor peak corresponding to peak A in Fig. 3-2
was seen. The elution time for the 28 kDa fragment was
unchanged from Fig. 3-2 and corresponded to peak C from this
figure.


Absorbance, 220 nm
62
Time after Injection,min
Fig. 3-4 HPLC purification of the microtubule-binding
fragment of MAP-2 digested initially without microtubules.
The inset shows a 12% (w/v) polyacrylamide gel before and
after the HPLC purification: lane 1, digest before
purification; lane 2, fraction 22; lane 3, fraction 23; lane
4, fraction 30; lane 5, fraction 31. Fractions were
collected every two minutes and were 1 ml in volume.


63
Discussion
The experiments described in this chapter were
designed to gain further insight about the microtubule
binding fragment of MAP-2. There is now general agreement
that initial proteolytic cleavage of MAP-2 yields two
fragments (Vallee 1980; Flynn et al. 1987). With thrombin,
these initial cleavage products corresponding to values of
240 kDa and 28 kDa based on electrophoresis, are guite
stable with regard to further degradation. All efforts to
seguence Immobilon-1inked MAP-2 and the similarly
immobilized 240 kDa projection arm fragment consistently
failed to yield any detectable levels of PTH-amino acids.
Nevertheless, acid hydrolysis and subseguent amino acid
analysis of these immobilized proteins demonstrated that
sufficient levels were clearly present for detection in the
gas-phase sequencer. This observation led me to believe
that intact MAP-2 showed no evidence of a free N-terminus,
and another group recently reported the same difficulty in
attempts to sequence MAP-2 (Kosik et al. 1988). These
observations suggest that the MAP-2 amino acid sequence, as
derived from nucleotide sequence data (Lewis et al. 1988),
does not provide a complete account of the MAP-2 primary
structure, and further work will be required to establish
the nature of the N-terminal modification blocking Edman
degradation.
A striking common structural feature in MAP-2 and tau
emerges from the combined findings of Aizawa et al. (1988)
and these studies. The former found that chymotryptic


64
cleavage of the bovine tau proteins yielded a microtubule-
binding fragment with the N-terminal sequence shown in Fig.
3-3, and I have now demonstrated that thrombin attacks at a
similarly accessible region in bovine MAP-2 (See also Fig.
3-3). It should be noted that both of these cytomatrix
proteins have four proline residues in exact registration,
and with the exception of the occurrence of a val-pro in the
MAP-2 sequence, each of the prolines in both cleavage sites
was preceded by a hydroxy-amino acid. Efforts to survey
other known sequences in the GenBank database have indicated
the uniqueness of these protease-accessible regions in tau
and MAP-2; however, Earnshaw et al. (1987) described a
centromere-binding protein containing three prolines in
exactly corresponding positions with little other structural
relatedness to tau and MAP-2. Also it should be noted that
the NF-M sequence in chicken contains a proline at every
third residue for 102 residues in the repeated sequence
(EXPXSP)17 (Zopf et al., 1987). The circular dichroism
spectral data of Hernandez et al. (1986) indicates that
uncleaved MAP-2 contains little, if any, alpha helical or
pleated-sheet secondary structure; yet, the preferential
action of the endoprotease thrombin at a single site
suggests that MAP-2 may display some "hinge-point" behavior
akin to the protease-accessible region of myosin. This
region may permit the projection arm to swing away from the
microtubule surface. Certainly, the observed sedimentation
coefficient of 4.5 (Hernandez et al. 1986) also suggests
that MAP-2 has extended a flexible structure. The roughly


65
spherical hemoglobin molecule, itself only one-third the
molecular weight of MAP-2, has an almost identical
sedimentation coefficient (Sanders et al. 1981).
Chymotryptic cleavage between Tyr-128 and Ser-129 in the tau
proteins may reflect a corresponding protease-accessible
site of structural discontinuity between microtubule-binding
and projection domains. These hinge point regions may be
very important in their presentation of the microtubule
binding sequences that actually interact with tubulin.


CHAPTER 4
THE MICROTUBULE-BINDING FRAGMENT OF MAP-2:
IDENTIFICATION OF AN ASSEMBLY-PROMOTING PEPTIDE
AND DISPLACEMENT OF HIGH-MOLECULAR-WEIGHT MAPs
Introduction
Microtubule-associated proteins (MAPs) exhibit one of
several properties: the ability to copolymerize with
tubulin during microtubule assembly, the capacity to utilize
tubulin or another MAP as substrates for enzyme-catalyzed
modification, or the use of microtubules as the
architectural framework for motility (Olmsted, 1986; Purich
and Kristofferson, 1984). The first property is shared by
the high-molecular-weight proteins (MAP-1 and MAP-2) as well
as the tau proteins, and these proteins remain associated
with reassembled microtubules during the course of
microtubule-protein purification. Recently, the cDNA-
derived amino acid sequences of the murine MAP-2 (Lewis et
al., 1988) and the murine tau (Lee et al., 1988) proteins
have been defined, and these proteins were both found to
contain a related triad of imperfectly repeated
octadecapeptide sequences in their tubule-binding regions.
Oligopeptide analogues of the repeated sequences in murine
tau and a 190 kD bovine adrenal gland MAP can promote
microtubule assembly as monitored by light scattering
techniques (Ennulat et al., 1989; Aizawa et al., 1989). I
wished to investigate whether the triad of imperfect
66


67
octadecapepti.de repeats of murine MAP-2 and a MAP-2 sequence
from the protease-accessible hinge region could promote
microtubule polymerization and mimic the action of MAPs.
While several peptides corresponding to sequences in
fibrous MAPs can stimulate microtubule assembly, very little
is known about whether these synthetic peptides constitute
the entire site necessary for the MAP binding to
microtubules. At the time little information on MAP-2
sequences reponsible for binding to microtubules or
promotion of tubulin polymerization was available. The
report of Lewis et al. (1988) showed a 100 residue
polypeptide consisting of the first two imperfect repeats
plus flanking sequences could cosediment with MAP-stabilized
microtubules. No information on the binding of small
peptides or promotion of tubulin polymerization was known.
The experiments described in this chapter attempt to define
an "active site" of MAP-2 by testing for sequences promoting
tubulin polymerization. The most likely candidates were the
repeated sequences since two of them were in the 100 residue
polypeptide of Lewis et al. (1988). These peptides were
chemically synthesized along with a hinge-region sequence
and tested for stimulation of tubulin polymerization.
If the repeated sequences are indeed the primary sites
of interaction, then those promoting tubule assembly in the
absence of MAPs may also displace MAPs from microtubules or
block their binding to microtubules. Moreover, I was
motivated to learn whether a particular peptide and MAP
display competitive binding behavior that would indicate the


68
peptide(s) binding to the same site as MAPs on the
microtubule. The effectiveness of all three MAP-2 repeated
peptide analogues in terms of MAP displacement from
microtubules was also checked. These experiments show that
peptides corresponding to the second repeated sequence of
MAP-2 can promote microtubule polymerization and displace
MAP-1 and MAP-2 from recycled microtubule-protein.
Materials and Methods
Materials
[32P]ATP (7000 Ci/mmol) and [3H]GTP (18 Ci/mmol) were
purchased from ICN along with ultrapure grades of ammonium
sulfate, sodium dodecyl sulfate (SDS), acrylamide, and bis-
acrylamide. Liquid scintillation cocktail 3a70 was obtained
from Research Products International. Acetate kinase was a
Boehringer Mannheim product, while phosphocellulose resin
and GF/F glass fiber filters were from Whatman. Anti-B
tubulin antibody was purchased from Amersham. DEAE-Sephadex
A-50 was purchased from Pharmacia; and bovine thrombin,
trifluoroacetic acid, Mes, Pipes, and Tris buffers,
dithiothreitol, guanosine triphosphate, bovine serum
albumin, EGTA, Triton X-100, phenylmethanesulfonylfluoride,
catalytic subunit of cAMP-dependent protein kinase, and goat
anti-murine IgG Texas red conjugate were from Sigma. t-BOC
amino acids and the phenylacetamidomethyl resin were from
Applied Biosystems International. Taxol was a gift supplied
by Dr. Matthew Suffness at the National Cancer Institute,
Bethesda Md.


69
Preparation of proteins
Isotonic bovine brain microtubule-protein was isolated
according to the method of Karr et al. (1979) and stored at
-80C after two cycles of assembly-disassembly. Hypotonic
bovine brain microtubule-protein was isolated by the method
of Shelanski et al. (1973). Tubulin was prepared according
to the method of Kristofferson et al. (1986) [32P]MAP-2
was purified by the procedure of Herzog and Weber (1978) as
modified by Flynn et al. (1987) except the purified protein
was concentrated by ammonium sulfate precipitation after gel
filtration chromatography. Unlabeled MAP-2 was prepared
identically except the phosphorylation reaction was omitted
prior to the gel filtration column.
Preparation of synthetic peptides
All peptides were made with an Applied Biosystems
synthesizer model 430A according to the method of Erickson
and Merrifield (1976) with t-BOC protected amino acids and
starting with a phenylacetamidomethyl resin. Peptides were
cleaved and deprotected using a mixture of hydrogen
fluoride, anisle, and dimethyl sulfide in a 9:1:0.3 ratio
(v/v) at -10"C for 2.5 hours. After evaporation the resin
was washed with cold diethyl ether and extracted into 1 M
acetic acid and then freeze dried. Purity was tested by
HPLC profile or by gas phase microsequencing. The peptides
were stored at -20C as a lyophilized powder. All synthetic
peptides except the mN peptide were made by Dr. Jan Pohl of
the microchemical facility at Emory University. The mN


70
peptide was synthesized by the Protein chemistry core
facility at the University of Florida.
Microtubule assembly with synthetic peptides
All assembly experiments were done with a GTP-
regenerating system (MacNeal et al., 1977) consisting of 2
units/ml of acetate kinase, 10 mM acetyl phosphate, and 0.1
mM [3H]GTP (20 Ci/ml). All assay mixes also contained 1 mM
dithiothreitol to maintain reduced sulfhydryls in the
peptides. The peptides were weighed out just before use and
dissolved in 100 mM Pipes, pH 6.8, 1 mM EGTA, 1 mM magnesium
sulfate with 1 mM dithiothreitol. Varying concentrations of
each peptide were added to 1.6 mg/ml pure tubulin and 0.4
mg/ml three-cycle microtubule-protein and incubated at 30C
for 30 minutes. The extent of microtubule assembly was
monitored by the rapid filtration assay of Maccioni and
Seeds (1978) as modified by Wilson et al. (1982).
Microtubules were diluted 20X into 100 mM Pipes, pH 6.8, 1
mM EGTA, 1 mM magnesium sulfate, 1% (v/v) glutaraldehyde,
10% (v/v) dimethylsulfoxide, 25% (v/v) glycerol, and 1 mM
ATP and kept at 30C until ready to assay. The diluted
mixture was then applied to Whatman GF/F filters on a vacuum
filtration device presoaked in the same buffer except no
glutaraldehyde was used. Each filter was then washed with
15 ml of the same buffer and the radioactivity was
solubilized in 1.5 ml 0.1 N NaOH for 30 minutes followed by
addition of scintillation cocktail.


71
Preparation of microtubule seeds and elongation assay
Seeds were prepared according to Kristofferson et al.
(1986). Tubulin at 5 mg/ml was assembled with 1 mM GTP in
80 mM Pipes, pH 6.8, 10 mM magnesium chloride, 1 mM EGTA, in
30% (v/v) glycerol at 37C for 30 minutes. The microtubules
were then sheared with 3 passes through a 22 gauge needle to
produce microtubule seeds. The seeds were diluted 100X into
0.5 mg/ml tubulin and varying concentrations of peptides.
After 30 minutes at 37C the samples were handled as
described in the preceding section for measuring tritiated
guanine nucleotides.
Electron microscopy
Microtubules were diluted 10X into a warmed solution of
1% (v/v) glutaraldehyde in microtubule-assembly buffer and
fixed for one minute at room temperature. The fixed samples
were placed on a carbon coated Formvar 400 mesh grid and
negatively stained with 1% (w/v) uranyl acetate. Grids were
observed on a JEOL 100 CX microscope at 50,000X
magnification. Samples with and without peptide were
processed identically.
Fluorescence microscopy
Microtubules formed in the presence of synthetic
peptides were diluted 10X into a warmed solution of 1% (v/v)
glutaraldehyde and 0.1% (v/v) Triton X-100 in microtubule-
assembly buffer and fixed for 2 minutes at room temperature.
The microtubules were diluted to 50,000 times their original
concentration and an aliquot of 100 /xl was centrifuged on to
a glass coverslip at 30 psi in a Beckman airfuge for 20


72
minutes. The coverslip was then fixed in -20C methanol for
4 minutes and blocked with 10 mg/ml bovine serum albumin in
phosphate buffered saline, pH 7.3, with 0.1% (v/v) Triton X-
100 for 10 minutes. The coverslip was then stained with a
murine anti-tubulin antibody at a dilution of 1:200 for 30
minutes followed by washing in the same buffer. A goat
anti-murine IgG secondary antibody conjugated with Texas red
fluorochrome was used at a dilution of 1:80 followed by
washing in phosphate buffered saline, pH 7.3, with 0.1%
(v/v) Triton X-100. The coverslips were mounted in 20 mM
Tris, pH 7.9, with 90% (w/v) glycerol and viewed with a
Zeiss epifluorescence microscope at 1000X power.
Isotonic microtubule experiments
Before use the isotonic bovine brain microtubule-
protein was carried through a third cycle of
assembly/disassembly, and the concentration of protein was
determined by the method of Bradford (1976). Synthetic
peptides were weighed out just prior to use and dissolved in
PEM buffer (100 mM Pipes, pH 6.8, ImM EGTA, 1 mM MgS04)
containing 1 mM dithiothreitol. Peptides were added at the
indicated concentrations to 0.8 mg/ml isotonic microtubule-
protein with 0.5 mM GTP and 1 mM dithiothreitol and
incubated at 37C for 20 minutes. The microtubules were
subsequently stabilized with 10 /M taxol for 10 minutes at
37 C. The samples, 250/ul final volume, were then
centrifuged for 8 minutes at 300,000 x g, 37C in a Beckman
TL 100.2 rotor. The pellets were dissolved in 8 M urea and
analyzed by gel electrophoresis.


73
Competition with radiolabeled MAP-2
All radiolabeled MAP-2 experiments were performed with
polypropionate airfuge tubes which were coated with 10 mg/ml
bovine serum albumin for 5 minutes and rinsed with PEM
buffer just prior to use. This treatment reduces
nonspecific binding of proteins to the walls of the
centrifuge tubes. The radiolabeled MAP-2 was clarified
prior to use for 20 minutes at 130,000 x g in a Beckman
airfuge to remove any aggregated or denatured protein.
Phosphocellulose-purified tubulin was incubated at 5 mg/ml,
37C with 1 mM GTP for 20 minutes and subsequently
stabilized with 50 xM taxol for an additional 10 minutes.
The microtubules were then diluted twenty-fold into a
solution containing 3 /xM radiolabeled MAP-2 with either
unlabeled MAP-2 or synthetic peptides for 20 minutes at
37 C. The solution also contained 10 /xM taxol and 1 mM GTP
to maintain microtubule stability. The samples, final
volume of lOOjxl, were then carefully loaded into coated
airfuge tubes with the aid of a microcapillary pipetter onto
a 50 /I layer of 20% (w/v) sucrose in PEM buffer warmed to
37C. The samples were centrifuged for 30 minutes at
130,000 x g and the supernatants removed and discarded. The
pellets were washed with 100 /xl of 10 mg/ml bovine serum
albumin in phosphate buffered saline, pH 7.3, containing
0.1% Triton X-100 and resuspended in 100 |il 8 M urea.
Aliquots of 25 /xl were taken for liquid scintillation
counting.


74
Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis was done by the
method of Bloom et al. (1985) omitting sodium dodecyl
sulfate in the separating and stacking gels and adding 2 M
urea to the separating gel. Gels were stained with
coomassie Brilliant Blue R-250 and scanned with an LKB
ultrascan densitometer.
Results
Peptide interactions with tubulin and microtubule-protein
To further analyze sequence(s) responsible for MAP-2
binding to tubulin within the 28 kDa fragment, four peptides
were synthesized. The first (mN = TPHTPGTPK) corresponded
to the N-terminus of the 28 kDa fragment from bovine MAP-2
that was determined previously by microsequencing (see Table
3-2). The others corresponded to the three octadecapeptide
repeats (m1 = VKSKIGSTDNIKYQPKGG, m2 = VTSKCGSLKNIRHRPGGG,
m3 = AQAKVGSLDNAHHVPGGG). Peptide mM was based on the
bovine sequence data while the murine MAP-2 sequence data
was used for m.,, m2, and m3, because no such data are yet
available for the bovine MAP-2. The high state of purity of
each peptide was confirmed on the basis of HPLC elution
profile analysis or gas-phase microsequencing.
First, I sought to determine whether any of these
peptides would influence the assembly of microtubule-protein
that contained both tubulin and MAPs. I worked with
recycled microtubule-protein to which sufficient pure
tubulin was added to lower the content of MAPs to about one-
fifth their normal level. This final composition was


75
approximately 5% MAPs and 95% tubulin by weight. This ratio
was chosen to accentuate any stimulatory effects of the
peptides on the assembly process, and no microtubule
polymerization occurred at the protein concentrations used
without peptide addition. To assay the extent of
microtubule assembly at different levels of peptides mN, m1,
m2, and m3, [3H]guanine nucleotide uptake was measured with
the glass fiber filter assay of Maccioni and Seeds (1978) as
modified by Wilson et al. (1982). Only peptide m2,
corresponding to the second repeat in MAP-2, stimulated
microtubule-assembly as evidenced by the data shown in Fig.
4-1. The level of m2 peptide reguired for polymerization
was very high. Concentrations greater than 250 /M were
needed to stimulate microtubule polymerization. When
peptides mN, m1, or m3 were employed individually, no
incorporation of guanine nucleotide was observed above
background levels. Moreover, in companion experiments, I
found that none of these peptides mixed individually with
peptide m2 altered the stimulation of microtubule assembly
by peptide m2. A range of concentrations from 0-1 mM was
tried for peptides m, and m3 with 0.5 mM or 0.75 mM m2
peptide but none showed any effect on the extent of
microtubule polymerization.
I also found that assembly of pure tubulin could be
stimulated by m2 only. Indeed, assembly with tubulin and m2
exhibits a typical time-course for the polymerization
process as shown in Fig. 4-2. An initial lag phase
indicative of subunit nucleation was seen followed by a


Incorporation, 10 cpm
77
Minutes
Fig. 4-2 Time course of peptide induced assembly.
Phosphocellulose-purified tubulin (1.0 mg/ml) was incubated
with m2 peptide (1.0 mM) for 30 min. at 37C. At the times
indicated, the amount of GTP incorporation was determined as
described in "methods1'.


78
rapid polymerization phase that plateaued around 30 minutes.
Without addition of m2 peptide, no tritium label is retained
on the glass fiber filters. I verified that the observed
polymerization resulted in microtubules by using electron
microscopy (see Fig. 4-3) and immunofluorescence microscopy
(see Fig. 4-4). The electron micrographs revealed a
morphology typical of microtubules composed solely of
tubulin. When tubulin (1 mg/ml) was incubated with and
without peptide m2 (1 mM) microtubules were observed only
in those micrographs of samples to which this peptide had
been added. The same was seen for the immunofluorescence
micrographs in Fig. 4-4 where panel A had the same
concentrations of protein and peptide as the electron
micrograph in Fig. 4-3 and panel B was without added
peptide. Panel A shows typical in vitro microtubules
stained with an anti-tubulin antibody. The concentration of
tubulin used was 1 mg/ml because it was clearly above the
critical concentration for peptide m2 induced assembly while
for tubulin alone it was just at the lower limit for
polymerization (see Fig. 4-5). Any molecule that shifts the
x-intercept to the left is a microtubule-stabilizer and any
molecule that shifts it to the right is a microtubule-
destabilizer. Clearly, m2 is a stabilizer of microtubule
polymerization. I also tested the action of several common
inhibitors of microtubule assembly to learn whether peptide
m2 induced assembly in a manner akin to normal assembly of
brain microtubules. Inclusion of colchicine (0.1 mM),


79
Fig. 4-3 Electron micrograph of peptide induced assembly.
Tubulin (1.0 mg/ml) and m2 peptide (1.0 mM) were incubated
for 30 min. at 37"C and then diluted into 1% glutaraldehyde
in microtubule assembly buffer warmed to 37"C. After
fixation for one minute, the sample was processed for
electron microscopy. An identical sample without m2 peptide
was also done but showed no microtubules. Magnification is
50,000X and the bar equals 0.25 Jim. Formvar coated grids,
uranyl acetate, and photographs were supplied by the
Electron Microscopy core facility


80
Fig. 4-4 Immunofluorescence of microtubules polymerized with
and without m2 peptide. Panel A shows the same tubulin and
peptide concentrations as Fig. 4-3 and panel B shows just
tubulin with no peptide addition. Both samples were stained
with anti-beta-tubulin followed by an anti-mouse IgG
conjugated with Texas red fluorochrome.


Incorporation, 10 cpm
81
Fig. 4-5 Critical concentration plot of peptide induced
tubulin polymerization. Varying concentrations of tubulin
plus microtubule seeds were mixed with or without m2 peptide
(1.0 mM) and assayed for GTP incorporation after 30 min. at
37 C.


82
calcium ion (2 mM) or podophyllotoxin (0.1 mM) resulted in
complete inhibition of peptide m2-induced assembly.
These observations indicate that only the peptide m2,
with a sequence corresponding to the second repeated region
of the microtubule-binding fragment MAP-2 could stimulate
tubulin assembly. Nonetheless, the possibility remained
that the other peptides could still promote elongation, but
not nucleation, of microtubule assembly. To investigate
this possibility, I added pre-formed microtubule seeds to
tubulin (0.5 mg/ml) and [3H]GTP in the presence or absence
of the peptides. Without any peptide additions, only a
minimal increase in guanine nucleotide incorporation was
observed; however, upon addition of peptide m2, significant
assembly was again observed. By contrast, peptides m1 and
m3 failed to cause any significant increase of labeled
guanine nucleotide incorporation into microtubules beyond
background levels (see Fig. 4-6) Thus, m2 is the only
peptide that can stimulate nucleation and elongation.
Displacement of MAPs from recycled microtubule-protein by
MAP-2 repeated sequence peptides
While only peptide-m2 promoted microtubule self-
assembly, I was interested in determining whether m1 and m3
might also bind to assembled tubules and displace MAP-2.
Therefore, the ability of the MAP-2 repeated sequence
peptides to displace high-molecular-weight MAPs from
assembled microtubules was assessed. For this purpose,
microtubule-protein isolated by the isotonic extraction
method (Karr et al., 1979) was used because such protein as


83
Fig. 4-6 Seeded assembly of tubulin with synthetic peptides.
Microtubule seeds were added to a solution containing 0.5
mg/ml tubulin (a level below the critical concentration).
Varying amounts of m1# m2, and m3, were added and
polymerization initiated by warming to 37C. The plotted
values correspond to radiolabel incorporation over a 30
minute period.


84
isolated from the gray matter of the brain is rich in both
MAP-1 and MAP-2. The isotonic microtubule-protein was
stabilized with taxol after 20 minutes of 1 mM GTP at 37C.
This method reduced the amount of abnormal microtubule
structures common with taxol-induced polymerization where
taxol is added at the start of polymerization. In this
experiment, taxol was added after 20 minutes of microtubule
assembly. After a 10 minute incubation, the peptides were
added and the samples kept at 37"C for an additional 20
minutes. In addition to the three repeats of MAP-2, two
other peptides were tested. The first (m^) was a glycine
substitution for a lysine in the m, peptide converting the
carboxyl terminal sequence to that of all the other repeats.
The second (m2') was the same as m2 plus the next three
residues present in the MAP-2 sequence. These additional
residues were RVK which made the peptide more electro
positive as well as possibly adding more structural
conformation. As seen in Fig. 4-7, SDS gel
electropherograms of the microtubule after assembly and
centrifugation, indicate that MAP-2 was only susceptible to
displacement by a 21-amino acid peptide m2' corresponding to
the m2 sequence above plus residues RVK at the C-terminus.
Interestingly, MAP-lb was selectively displaced by peptide
m2, and all high-molecular-weight MAPs were removed from
microtubules in the presence of peptide m2' Densitometry
tracings of lanes 2, 5, and 6 indicate the profiles of MAPs
with m2, m2', and no peptide, respectively (Fig. 4-8).
Because the 21-amino acid peptide was more effective in


85
1 2 3 4 5 6
MAP-1 MAP-2 a, b B S3 iS H S S=j
rri! m2 m3 m; m£ none
Fig. 4-7 Effects of MAP-2 peptides on MAP binding to
microtubules. Coomassie Blue staining of proteins in
pelleted microtubule fractions after electrophoresis on a 4%
polyacrylamide gel: (Lane 1) m1; (lane 2) m?; (lane 3) m3;
(lane 4) m1 ; (lane 5) m2'; (lane 6) no peptide. All
peptides were added to a concentration of 2.0 mM.


86
Fig. 4-8 Densitometry of the coomassie blue stained gel.
Upper trace represents lane 6, middle trace lane 2, and the
lower trace is lane 5 of the gel depicted in Fig. 4-7.


87
displacing MAPs, it was compared to the m2-peptide with
regard to the promotion of microtubule assembly using
tritiated GTP incorporation as a measure of polymerized
protein (see Fig. 4-9). I found that tubulin polymerization
was considerably more effective in the presence of m2',
especially at lower peptide concentrations. At the greatest
concentration of peptide, the level of tritiated GTP
incorporation was the same for both m2 and m2'. Together,
these observations suggest that only peptides corresponding
to the second repeated sequence can displace MAPs from
assembled microtubules. The data also indicate that both
MAP-1 and MAP-2 can be displaced by peptide-m2', suggesting
further that this peptide may bind to common, or closely
overlapping, sites on microtubules. Peptides m1, m2, and m3
were otherwise without effect, as was an analogue of m1
containing a gly in place of lys toward the C-terminus.
This m1 analogue was synthesized because the lysine residue
was disrupting a possible beta turn structure. In m2 and m3
as well as all three repeats of tau, the carboxyl termini
are proline-(glycine)3. This structure was hypothesized to
be important for microtubule-assembly since both the first
two tau repeats contained this carboxyl terminal tail and
could promote tubule polymerization. The first repeat of
MAP-2 was very similar to the first repeat of tau except for
the glycine difference; however, the m1 analogue failed to
promote tubulin polymerization and also failed to displace
high-molecular-weight MAPs.


88
PEPTIDE CONCENTRATION (mM)
Fig. 4-9 Comparison of the stimulation of tubulin
polymerization by peptides m2 and m2'. The incorporation of
tritiated GTP into microtubule-polymer was measured to
examine the assembly promoting activity of m? and the
extended analogue m2' Phosphocellulose-purified tubulin
was used at 1 mg/ml. The open circles represent m2 and the
closed circles m2' .


89
A gradient of m2' peptide was used to determine the
effective concentration range of displacing the MAPs. The
results shown in Fig. 4-10 demonstrate that the extended
second repeated sequence peptide m2' removes MAP-1 and MAP-
2 from microtubules in a concentration-dependent manner.
Again, densitometry was used to gauge the extent of MAP
depletion in the assembled tubule fraction, and the
concentration of peptide-m2' that displaces 50% of MAP-2 was
about 1.5-2.0 mM (data not shown). This level of peptide-
m2' is about four times the concentration needed to promote
tubulin polymerization in the absence of MAPs or
microtubule-stabilizing agents.
Radiolabeled MAP-2 binding to microtubules
While the findings presented in Figs. 4-7 and 4-10
provide clear evidence of MAP displacement, a quantitative
displacement/binding assay was developed to more accurately
measure the MAP displacement. MAP-2 was incubated with
3',51-cyclic-AMP-stimulated protein kinase and [c-32P]ATP
under conditions that have been found to result in the
incorporation of about 1-1.5 phosphoryl groups per MAP-2
molecule (Flynn et al., 1987). First I determined the
concentrations of tubulin and MAP-2 necessary for saturation
of binding to microtubules. The level of tubulin used was
0.25 mg/ml which was taxol-stabilized like the isotonic
microtubule-protein described in the previous section. At
this low level of tubulin, 1-5 ¡jlM of 32P-MAP-2 was seen to
saturate the available binding sites on the microtubules.
This is depicted in Fig. 4-11.


90
MAP-1 MAP-2a,b
as t* ** ** :
0 0.5 1.0 1.5 2.0 2.5
Fig. 4-10 Effects of increasing the m2' concentration on
high-molecular-weight MAP binding to microtubules. Peptide
m2' was added to isotonic microtubule-protein to the final
mM concentration indicated at the bottom of each lane.
After centrifugation, the pellet fractions were analyzed by
gel electrophoresis. The coomassie Blue staining of a 4%
polyacrylamide gel is shown.


Full Text
INTERACTIONS OF MICROTUBULE-ASSOCIATED PROTEIN-2
WITH MICROTUBULES AND NEUROFILAMENTS
BY
JOHN CHARLES JOLY
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
1990

ACKNOWLE DGEMENTS
This work was made possible due to the love and support
of my parents. Their support through my undergraduate and
graduate studies enabled me to complete this long journey.
I must thank members of the Purich lab; especially Greg
Flynn for giving me my start in the lab and collaborating on
the work in Chapter 2, and Jim Angelastro for constantly
patient help with the flipping HPLC. Also Alexandra
Ainzstein for carrying out one of the digestion experiments
in Chapter 3. I am also greatly indebted to Dan Purich for
support through these years, for tolerating my antics and
for his gracious help in aiding my postdoctoral hunt.
Without his help I am sure I would not have enjoyed this
last year as much as I did.
ii

TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS ii
LIST OF TABLES V
LIST OF FIGURES vi
ABBREVIATIONS viii
ABSTRACT ix
CHAPTERS
1 INTRODUCTION 1
An Overview of the Cytoskeleton 1
Microtubules 2
Neurofilaments 6
Microtubule-associated Proteins 12
Proposal 26
2 INTERACTIONS OF MAP-2 WITH TUBULIN AND NF-L 29
Introduction 29
Materials and Methods 30
Results 33
Discussion 42
3 THE MICROTUBULE-BINDING FRAGMENT OF MAP-2:
LOCATION OF THE PROTEASE-ACCESSIBLE SITE 46
Introduction 46
Materials and Methods 47
Results 51
Discussion 62
4 THE MICROTUBULE-BINDING FRAGMENT OF MAP-2:
IDENTIFICATION OF AN ASSEMBLY-PROMOTING PEPTIDE
AND DISPLACEMENT OF HIGH-MOLECULAR-WEIGHT MAPS .. 66
Introduction 66
Materials and Methods 68
Results 74
Discussion 97
iii

5 CONCLUSIONS AND FUTURE DIRECTIONS
103
Interactions with Neurofilaments 103
Structure of MAP-2 107
MAP-2 Sequence Interactions with Microtubules .. 108
REFERENCES 114
BIOGRAPHICAL SKETCH 124
iv

LIST OF TABLES
Table page
1-1 Major classes of microtubule-associated
proteins 13
3-1 Amino acid composition of the 28 kDa
MAP-2 fragment 55
3-2 Amino terminal sequence analysis of the
28 kDa fragment 57
v

LIST OF FIGURES
Figure page
1-1 Comparison of the carboxyl termini of
both a- and B-tubulin from chick brain .... 7
1-2 Structural organization of the neuro¬
filament triplet protein 10
1-3 Comparison of the carboxyl termini of
murine MAP-2 and murine tau protein 25
1-4 Summary of MAP-2 structure in relation to
a microtubule 27
2-1 Thrombin digestion of radiolabeled MAP-2 .. 35
2-2 Binding of MAP-2 or MAP-2 fragments to
neurofilaments or tubulin 37
2-3 Autoradiogram of MAP-2 and MAP-2 fragments
binding to cytoskeletal protein 38
2-4 MAP-2 binding to purified neurofilament
triplet protein or the L subunit of
neurofilaments 40
2-5 Autoradiogram of MAP-2 and MAP-2 fragment
binding to neurofilament triplet protein
and L subunit 41
2-6 Determination of the isoelectric point of
the 28 kDa fragment of MAP-2 42
3-1 Purification of heat-stable microtubule¬
binding fragment and tau 52
3-2 HPLC purification of the microtubule-binding
fragment of MAP-2 digested with microtubules
present 54
3-3 Comparison of proteolytic fragmentation
patterns and the amino terminal sequences of
the microtubule binding fragments of MAP-2
and tau 58
vi

3-4 HPLC purification of the microtubule-binding
fragment of MAP-2 digested initially without
microtubules 62
4-1 Stimulation of microtubule assembly with
synthetic peptides 76
4-2 Time course of peptide induced assembly .... 77
4-3 Electron micrograph of peptide induced
assembly 79
4-4 Immunofluorescence of microtubules polymer¬
ized without and with m2 peptide 80
4-5 Critical concentration plot of peptide
induced tubulin polymerization 81
4-6 Seeded assembly of tubulin with synthetic
peptides 83
4-7 Effects of MAP-2 peptides on MAP binding to
microtubules 84
4-8 Densitometry of the coomassie blue stained
gel 86
4-9 Comparison of the stimulation of tubulin
polymerized by peptides m2 and m2' 87
4-10 Effects of increasing the m2' concentration
on high-molecular-weight MAP binding to
microtubules 89
4-11 32P-MAP-2 binding to taxol-stabilized
microtubules 91
4-12 Displacement of trace phosphorylated MAP-2
from taxol-stabilized microtubules by
unlabeled MAP-2 92
4-13 Displacement of trace phosphorylated MAP-2
from taxol-stabilized microtubules by
peptide m2' 94
4-14 Radiolabeled MAP-2 binding to taxol-
stabilized microtubules in the presence and
absence of 1.5 mM m2' peptide 95
4-15 Double reciprocal plot of MAP-2 binding .... 96
5-1 The octadecapeptide repeats of murine MAP-2,
murine tau, bovine tau, and a corresponding
sequence of the 190 kDa adrenal gland MAP .. 112
vii

ABBREVIATIONS
ATP, adenosine triphosphate
cAMP, cyclic adenosine monophosphate
cDNA, complementary DNA
DEAE, diethylaminoethyl
GTP, guanosine triphosphate
HPLC, high pressure liquid chromatography
MAP, microtubule-associated protein
mRNA, messenger RNA
NF, neurofilament
pi, isoelectric point
PMSF, phenylmethanesulfonyl fluoride
SDS, sodium dodecyl sulfate
viii

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
INTERACTIONS OF MICROTUBULE-ASSOCIATED PROTEIN-2
WITH MICROTUBULES AND NEUROFILAMENTS
by
John Charles Joly
May, 1990
Chairman: Dr. D. L. Purich
Major Department: Biochemistry and Molecular Biology
Bovine brain microtubule-associated protein-2 (MAP-2)
is a 280 kDa protein that binds to microtubules and
neurofilaments. MAP-2 was fragmented by thrombin into a 240
kDa projection domain and a 28 kDa microtubule-binding
domain. The 28 kDa cleavage fragment possessed a
neurofilament binding site for the L subunit of
neurofilaments. The thrombin cleavage site of MAP-2 was
very similar to the chymotryptic cleavage site of the
microtubule-associated protein tau. The 28 kDa microtubule¬
binding fragment was derived from the carboxyl terminus of
the intact protein and the 240 kDa projection domain was
from the amino terminus. The microtubule-binding fragment
was very rich in lysine and arginine residues and its
isoelectric point was approximately 10.0. The microtubule¬
binding fragment contained three octadecapeptide imperfect
repeats located fifty residues from the thrombin cleavage
site. These sequences were chemically synthesized to assay
ix

for promotion of tubulin polymerization. Only the second
promoted tubulin polymerization in vitro, yielding
microtubules of normal morphology. The time course of
peptide-induced microtubule assembly was similar to
microtubule-protein in vitro. This octadecapeptide
displaced MAP-lb from MAP-containing microtubules. The
addition of the next three amino acids in the MAP-2 sequence
to the carboxyl terminus of the peptide increased its
ability to promote tubulin polymerization at lower
concentrations and displaced all high-molecular-weight MAPs,
MAP-la,b and MAP-2a,b. This extended peptide displayed
competitive binding with radiolabeled MAP-2 to taxol-
stabilized MAP-free microtubules, suggesting the peptide
bound to the same site on microtubules as MAP-2. The
dissociation constant for MAP-2 binding was 3.4 /iM in the
absence of the extended peptide and 14 nK in the presence of
1.5 mM peptide. The estimated inhibition constant for the
extended peptide is 0.5 mM, about 100 times lower than for
the K,,, of MAP-2. These observations suggested that the
second repeated sequence of MAP-2 represents an important
recognition site for MAP-2 binding to microtubules and that
other structural features within MAP-2 may reinforce the
strength of MAP-microtubule interactions.
x

CHAPTER 1
INTRODUCTION
An Overview of the Cvtoskeleton
The cytoskeleton of eukaryotic cells is a dynamic
organelle responsible for maintaining cell shape and
rigidity, cell motility, and intracellular vesicle transport
and trafficking. The cytoskeleton consists of three main
types of filaments: microfilaments, intermediate filaments,
and microtubules. Microfilaments are seven nanometers in
diameter and are composed primarily of actin but also
contain actin-binding proteins. Intermediate filaments are
ten nanometers in diameter and vary in composition depending
on the particular cell type. In brain tissue, the neuronal
cell intermediate filaments are made of neurofilament
proteins while in glial cells, the intermediate filaments
are composed of glial fibrillary acidic protein.
Microtubules are 24 nanometer diameter structures composed
mainly of the heterodimer tubulin. In addition to tubulin,
there are cell specific microtubule-associated proteins
(MAPs) that bind to microtubules.
When specific cytoskeletal filaments are mixed in
vitro, they interact with each other. If bovine spinal cord
neurofilaments are mixed with bovine brain microtubules, the
viscosity of the resulting solution increases greatly and a
gel is produced (Runge et al., 1981). This is thought to be
1

2
a relevant and physiological interaction based on
microscopical examination of neuronal axoplasm which shows
neurofilaments and microtubules running parallel to each
other in neurite processes. The work of Hirokawa and his
coworkers over the years has demonstrated through microscopy
techniques that projections exist between the filamentous
structures (Hirokawa et al., 1985; Hirokawa 1982). These
projections could be cross-bridges connecting the two types
of filaments. Possible candidates for the cross-bridges are
MAPs. Both microtubule-associated protein-2 (MAP-2) and tau
proteins are MAPs and can bind to neurofilaments as well as
microtubules (Letterier et al., 1982; Heimann et al., 1985;
Miyata et al., 1986). It is the role of MAPs, specifically
MAP-2 and its interactions with microtubules and
neurofilaments, that is the focus of this thesis.
Microtubules
Microtubule Structure and Function
Microtubules are the most dynamic component of the
cytoskeleton and exist in all eukaryotic cells except
enucleated erythrocytes. The microtubule core is formed
from alpha and beta tubulin heterodimers arranged into
parallel rows, extending the length of the tubule, termed
protofilaments. Isolated microtubules and those observed in
sectioned cell specimens contain thirteen protofilaments,
while most microtubules assembled in vitro possess fourteen
protofilaments (McEwen and Edelstein, 1980). The tubulin
subunits arrange themselves in a head-to-tail fashion along
the protofilaments yielding the distinct polarity in

3
microtubules. This polarity was first observed by Rosenbaum
and Child (1976) and Witman (1975), who demonstrated biased
addition to microtubules in vitro. One end of the
microtubule polymer displays an increased rate of addition
compared to the other end. This characteristic polarity of
microtubules can be determined by interactions with
microtubule-binding proteins. One method is based on the
interaction of dynein with assembled microtubules (Haimo et
al., 1979; Haimo, 1982). Dynein is a large protein found in
flagella of Tetrahymena that binds to microtubules and
hydrolyzes adenosine triphosphate to produce the whip-like
motion during movement. The main globular head of the
dynein molecule tilts at an angle of 55° in the direction of
the end of preferred growth of the microtubule. Another
method to distinguish microtubule polarity takes advantage
of special in vitro solution conditions that favor formation
of microtubule walls decorated with additional
protofilaments. The extra protofilaments align themselves
into hook-shaped sheets and curve in one specific direction
depending on the polarity; either clockwise or
counterclockwise (Burton and Himes, 1978).
This intrinsic polarity of microtubules enables
microtubules to perform vectorial functions. When
intracellular vesicles travel along a microtubule in an
axon, the vesicle generally moves in one direction. Rarely
will a vesicle change its direction after starting its
journey, and most vesicles move toward the cell body in a
neuron (Hollenbeck and Bray, 1987). The polarity of a

4
microtubule is clearly a vital property for interactions
with other cytoskeletal components as well as for their
proper function. In a non-neuronal cell, microtubules are
capped at their minus end near the centrioles and extend the
plus end to the cell margin, yielding an overall radial
polarity in the cell.
Some other interesting properties of microtubules are
that they self-assemble in vitro in the presence of GTP at
warm temperatures and that they depolymerize at cold
temperatures or upon the addition of calcium ions. Each
alpha/beta dimer binds two moles of GTP per mole tubulin,
but each heterodimer has two types of nucleotide binding
sites (Weisenberg et al., 1968; Berry and Shelanski, 1972).
The beta subunit has an exchangeable nucleotide binding site
that readily exchanges GDP for GTP. The alpha subunit has a
nonexchangeable binding site that exchanges GDP for GTP very
slowly. Guanosine triphosphate nucleotides are hydrolyzed
only upon or after heterodimer incorporation into the
polymeric tubule.
Both in vitro and in vivo microtubule dynamics have
been monitored through the use of tubulin modified with a
fluorescent tag. This tag can be a direct covalent
attachment of a fluorochrome or by using an anti-tubulin
antibody with a secondary antibody labeled with a
fluorescent probe. In most cases examined the microtubules
have been shown to be highly dynamic structures rapidly
polymerizing and depolymerizing depending on the surrounding
conditions (Mitchison and Kirschner, 1984; Kristofferson et

5
al., 1986; Sammak et al., 1987). In vitro, the addition of
MAPs reduces microtubule dynamics (P.S. Yamauchi, personal
communication).
Microtubules are essential for proper mitotic function.
When the mitotic spindle poison colchicine is added to
cells, mitotic arrest is seen; particularly at metaphase
prior to microtubule depolymerization in anaphase which
achieves chromosome movement toward the spindle poles. The
chromosomes are connected to the microtubules through
kinetochores which cap the microtubules at one end, while
the opposite end is capped by the spindle pole or centriole.
During interphase the centrioles seem to act as microtubule
organizing centers and are located just outside the nucleus.
Some other important functions of microtubules are the
maintenance of cell anisometry and promotion of cell shape
changes. Nerve axons and retinal rod cells rely on the
microtubule network to support their distinctive cell
morphology, especially the neurite processes (Heidemann et
al., 1986). Conversely, the activation of platelets and the
action of polymorphonuclear leukocytes depend on changes in
the microtubule-cytoskeleton (Malawista, 1986).
Microtubules also provide the basic framework for the cell
motility machinery as they are essential components of
flagella and cilia.
Tubulin
The main component of microtubules is tubulin. This
protein exists as a heterodimer of 100 kDa with each subunit
possessing a molecular mass of approximately 50 kDa. There

6
is considerable homology between the a and 6 subunits
suggesting a common ancestral origin (Valenzuela et al.,
1981). Both a and B tubulin are very conserved across
species lines suggesting that there are stringent
requirements on the structure of tubulins over a reasonably
great phylogenetic range (Cleveland and Sullivan, 1985).
Both subunits contain a glutamate-rich carboxyl terminus
that is thought to be responsible for MAP binding to
microtubules (see Fig. 1-1). When the carboxyl termini of
both subunits are proteolytically removed with subtilisin,
the tubulin can self-assemble without MAP binding (Serrano
et al., 1984). In fact, when these tubules are sedimented
and the pellet fraction analyzed, the MAPs are found only in
the supernatant fraction. The presence of high
concentrations of salt (0.4 M -0.6 M) disrupts MAP binding
to microtubules indicating the interaction of MAPs with
microtubules is dependent on the ionic interactions. Thus
the glutamate rich carboxyl termini are thought to be the
binding sites for MAPs which in turn contain positively
charged residues (Littauer et al., 1986).
Neurofilaments
Neurofilament Structure
Neurofilaments are one member of the intermediate
filament protein family of which there are five main
components: (1) acid keratins, (2) basic keratins, (3) glial
fibrillary acidic protein, vimentin, peripherin, and
desmin, (4) neurofilaments and alpha-internexin, and (5)
lamins. Keratins are found in epithelial cells and their

Carboxyl Termini of a- and 6-tubulin
6 Asp-Glu-Gln-Gly-Glu-Phe-Glu-Glu-Glu-Gly-Glu-Glu-Asp-Glu-Ala
a Glu Gly-Glu-Gly-Gly-Glu-Glu-Gly-Glu-Glu Tyr
Fig. 1-1 Comparison of the carboxyl termini of both a- and 6-tubulin from chick brain
(Valenzuela et al., 1981). Note the high glutamate content of both sequences.

8
derivatives such as skin and nails (Steinert and Roop,
1988). Desmin filaments are found mostly in muscle cells
while vimentin filaments are located in mesenchymal cells
(Steinert and Roop, 1988). Glial fibrillary acidic protein
is the basic building block of glial filaments which are
found in glial cells (Steinert and Roop, 1988). Peripherin
is located in the neurons of the peripheral nervous system
(Portier et al., 1984) while neurofilament proteins are
found in most neuronal cells (Steinert and Roop, 1988).
These filaments are composed of three subunits. The main
core of the filament is made of the L subunit, a 70 kDa
protein. The other two subunits are the M subunit and H
subunit which are 150 kDa and 210 kDa respectively. Both of
these subunits contain multiple phosphates on serine and
threonine residues.
The neurofilament proteins have some common structural
features with each other and all other intermediate filament
proteins. Each has a 40 kDa conserved rod domain believed
to be derived from a common ancestral gene (Weber et al.,
1983). This common rod-shaped region is rich in alpha
helical content and possesses a very conserved epitope that
reacts with a mouse monoclonal antibody that recognizes all
intermediate filament proteins (Pruss et al., 1981). This
40 kDa region has been conserved and is essential for
intermediate filament assembly (Steinert et al., 1981;
Geisler and Weber, 1981). Each intermediate filament
protein contains hypervariable regions that flank the
central rod region. These hypervariable regions form the

9
amino and carboxyl termini of the proteins also known as the
head and tail regions respectively. It is the variation in
these regions that distinguishes each individual
intermediate filament protein. The difference between
neurofilament proteins and other intermediate filament
proteins like desmin and vimentin is the tail region.
Normally the tail region is only approximately 5 kDa in mass
but is over 55 kDa in the H subunit of neurofilaments and 50
kDa in the M subunit. It is the tail regions of the M and H
subunits (see Fig. 1-2) that contain many of the
phosphorylation sites found in these proteins (Julien and
Mushynski, 1983). The tail region of the L subunit is
considerably shorter than the M or H subunits and is rich in
glutamate content which may be important in MAP binding
similar to the glutamate rich carboxyl termini of alpha and
beta tubulin. The tail regions are considerably less
conserved across species lines.
Neurofilament Function
The neurofilament proteins are found mostly in axons as
opposed to the cell body and dendrites. Once assembled into
polymers, neurofilaments do not easily dissociate (Giesler
and Weber, 1981; Moon et al., 1981). In fact, they are
insoluble in aqueous buffers and their purification relies
on solubilization with high concentrations of urea
(Tokutake, 1984). Upon removal of the urea by dialysis, the
filaments reassemble. Filament formation after dialysis
from urea is not restricted to the neurofilament triplet
(i.e. neurofilaments composed solely L, M, and H subunits),

10
NF-L
Coilia Coil 1b Coil 2 Tail a E segment
NF-m !â– â–  k
Colli Coil 2 Talla El KSP1 E2 KSP2 KE including SP segments
nf-h ^\Pw^yyy/y///y^/////wm^////Ay//^
Coil 1 Coil 2 Talla E and KSP íagmant* KEP segment
Fig. 1-2 Structural organization of the neurofilament
triplet proteins. All three proteins contain coil-coil
regions common to all intermediate filament proteins.
Differences are seen in the amino terminal heads and
carboxyl terminal tails. The phosphorylation sites in NF-M
and NF-L are in the KSP-rich sequences.

11
but for each individual subunit when purified from the other
two subunits can also be assembled by this method although
the filaments are not as structurally intact for the M and H
subunits (Tokutake et al., 1984). Unlike the case of facile
microtubule assembly/disassembly, intermediate filament
proteins form long-lived structures that provide the only
known function of these proteins, namely stabilization and
maintenance of the axon's structural integrity and caliber.
Neurofilaments increase the size of an axon by adding to the
volume occupied by the cytoskeleton (Lasek et al., 1983).
The contribution to axonal diameter or caliber, has a direct
effect on axonal function because the larger the axonal
diameter, the faster the action potential travels down the
axon. Also, this increase in size permits specialization of
nerve cells because the largest axons have the ability to
excite several target nerve cells at one time (Zucker,
1972). This can synchronize an entire population of cells
to act coordinately as with muscle cells to produce
movement. Axon size is very important to organisms which
must coordinate large muscles to produce rapid and essential
processes necessary to organismal survival. With a minimum
number of junctions, axon size is very important and
expression of the neurofilament genes can be crucial for
proper organismal behavior and survival (Bullock and
Horridge, 1965). Interestingly, some axons contain little
neurofilament proteins but still function properly
apparently as a result of increased microtubule content that

12
can replace neurofilaments in increasing the size of the
axon (Morris and Lasek, 1984).
While neurofilaments are required architecturally for
proper axon function, they are not required for such axonal
dynamics as axonal transport which is dependent on
microtubules. When an axon is disrupted by the neurotoxin
B,B'-iminodipropionitrile, the axonal inner structure is
rearranged with neurofilaments segregating to the outer
regions of the axon diameter and microtubules and organelles
segregating toward the center (Papasozomenos et al., 1981).
This model system has been used to examine interactions with
microtubules and neurofilaments where it has been shown that
cross-links exist between microtubules and neurofilaments as
well as within neurofilament networks themselves. An
antibody specific for MAP-2 has been found to co-localize
with both groups of filaments in iminodipropionitrile-
treated neurons implicating MAP-2 as a cross-linker within
and between these cytoskeletal systems (Papasozomenos et
al., 1985).
Microtubule-Associated Proteins
Introduction
MAPs are a varied class of proteins that have been
classified on the basis of their binding or modification of
microtubules. When one considers the diversity of
microtubule functions, it is not surprising that there
exists a great number of proteins that regulate temporal,
spatial, and metabolic controls of microtubule processes
(see Table 1-1). The first attempt to characterize proteins

Table 1-1
Major classes of microtubule-associated proteins
Protein
Subspecies
Subunit mass
(kDa)8
Primary
source
Properties
MAP 1
1A, IB
350
brain
thermolabile;projec-
tion on microtubule
Light chains
28,30
brain
associated with MAP-1
MAP 1C
—
350
brain
cytoplasmic dynein
MAP 2
2A,2B
270
brain
thermostable;proj ec-
tion on microtubule;
separable into pro¬
jection (235 kDa)
and binding (35 kDa)
domains phosphory-
lated;binds calmodu¬
lin
Type II CAMP- 53,39
dependent protein
kinase
Tau 3-5 55-62 brain
associated with MAP-2
projection domain
thermostable;number of
peptides depends on
age and species;
phosphorylated;binds
calmodulin
MAP 3
180
brain

Table 1-1 Continued
Protein
Subspecies Subunit mass
Primary
Properties
(kDa)a
source
MAP 4;210-kDa
HeLa MAP; 205-kDa
Drosophila MAP
3-4
200-240 depending
on species
cultured mammalian
cells; mouse tissues
(MAP 4); Drosophila
(205-kDa)
thermostable
125-kDa MAP
—
125
cultured mammalian cells
Chartins
69,72,80
cultured mammalian
cells;primary neurons
subspecies
thermolabile;phosphor-
ylated
STOPS
microtubules
140,72,56
brain
stable
associated with cold-
Sea urchin
MAPS
“ —
37,78,80
150,200,235
sea urchin eggs;
sea urchin spindles
spindle localization
Kinesin
""
110
134
squid axoplasm
sea urchin eggs
moves particles on
microtubles
Dynamin
100
calf brain
microtubule-activated
ATPase producing
movement of micro¬
tubules, bundles
microtubules
denatured mass of major polypeptides in each class as determined by SDS-PAGE.

15
associated with microtubule-networks was by Gibbons (1965)
who demonstrated that dynein could be selectively released
and then rebound to axonemes of Tetrahymena. Later,
Weisenberg (1972) observed that microtubules can be
assembled in vitro from crude brain extracts, and this
observation led to the identification of proteins associated
with polymerized microtubules. Through the use of
repetitive cycles of temperature-dependent assembly and
disassembly (Shelanski et al., 1973), quantitative MAPs or
MAPs that bind to microtubules in a defined molar ratio were
identified. The two most obvious classes of MAPs in this
category were the high-molecular-weight MAPs, MAP-1 and MAP-
2, and a family of polypeptides known as tau which has a
molecular mass of 55 to 68 kDa.
Unfortunately, the use of temperature-dependent cycles
has obscured the discovery of many less abundant, but
potentially very significant MAPs. During the course of
several temperature-dependent cycles much protein is lost,
both tubulin and MAPs. Only appoximately fifteen percent of
the microtubule-protein is yielded from bovine brain tissue
after three cycles. Also the use of nucleotides, both
adenosine- and guanosine triphosphate, increases the yield
of microtubule-protein by increasing the amount of tubulin
that polymerizes, but also influences MAP associations with
microtubules. For instance, the inclusion of adenosine
triphosphate in the first warming of the brain tissue
extract releases MAP-1C, a cytoplasmic retrograde dynein,
from the microtubule-lattice (Paschal et al., 1987b). This

16
protein was not characterized as a microtubule-associated
protein for years because it was routinely discarded during
the cycling of microtubule-protein.
One of the high-molecular-weight MAPs already mentioned
is MAP-1 which is actually a family of polypeptides at
appoximately 350 kDa. There are two closely spaced bands on
denaturing polyacrylamide gels that are MAP-la and MAP-lb.
Even though they are similar in molecular weight they
exhibit different monoclonal antibody reactivities and
produce different peptide digest patterns. These proteins
exhibit no preferential localization in brain tissue but
their purification from white matter as a family takes
advantage of the fact that other high-molecular-weight
contaminants (i.e. MAP-2) are enriched in gray matter
(Vallee, 1986). There is no available purification to date
for separating the subspecies of this family. MAP-1C on the
other hand has been purified to homogeneity and found to be
a microtubule-activated ATPase that can translocate
microtubules on glass slides and can translocate vesicles
along microtubules in an ATP-dependent fashion (Shpetner et
al., 1988). This very large protein has a native molecular
mass of approximately 450 kDa and scanning transmission
electron microscopy revealed that MAP-1C has a morphology
and mass of a two-headed dynein (Vallee et al., 1988).
Additionally, ultraviolet irradiation in the presence of
vanadate cleaved the protein into two fragments of about the
same size as those produced from flagellar dynein (Paschal
et al., 1987a).

17
Another microtubule-activated ATPase that can
translocate microtubules as well as intracellular vesicles
is kinesin. Kinesin was first discovered during studies of
organelle transport in the giant sguid axon, where
microtubules serve as tracks for the movements of organelles
(Vale et al., 1985a). Kinesin isolated from sguid axoplasm
can induce movement of carboxylated latex beads along
purified microtubules or gliding of microtubules on glass in
the presence of ATP (Vale et al., 1985b). The direction of
movement along the microtubules was anterograde or towards
the nerve terminal in an axon. This is exactly the opposite
direction of movement as MAP-1C (Paschal and Vallee, 1987).
Kinesin has since been found in a variety of organisms and
cell types from mammalian brain tissue (Brady, 1985) to
Drosophila melanoqaster (Saxton et al., 1988). This wide
distribution of kinesin suggests it may be involved in a
variety of microtubule-based motility systems in different
cell types. Immunolocalization studies in sea urchin eggs
and some mammalian cultured cells have demonstrated kinesin
is located in the mitotic spindle suggesting a role for
kinesin in mitosis (Scholey et al., 1985).
Kinesin is composed of two heavy chains of 120-124 kDa
and two light chains of 62-64 kDa and is a highly elongated
molecule with an axial ratio of appoximately 20:1 (Bloom et
al., 1988). Rotary shadowing of kinesin shows it is a rod¬
shaped molecule approximately 80 nanometers long. One end
of each kinesin molecule contains a pair of globular domains
while the opposite end is fan shaped (Hirokawa et al.,

18
1989). Monoclonal antibodies against the heavy chains stain
the globular structures while antibodies versus the light
chains stain the fan-shaped end (Hirokawa et al., 1989). A
60 kDa amino terminal section of the heavy chain corresponds
to the globular head region and contains the nucleotide-
dependent microtubule binding activitity and is thought to
the motor domain (Yang et al., 1989).
Among the lesser known MAPs is the MAP-4 class which
consists of a series of MAPs with molecular masses around
200-240 kDa depending on the species. It is found in almost
all cultured mammalian cells, in mouse tissues, and in
Drosophila melanogaster. MAP-4 shares the property of
thermostablity with MAP-2, a feature that is rare in
proteins of their size. The overall structure and binding
to microtubules of a few MAP-4 types are just starting to be
analyzed. Another group of not very well characterized MAPs
are STOPs which confer cold-stability on microtubules
(Margolis and Rauch, 1981). Normally, microtubules
depolymerize when exposed to cold temperatures; in the
presence of STOPs, the microtubules are stable. These
tubules can be depolymerized by calmodulin and low calcium
concentrations, and it has been shown that these proteins
are all retained on a calmodulin affinity resin (Job et al.,
1982) .
Tau Proteins
Among the best characterized MAPs to date are the tau
proteins, a family of closely related polypeptides. On
denaturing polyacrylamide gels the proteins exhibit

19
molecular masses of 55 to 70 kDa with usually four or five
distinct bands appearing. Tau is a phosphoprotein with
serines and threonines primarily phosphorylated. It is
located in neuronal tissues and restricted to axons of
neurons. Tau has been found in paired helical filaments and
in plaques from Alzheimer brain patients (Goedert et al.,
1988). It has the remarkable properties of being soluble in
2.5% (v/v) perchloric acid and being insoluble in 25% (v/v)
glycerol (Lindwall and Cole, 1984). Tau also is heat-
stable, and its purification takes advantage of this
property.
The source of heterogeneity in tau was unclear until
recently when it has been demonstrated that the different
tau polypeptides are the result of alternative splicing from
one mRNA transcript (Himmler, 1989). By the selective
splicing of specific exons from the transcript, a specific
tau translation product is synthesized. This work was
performed with bovine brain mRNA. The first tau sequence to
be identified was the murine system by Lee et al. (1988) who
showed two tau transcripts were made in vitro with differing
carboxyl termini. An interesting structural feature of the
transcripts noticed in the murine tau investigation was the
presence of three imperfect octadecapeptide repeats in the
center of the molecule. Each repeated sequence ended with a
proline followed by three glycines. All the repeats
contained serines and threonines, and they were rich in the
basic amino acids arginine and lysine. These investigators

20
hypothesized the repeats were important for tau binding to
microtubules.
Another group of investigators showed a proteolytic
fragment of tau could bind to microtubules (Aizawa et al.,
1988) . A 14 JcDa chymotryptic fragment present in each
bovine tau polypeptide was found to bind to microtubules.
The amino terminus of this fragment was determined by Edman
sequencing which localized where the fragment was derived
from after comparison with the murine cDNA predicted
sequence. This proteolytic fragment contained two of the
three repeats believed to be involved in microtubule
binding. This was the first indication that not all the
repeats were necessary for tau binding to microtubules. An
interesting feature of bovine tau protein not elucidated by
the protein chemistry work of Aizawa et al. (1988) was shown
by the cDNA sequencing work of Himmler et al. (1989). They
determined that all the bovine forms of the tau polypeptides
contain four repeats rather than three as seen in the murine
forms. It is unclear what the significance of having four
repeats versus three repeats is although tighter or better
binding could be the result.
Some interesting work with the repeats of tau has been
done in vitro. Ennulat et al. (1989) has shown that
synthetic peptides corresponding to the first and second
repeats of murine tau protein can promote the polymerization
of tubulin. The third repeat has also been tested but
failed to promote tubulin polymerization into microtubules.
Himmler et al. (1989) also has demonstrated that a

21
polypeptide consisting of the four repeats can cosediment
with taxol-stabilized microtubules and he demonstrated that
just two repeats fused together also possessed this ability.
The importance of the cosedimentation data cannot be
overlooked, but the work of Ennulat et al., (1989) is more
significant because this was the first demonstration of a
synthetic peptide of a microtubule-associated protein
performing the same function as a MAP.
Interestingly, Aizawa et al. (1989) found a sequence
that is similar to one of the repeats of tau in the 190 kDa
adrenal gland-specific microtubule-associated protein. This
sequence could also promote the polymerization of tubulin in
vitro. An emerging theme in microtubule cytoskeletal
research is that a group of similar sequences in some MAPs
are responsible for promoting MAP-tubule interactions.
Microtubule-Associated Protein-2
Microtubule-associated protein-2 (MAP-2) is a very
large protein specific for neuronal tissue and restricted to
dendrites. Its molecular mass on denaturing gels is
approximately 280 kDa but the predicted mass from its cDNA
is only about 200 kDa (Lewis et al., 1988). MAP-2 is very
similar to tau in that it is heat-stable, a phosphoprotein,
and the murine form contains a trio of imperfect
octadecapeptide repeats. MAP-2 also shows heterogeneity on
denaturing gels splitting into two high-molecular-weight
forms, MAP-2a and MAP-2b. It is unknown what the cause of
this heterogeneity is. The only known function of MAP-2 is
to polymerize tubulin into microtubules.

22
MAP-2 can be phosphorylated by a variety of kinases.
The cAMP-dependent protein kinase, calmodulin dependent
protein kinase, calcium/phospholipid-dependent protein
kinase, and protein kinase C have all been shown to use MAP-
2 as a substrate (Goldenring et al., 1985; Tsuyama et al.,
1986; Akiyama et al., 1986). MAP-2 can also be
phosphorylated by non-neuronal specific kinases such as the
insulin receptor kinase and an epidermal growth factor
stimulated kinase (Kadowaki et al., 1985; Hoshi et al.,
1988). When MAP-2 is isolated by standard cycling
procedures from brains it contains about 10 moles phosphate
per mole of MAP-2 (Tsuyama et al., 1986). About ten more
phosphates can be added with exogenous cAMP-dependent
protein kinase. Rat brain MAP-2, isolated immediately
following rapid in vivo heat-treatment, contains
approximately 46 moles of phosphate per mole of MAP-2
(Tsuyama et al., 1987). Such microwave treatment reduces
the activity of phosphoprotein phosphatases. The
phosphorylation state of MAP-2 has a direct influence on its
function. The more phosphorylated the protein is, the less
its affinity for tubulin is and the ability to promote
polymerization is reduced (Murthy and Flavin, 1983).
MAP-2 has very little organized secondary structure.
Circular dichroic measurements of MAP-2 revealed it
contained very little alpha-helix or beta-sheet (Hernandez
et al., 1986). This same study determined that MAP-2
possessed a highly elongated structure as analyzed by gel
filtration chromatography and analytical

23
ultracentrifugation. In addition, the predicted structure
from the cDNA studies of Lewis et al., (1988) also revealed
little organized secondary structure.
Surprisingly, when MAP-2 is digested with various
endoproteases, only two major fragments are usually
produced: the first, a small fragment approximately 28-36
kDa in mass and the second, a large 240 kDa fragment. The
small fragment contains the microtubule-binding site of MAP-
2 (Vallee, 1980; Flynn et al., 1987), and the large fragment
is known as the projection domain because it is seen in
electron micrographs before protease treatment protruding
from the microtubule wall but is absent after protease
digestion (Vallee and Borisy, 1978). This limited digest
pattern with several proteases suggests some higher order of
structure. A completely random structure would give greater
heterogeniety in protease digests. If trypsin or
chymotrypsin are employed, the digest patterns are more
complex but the microtubule-binding products are 34-36 kDa.
The projection domain contains a majority of the
phosphorylation sites. The significance of these
phosphorylation sites in the projection domain remains
unclear. One known function of the projection domain is
that it contains a binding site for the regulatory subunit
of cAMP-dependent protein kinase (Vallee, 1986). This
suggests that MAP-2 may be associated with a protein kinase.
This kinase could phosphorylate the microtubule-binding
domain thereby modulating the affinity of MAP-2 for
microtubules.

24
The entire cDNA structure of murine MAP-2 recently was
reported by Lewis et al. (1988), and it showed that MAP-2
possessed three imperfect octadecapeptide repeats similar to
murine tau. Secondary structure predictions from the cDNA
confirmed the findings of Hernandez et al. (1986) showing
MAP-2 contained little alpha-helical content or beta-pleated
sheet content. A synthetic polypeptide of 100 amino acids
of MAP-2 sequence containing the first and second repeats
was shown to bind to MAP-stabilized microtubules and could
cycle with these microtubules (Lewis et al., 1988). This
report revealed extensive homology of MAP-2 with tau
especially in the carboxyl termini of both proteins as
depicted in Fig. 1-3. It will be interesting to know the
bovine MAP-2 sequence to see if it contains three
octadecapeptide repeats like murine MAP-2 or four repeats as
in the bovine tau proteins.
Recently a third form of MAP-2 has been found in
addition to MAP-2a and b (Garner et al., 1988). This
protein is a 70 kDa heat-stable MAP that cross-reacts with
MAP-2 antibodies but is only expressed in neonatal and
juvenile rats. Northern blots of different developmental
stages in rat show a 6 kilobase mRNA in neonatal brain
tissue and a 9 kilobase mRNA in adult brain tissue when
probed with a MAP-2 specific cDNA (Garner and Matus, 1988).
When this 70 kDa protein, now termed MAP-2c, was sequenced,
the sequence was the same as MAP-2a and b except for a 1,372
amino acid deletion corresponding to the central section of
the adult form (Papandrikopoulou et al., 1989). This was

1610
PPSYSSRTPGTPGTPSYPRT
* * * * * *
123
GERSGYSSPGSPGTPGSRSR
1670
DLKNVKSKIGSTDNIKYOPK
***** ****** * * **
183
DLKNVRSKIGSTENLKHOPG
1730
KLDFKEKAOAKVGSLDNAHH
***** * * ***** *
243
KLDFKDRVOSKIGSLDNITH
1630
PGTPKSGILVPSEKKVAIIR
**** *
143
TPSLPTPPTREP-KKVAWR
1690
GGQVQIVTKKIDLSHVTSKC
** **** * *** *****
203
GGKVOIVYKPVDLSKVTSKC
1750
VPGGGNVKIDSQKLNFREHA
****** *** ** *** *
263
VPGGGNKKIDTHKLTFRENA
1650
TPPKSPATPKQLRLINQPLP
******* * * *
163
TPPKSPASKSRLQTAPVPMP
1710
GSLKNIRHRPGGGRVKIESV
*** ** * **** * *
223
GS LGNIHHKPGGGOVEVKSE
1770
KARVDHGAEIITQSPSRSSV
** ****** ** *
283
KAKT DHGAEIV Y KS PW S GD
1790
AS PRRLSNVSSSGSINLLES
*** ********** *
303
TS PRHLSNVSSTGSIDMVDS
1810
PQLATLAEDVTAALAKQGL
******* * * ******
323
PQLATLADEVSASLAKQGL
Fig. 1-3 Comparison of the carboxyl termini of murine MAP-2 (upper sequence) and murine
tau protein (lower sequence). The asterisks indicate an exact match and the underlined
sequences refer to the imperfect octadecapeptide repeats. The hyphen at position 155 of
tau represents a space created to allow for better alignment of the sequences.

26
due to alternative splicing of the mRNA from one gene of
MAP-2. The protein still contained the carboxyl terminus
ofadult MAP-2 with the triad of imperfect repeats. It is
currently unclear what the significance of the embryonic and
adult forms is, although a reduced degree of cytoskeletal
cross-linking during axonal and dendritic growth with the
embryonic form is a possibility (Papandrikopoulou et al.,
1989) .
Altogether, the findings on the structural organization
of MAP-2 can be depicted as shown in Fig. 1-4. This figure
shows the section spliced out of mature MAP-2 creating the
MAP-2c form. The binding site for the regulatory subunit of
cAMP-dependent protein kinase is located at the amino
terminus and the proposed microtubule-bundling domain is
located at the extreme carboxyl terminus of the molecule.
When this bundling sequence is removed, MAP-2 loses its
ability to cause microtubule bundling (Lewis et al., 1989).
This sequence contains homology to leucine zipper proteins
and can be functionally replaced by these leucine zippers
such as the one contained in GCN4. It has been hypothesized
that the bundling domain at the carboxyl terminus of MAP-2
interacts with another MAP-2 carboxyl terminus causing the
microtubules to bundle.
Proposal
The aim of this study was to determine how MAP-2
interacted with two different cytoskeletal components,
neurofilaments and microtubules. This was to be
accomplished through the use of proteolytic digests to

27
MAP-2 Structure:
Microtubule
Protein Kinase
R2C2
Binding Site
Hinge Region
rv
M, â– 
M2-i
1 -
m3
1 1
Absent in MAP-2C •
\ ü
Microtubule x Projection
Binding
Fragment
Bundling Domain
Arm
M1 =VKSKIGSTDNIKYQPKGG
M 2 = VTSKCGSLKNIRHRPGGG
M3=AQAKVGSLDNAHHVPGGG
Fig. 1-4 Summary of MAP-2 structure in relation to a
microtubule. The amino terminus of MAP-2 contains a binding
site for the regulatory (R) subunit of cAMP-dependent
protein kinase. The carboxyl terminus contains the
imperfect repeats as well as a proposed bundling sequence.
The hinge region contains a protease sensitive site.

28
determine which fragment(s) of MAP-2 could interact with the
different polymers. Once the fragment was determined, a
structural characterization of its biochemical properties
was to be carried out. This included a determination of its
isoelectric point, amino acid composition, and amino
terminal analysis. With the availability of the entire
seguence of murine MAP-2, internal sequences responsible for
promoting tubulin polymerization in vitro were to be
identified. In addition to tubulin polymerization, MAP-2
sequences responsible for displacement of high-molecular-
weight MAPs from microtubules were to be identified.

CHAPTER 2
INTERACTIONS OF MAP-2 WITH TUBULIN
AND NF-L
Introduction
Neuronal cytoplasm is highly organized, and both
microtubules and neurofilaments run parallel with respect to
the axon's longitudinal axis in a manner suggesting
microtubule-to-neurofilament cross-linking (Wuerker and
Palay, 1969; Ellisman and Porter, 1980; Hirokawa, 1982). In
vitro observations indicate that microtubules interact with
neurofilaments, and MAPs can enhance the attainment of high
solution viscosity and/or gelation (Runge et al., 1981;
Aamodt and Williams, 1984b; Minami and Sakai, 1983;
Letterier et al., 1982). Aamodt and Williams (1984a) used
falling-ball viscosmetry to demonstrate the occurrence of an
optimal MAP level in plots of viscosity/gelation versus MAP
concentration; they likened this MAP concentration profile
to that of bivalent antibody cross-linking in
immunoprecipitin formation. Previous studies have traced
this apparently optimal MAP profile to the presence of
endogenous GTPase activity, and the inhibition of cross¬
linking/gelation at high MAPs can be eliminated with a GTP-
regenerating system (Flynn and Purich, 1987). The
requirement of GTP is to maintain microtubule stability as
microtubules will disassemble after all the GTP is converted
to GDP. Nonetheless, high-molecular-weight MAPs do bind to
29

30
microtubules (Kim et al.f 1979; Vallee, 1982; Purich and
Kristofferson, 1984) and neurofilaments (Runge et al., 1981;
Aamodt and Williams, 1984b; Minami and Sakai, 1983;
Letterier et al., 1982), and some interactions of the 280
kDa neuronal MAP-2 have been explored by limited proteolytic
fragmentation. Tubule binding is restricted to a 34-36 kDa
tryptic or chymotryptic fragment of MAP-2, and the remaining
240 kDa component corresponds to the lateral projections
observed in electron micrographs of microtubules decorated
with MAP-2 (Kim et al., 1979; Vallee and Borisy, 1977).
During the course of studies on neurofilament-
microtubule-MAP-2 interactions I sought to localize the
site(s) of neurofilament binding with respect to the tubule¬
binding and -projection domains of MAP-2. A thrombin
cleavage technique was developed to obtain these MAP-2
fragments in higher yields than that obtained with trypsin
or chymotrypsin. Interestingly, a 28 kDa tubule-binding
domain was found to contain a neurofilament-binding site.
My studies also suggest that this binding interaction has
considerable ionic character, as suggested by isoelectric
point determinations of the MAP-2 fragment.
Materials and Methods
Materials
Bovine thrombin (catalog number, T 4648) and the
catalytic subunit of cAMP-dependent protein kinase were
purchased from Sigma. Ultrapure ammonium sulfate and urea
were purchased from Schwarz-Mann, and carboxymethyl Sephadex
from Calbiochem. [32P]ATP (specific activity > 7000

31
Curies/mmol) was an ICN product, and Ampholines were
obtained from LKB. Assembly buffer for preparation of
microtubule-protein contained 0.1 M piperazine-N, N'-bis[2-
ethanesulfonic acid], 1 mM ethyleneglycol-bis[B-aminoethyl
ether]-N,N,N1,N1-tetracetic acid, and 1 mM magnesium
sulfate.
Preparation of proteins
Bovine brain microtubule-protein was prepared by the
procedure of Shelanski et al., (1973). Neurofilaments were
prepared from fresh bovine spinal cord by the method of
Delacourte et al., (1980) as modified by Letterier et al.,
(1982). Neurofilament triplet protein was prepared as
described by Tokutake et al., (1983), and the NF-L subunit
of neurofilaments was purified according to the method of
Geisler and Weber (1981). Tubulin was separated from MAPs
by the phosphocellulose method of Weingarten et al., (1975).
MAP-2 preparation and phosphorylation
MAP-2 was purified by the method of Herzog and Weber
(1978), concentrated by ammonium sulfate precipitation, and
phosphorylated by the catalytic subunit of cAMP-dependent
protein kinase prior to gel filtration chromatography.
Typically 500 units of kinase was dissolved in 0.025 ml
dithiothreitol (50 mg/ml), incubated at room temperature for
10 minutes, and used immediately with 1.5 millicuries
[32P]ATP, 0.02 mM unlabeled ATP, for 30 minutes at 37°C in
the presence of approximately 40 mg heat-stable MAPs. The
MAPs were separated on a BioGel A-1.5M column with the MAP-
2 fractions pooled and concentrated in a dialysis bag

32
against dry carboxymethyl Sephadex at 4°C. This purified
[32P]MAP-2 was clarified by centrifugation at 130,000 x g
for 25 minutes in a Beckman Airfuge prior to digestion and
incubations with cytoskeletal proteins.
Digestion of MAP-2 with thrombin
Purified and radiolabeled MAP-2 (50,000 CPM//xg) was
incubated at 0.4 mg/ml with 4 units/ml thrombin. To
determine the optimum time of digestion, aliquots were taken
at 5 minute intervals from 0 to 30 minutes. Once an optimum
time of 30 minutes was determined, all digestions of MAP-2
prior to incubation with cytoskeletal proteins were
conducted for 30 minutes and quenched by the addition of 1
mM phenylmethylsulfonyl fluoride and incubation on ice.
Sedimentation of polymerized protein
Thrombin-digested or undigested MAP-2 was incubated
with neurofilaments for 10 minutes at 4°C, or microtubules
for 30 minutes at 37°C. The incubations were then layered
over 20% (w/v) sucrose in assembly buffer and centrifuged at
130,000 x g for 20 minutes. Supernatant fractions were
discarded and the pellet fractions were washed with 1 mg/ml
bovine serum albumin and 0.1% (v/v) Triton X-100 and
resuspended in 8 M urea. Aliquots of both supernatant and
pellet fractions were analyzed for radioactivity by liquid
scintillation spectrophotometry, and an equal number of
counts were loaded on 7-17% (w/v) polyacrylamide gels.
Polyacrylamide gel electrophoresis
Sodium dodecyl sulfate gel electrophoresis was carried
out as described by Laemmli (1970), and nonequilibrium pH

33
gel electrophoresis (NEPHGE) was performed by the method of
Roberts et al., (1984). For the NEPHGE gels radiolabeled
MAP-2 was digested with thrombin and the digestion was
quenched by the addition of an equal volume of 9.3 M urea,
0.5% (w/v) dithiothreitol and 2% (v/v) Nonidet NP-40 in 5 mM
potassium carbonate. The digests were loaded onto "NEPHGE"
tube gels with 1% (v/v) Ampholine 9-11 and 2% (v/v)
Ampholine 3.5-10, and run for 2000 volt-hours. Gels were
stained with coomassie blue, destained, dried under vacuum,
and exposed to Kodak X-AR5 film at -80°C.
Results
Binding to microtubules and neurofilaments
In view of the high molecular weight of MAP-2,
proteolytic fragmentation by trypsin or chymotrypsin has
proven to be useful in defining the MAP-2 domain(s)
interacting with other cytoskeletal components (Olmsted,
1986). Vallee (1980) first demonstrated that MAP-2 can be
fragmented into 35 and 240 kDa components by chymotrypsin or
trypsin. The smaller fragment contains the microtubule¬
binding domain, and the larger is designated as the
projection-arm domain. While these protease cleavage
products have been very useful in many investigations of
microtubule self-assembly, chymotryptic and tryptic cleavage
do not yield stable limit polypeptides, and the stability of
such proteolytic fragments is quite limited, leading to the
loss of the initially cleaved domains and the ability of
these fragments to stimulate microtubule assembly. In a
survey of the action of other proteases, I observed that

34
thrombin, an arginine-specific serine protease,
predominantly yielded MAP-2 fragments of 28 kDa and 240 kDa.
MAP-2 cleavage can be readily assessed by SDS gel
electrophoresis of [32P]MAP-2 because this protein is
extensively phosphorylated (Theurkauf and Vallee, 1983). As
seen in Fig. 2-1 this 28 kDa fragment is very stable and
very resistant to further digestion over the time points
shown. Indeed, phenylmethanesulfonyl fluoride at 1.0 mM
final concentration blocked any further degradation over a
five to seven day period at 4°C.
This development has allowed for probing with much
greater ease the interactions of MAP-2 fragments with
neurofilaments and microtubules. The basic approach is to
determine which proteins or protein fragments cosediment
with assembled microtubules or neurofilaments using
ultracentrifugation and subsequent electrophoretic analysis.
First MAP-2 was enzymatically phosphorylated with the cAMP-
dependent protein kinase to a level of 1 mole of added
phosphate per mole of MAP-2 based on the conditions of
Letterier et al., (1982). After purification of the
radiolabeled protein by gel filtration chromatography, the
MAP-2 was concentrated, and aliquots were digested with
thrombin. Next, the thrombin-digested fragments (indicated
by the plus sign) or undigested MAP-2 (indicated by the
minus sign) were incubated with microtubules or
neurofilaments under the conditions listed in Fig. 2-2. The
polymerized and unpolymerized cytoskeletal proteins were
separated into pellet [p] and supernatant [s] fractions by

35
Fig. 2-1 Thrombin digestion of radiolabeled MAP-2.
[32P]MAP-2 was incubated at 37 °C with 4 units/ml thrombin
for the indicated time in minutes. The digestion was
quenched by heating at 100°C for 5 minutes in the presence
of sodium dodecyl sulfate and the products resolved on a 15%
(w/v) polyacrylamide gel. The gel was then dried under
vacuum and expose to Kodak X-AR 5 film.

36
ultracentrifugation. Because the projection and tubule¬
binding domains do not contain identical phosphorylation
sites, a constant total amount of radioactivity was applied
for each electrophoretic sample. Lanes 1-4 of the coomassie
stained gel in Fig. 2-2 and the corresponding lanes of the
autoradiogram in Fig. 2-3 demonstrate that only the 28 kDa
thrombin-produced fragment of MAP-2 binds to neurofilaments.
The next four lanes in both Fig. 2-2 and Fig. 2-3
demonstrate that this thrombin fragment behaves as the so-
called microtubule-binding domain of MAP-2 as it binds to 1
mg/ml of taxol-stabilized microtubules composed solely of
tubulin. In the absence of neurofilaments or microtubules
the 28 kDa fragment remained in the supernatant fraction
even after ultracentrifugation as seen in lanes 9-12 of Fig.
2-3. Indeed, the entire pellet fraction was used for the
electrophoretic analysis in lanes 10 and 12 of Fig. 2-3, and
virtually no high-molecular-weight or fragmented MAP-2
cosedimented without neurofilaments present. These
observations verified that the fragment is only sedimentable
as a result of interactions with either neurofilaments or
microtubules.
Next, I sought to determine the neurofilament
protein(s) interacting with MAP-2 or the 28 kDa fragment.
Earlier work by Miyata et al., (1986) and Heimann et al.,
(1985) demonstrated MAP binding to the low-molecular-weight
subunit of neurofilaments. A second series of binding
assays were conducted and Fig. 2-4 shows the coomassie blue
staining pattern and Fig. 2-5 shows the corresponding

37
NF NoNF
Protein Tubulin or Tb
M /++ —\/+ +—V+ + - M
\S p s pAs p S paS P s P/
12 34 56 78 9 10 II 12
Fig. 2-2 Binding of MAP-2 or MAP-2 fragments to
neurofilaments or tubulin. A coomassie blue stained
gradient 7-17% (w/v) polyacrylamide gel is shown.
Radiolabeled MAP-2 was incubated with (+) or without (-)
thrombin as described in Fig. 2-1, quenched with 1 mM
phenylmethylsulfonyl fluoride and incubated with 2 mg/ml
neurofilament protein or 1 mg/ml taxol-stabilized tubulin
with 1 mM guanosine triphosphate. Samples were then handled
as described in "Methods”. M refers to molecular weight
markers, s to supernatant fraction, and p to pellet
fraction.

38
NF No NF
Protein Tubulin or Tb
4 + -A4 + _A/h+--\
r nr i»i i -2°°k
—116K
-97.4 K
-66.2K
-42.7 K
4
\S p S P/\S p S P/\S p S P/
Fig. 2-3 Autoradiogram of MAP-2 and MAP-2 fragment binding
to cytoskeletal protein. The coomassie blue stained
polyacrylamide gel shown in Fig. 2-2 was soaked in 25% (v/v)
glycerol for 30 minutes after destaining and dried under
vacuum. The dried gel was exposed to x-ray film for 4 hours
without intensifying screens.

39
autoradiogram. Lanes 3, 4 and 7, 8 of Fig. 2-5 verified
that uncleaved MAP-2 binds to the neurofilament triplet and
to filaments composed solely of L subunit. The data shown
in lanes 1, 2 and 5, 6 of Fig. 2-5 extend the earlier
observations by clearly demonstrating the binding of the 28
kDa fragment to the neurofilament triplet and L component of
neurofilaments. It should be noted that only trace levels
of tubulin are evident in the 50-55 kDa molecular weight
range in the gels shown in Fig. 2-2. Much higher levels of
tubulin are required for binding of MAP-2 to assembled
tubules. This suggests that binding depends on presence of
neurofilaments and does not require tubulin or assembled
microtubules for binding.
Determination of the isoelectric point of the 28 kDa
fragment
Tubulin and neurofilament proteins contain negatively
charged regions that may be important in MAP binding
(Olmsted, 1986). The data presented in the preceding
section demonstrate that the 28 kDa fragment of MAP-2 binds
to both cytoskeletal organelles. Accordingly, I tried to
use conventional isoelectric focusing to determine the
isoelectric point of the radiolabeled fragment. This
consistently failed at all ranges of ampholytes, and the
fragment never migrated into the first dimension of a two
dimensional gel. This is indicative of very basic proteins.
In order to estimate the isoelectric point of very basic
neurofilament subunit proteins the Nonequilibrium pH gel
electrophoresis technique is used (Roberts et al., 1984).
When this technique was tried, the fragment readily migrated

40
NF NF
Triplet Low NoNF
M /++ \/++ \4+__\ M
\s P s PAS p S P7\S p s P /
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 2-4 MAP-2 binding to purified neurofilament triplet
protein or the L subunit of neurofilaments. A coomassie
blue stained gradient 7-17% (w/v) polyacrylamide gel is
shown. Radiolabeled MAP-2 was incubated with thrombin as
described in Fig. 2-2 and in "Methods", and incubated with
either 2.2 mg/ml neurofilament triplet or 1.1 mg/ml L
subunit at 4°C for 10 minutes. Nomenclature is the
in Fia. 2-2.
same as

41
NF NF No
Triplet Low NF
4 + —^4 + --^4+--\
e ”i:ri ? ~2ook
—116K
-97.4 K
—66.2K
-42.7 K
\s p s p/\s p s p/\s psp/
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 2-5 Autoradiogram of MAP-2 and MAP-2 fragment binding
to Neurofilament triplet protein and L subunit. The
coomassie blue gel shown in Fig. 2-4 was soaked in 25% (v/v)
glycerol for 30 minutes after destaining and dried under
vacuum. The dried gel was exposed to x-ray film for 4 hours
without intensifying screens.

42
into the first dimension of the gel as seen in Fig. 2-6, and
it migrated between two very basic isoelectric point
markers, ribonuclease (pl=9.3) and lysozyme (pl=10.5-11.0).
The thick arrow denotes the position of the 28 kDa fragment
in the gel. From this migration pattern, the isoelectric
point of the 28 kDa fragment of MAP-2 was estimated to be
approximately 10. The migration of the trace-labeled
fragment in both dimensions also indicated that the
phosphorylation conditions do not lead to significant
heterogeneity in overall ionic charge or molecular weight.
Furthermore, the dephosphorylated form will necessarily
display an even higher isoelectric point. The large smear
at the top right side of the gel is the projection domain
and further breakdown products of this domain. These
fragments are acidic and do not readily migrate into the
gel.
Discussion
The findings presented in this chapter indicate that
neurofilaments, a specific class of intermediate filaments,
interact with MAP-2 in the region corresponding to the 28
kDa microtubule-binding domain. In earlier work with actin,
Sattilaro (1986) reached a similar conclusion about the
binding of chymotryptic fragments of MAP-2 to polymerized
actin. Likwise, this same fragment of MAP-2 constitutes the
microtubule-binding domain (Vallee, 1980). Thus, all three
major cytoskeletal classes (i.e. microfilaments,
intermediate filaments, and microtubules) interact with a
common structural region of MAP-2. In view of our estimated

43
NEPHGE
Fig. 2-6 Determination of the isoelectric point of the 28
kDa fragment of MAP-2. An autoradiogram of a two
dimensional gel is shown. The first dimension is
Nonequilibrium pH gel electrophoresis and the second
dimension is sodium dodecyl sulfate polyacrylamide gel
electrophoresis with a 7-17% (w/v) gradient of acrylamide.
Protein molecular weight/isoelectric point reference
standards were: 1) phosphorylase b; 2) bovine serum albumin;
3) carbonic anhydrase; 4) soybean trypsin inhibitor; 5)
ribonuclease and 6) lysozyme.

44
isoelectric point value of 10 for the 28 kDa component, all
of these MAP interactions must be strongly influenced by
electrostatic charge. Vallee (1982) has clearly
demonstrated that 0.35 M sodium chloride can remove MAPs
from taxol-stabilized microtubules, and this observation is
also in harmony with the notion of ionic interactions
between tubules and MAPs. Furthermore, it has been shown
that increased phosphorylation of MAP-2 reduces its ability
to promote tubulin polymerization (Murthy and Flavin, 1983).
My use of trace labeling of heat-stable MAPs with
[32P]ATP and protein kinase reinforces the general utility
of this method as first applied by Heimann et al., (1985).
The radiolabeled low-molecular-weight thrombin fragment of
MAP-2 is more stable than that obtained with trypsin and
chymotrypsin, and this stability may also facilitate studies
of the stoichiometry and dissociation constants for fragment
binding to microtubules, neurofilaments, or actin. Other
more approximate methods using intact MAPs and either
densitometry or pelleting of assembled cytoskeletal elements
(Miyata et al., 1986; Heimann et al., 1985) still require
refinement and/or verification.
Finally, the thrombin results underscore the facility
and generality of serine protease of MAP-2 into low- and
high-molecular-weight fragments. This behavior is
reminiscent of proteolytic action on myosin. Furthermore,
intact MAP-2 has an isoelectric point of 5.4 (Berkowitz et
al., 1977), whereas I found that the tubule/filament-binding
domain has a value of 10. This suggests that there must be

45
significant acidic and basic charge localization in the
high- and low-molecular-weight fragments, respectively, of
MAP-2. Nonetheless, the biological significance of this
protein design feature awaits further understanding of the
role of MAP-2 in the cytomatrix.

CHAPTER 3
THE MICROTUBULE-BINDING FRAGMENT OF MAP-2:
LOCATION OF THE PROTEASE-ACCESSIBLE SITE
Introduction
The thrombin protease digestion of MAP-2 shown in Fig.
2-1 revealed a very stable 28 kDa fragment. This fragment
possessed the sequences responsible for cosedimentation of
MAP-2 with microtubules as well as neurofilaments. Since
this fragment contained the "active site" of MAP-2, I wanted
to learn more about its biochemical properties. It was
already known the fragment had a very basic isoelectric
point and it was suspected that the basic residues were
involved in the binding to the anionic termini of both alpha
and beta tubulin. A second interesting point concerning the
structure of the fragment was the specificity and stability
of the protease digestion products. The autoradiogram of
Fig. 2-1 showed no heterogeneity in the 28 kDa microtubule¬
binding fragment and limited heterogeneity in the very
protease-sensitive 240 kDa domain. However, limited
heterogeneity in the larger fragment could be observed by
the low resolving power of the 15% (w/v) polyacrylamide gel
for large molecular weight proteins. Nevertheless, the
specificity of the digestion in producing the 28 kDa
fragment was impressive. This specificity implied some
higher order structure in the MAP-2 molecule yet a previous
46

47
report on the circular dichroism spectrum of MAP-2 revealed
little alpha-helical or beta-pleated sheet content. If
there was no higher order structure, I would expect to see a
far more complex pattern of digestion. In order to resove
this paradox a large scale purification of the microtubule¬
binding fragment of MAP-2 was undertaken.
In addition, the structure of MAP-2 was probed by
protease digests with and without the presence of
microtubules. The low-molecular-weight digestion products
were isolated and their amino terminal composition checked
by Edman sequencing.
Materials and Methods
Materials
[32P]ATP (7000 Ci/mmol) was purchased from ICN along
with ultrapure grades of ammonium sulfate, sodium dodecyl
sulfate (SDS), acrylamide, and bis-acrylamide. Immobilon
was obtained from Millipore Corporation and coomassie
brilliant blue R-250 was from Serva. DEAE-Sephadex A-50 was
purchased from Pharmacia; and bovine thrombin,
trifluoroacetic acid, Mes buffer, and phenylmethane-
sulfonylfluoride (PMSF) were from Sigma.
Preparation of proteins
Bovine brain microtubule protein was prepared by the
procedure of Shelanski et al. (1973). MAP-2 was purified by
the method of Herzog and Weber (1978) and radiolabeled as
previously described (Flynn et al. 1987).

48
Preparation of heat-stable microtubule-binding fragments
Heat-stable microtubule-binding fragments were prepared
according to Vallee (1986) with the following modifications.
Thrombin was used instead of chymotrypsin at 8 U/ml and 37°C
for 30 minutes to digest thrice cycled bovine microtubule-
protein at a concentration of 5 mg/ml. PMSF was added to 1
mM at the end of the digestion to stop proteolysis. The
assembled tubules were sedimented at 100,000 x g for 75
minutes at 30°C and the pellet was resuspended in 0.75 M
NaCl and 1 mM dithiothreitol. After homogenization and
incubation on ice for 30 minutes the protein was heated in a
boiling water bath for nine minutes followed by cooling on
ice for 20 minutes. The resulting slurry was centrifuged
for 30 minutes at 15,000 x g at 4°C. The supernatant
fraction contained heat-stable microtubule-binding fragments
from tau and MAP-2. These heat-stable binding fragments
were concentrated by ammonium sulfate precipitation and then
dialyzed against microtubule assembly buffer (100 mM Mes, pH
6.8, 1 mM EGTA, and 1 mM magnesium sulfate) at 4°C with 1 mM
PMSF, and passed over a 1 ml DEAE-Sephadex A-50 column
equilibrated in the same buffer. The breakthrough fractions
were pooled and precipitated with 60% (w/v) ammonium
sulfate. After sedimentation, the precipitate was
resuspended in assembly buffer and used for HPLC analysis or
Immobilon blotting.

49
Preparation of the 28 kDa fragment of MAP-2 without
microtubules present during digestion
Purified MAP-2 corresponding to 0.6 mg at a
concentration of 2.0 mg/ml was digested with 5 U/ml thrombin
for 30 minutes at 37°C. Digestion was quenched with 2 mM
PMSF and cooling on ice for 10 minutes. The digestion
products were passed over a 1 ml DEAE-Sephadex A-50 column
and handled as described in the preceding section.
HPLC purification of the microtubule-binding fragment of
MAP-2 and its amino acid analysis
High performance liquid chromatography was carried out
on a Hewlett Packard Model 1090a chromatograph, equipped
with a diode array detector. The ammonium sulfate
concentrated, microtubule-binding fragments were clarified
by centrifugation at 3000 x g for 5 minutes prior to loading
on a Waters Associates C-18 column, equilibrated in 0.1%
(v/v) trifluoroacetic acid. The protein was eluted with a
linear gradient from 0-50% (v/v) acetonitrile with 0.1%
(v/v) trifluoroacetic acid at a flow rate of 0.5 ml/min and
1 ml fractions were collected. The elution profile was
monitored at a wavelength of 220 nm because the fragments
are very low in aromatic amino acid content. Initially,
polyacrylamide gel electrophoresis was used to check the
composition of material in each peak. Fractions containing
the MAP-2 microtubule-binding domain were pooled, dialyzed
against 100 mM ammonium bicarbonate, lyophilized, and then
hydrolyzed in 6 N HC1 for 24 hours at 110°C. Samples were
analyzed with a Beckman Model 6300 Amino Acid Analyzer.

50
SDS electrophoresis and blotting
Immobilon was handled according to the manufacturer's
instructions prior to electroblotting. The
polyvinylidenedifluoride membrane was wetted in 100% (v/v)
methanol for 5 minutes followed by soaking in distilled
water for another 5 minutes and then it was allowed to dry.
Ultrapure grades of SDS, acrylamide, and bis-acrylamide were
used to avoid blocking the N-terminus. The electrophoretic
samples for sequencing were heated to 80°C for 5 minutes
after adding Laemmli sample buffer which contained ultrapure
SDS but no bromophenol blue dye. As an indicator of when
the electrophoresis was finished molecular weight markers
were run in adjacent lanes with dye present. To scavenge
any radicals that could possibly react with the samples,
0.1% (v/v) thioglycolate was added to the top chamber
buffer. A 12% (w/v) acrylamide SDS-gel containing heat-
stable microtubule-binding fragments was electrophoretically
transferred to the membrane in 10 mM CAPs, pH 10.0, 10%
(v/v) methanol for 6 hours at 70 volts. The membrane was
stained with Coomassie Brilliant Blue R-250, destained in
50% (v/v) methanol-10% (v/v) acetic acid. The blot was air
dried and stored at -20°C in the dark until the sequencer
was available. The band of interest was excised with a
razor blade and sequenced in a gas-phase protein sequencer
(Applied Biosystems 470A protein sequencer) with on-line
phenylthiohydantoin analyzer at the Protein Chemistry core
facility.

51
Results
Site of thrombin cleavage
In order to gain more information about this site of
facile thrombin cleavage, a high-yield isolation method was
developed for amino acid analysis and sequencing
experiments. I again employed thrombin, but digested
assembled three-cycle microtubule-protein (i. e.. tubulin and
MAPs), followed by heat-treatment of the resulting pellet to
remove all tubulin and heat-labile MAPs. Then, DEAE-
Sephadex ion-exchange chromatography was used to separate
the heat-stable microtubule-binding fragments from any high
molecular weight digestion products. This ion-exchange
chromatography step was a key part of the purification and
took advantage of the basicity of the microtubule-binding
fragments as they did not interact with the resin while any
incompletely digested MAPs and any contaminating projection
domain did interact with the resin very strongly. This is
shown in Fig. 3-1. The breakthrough fractions from the
DEAE-Sephadex chromatography contained the microtubule¬
binding fragments of the heat-stable proteins MAP-2 and tau
(see lane 5) resulting in greater heterogeneity than that
seen in Fig. 2-1 where purified MAP-2 was digested. I also
found that a tau monoclonal antibody recognized the upper
bands in lane 5, identifying them as putative digestion
products of tau.
At this point in the purification scheme, I observed
four closely spaced bands on a gel ranging from 28 to 36

52
M 1 2 3 4 5
Fig. 3-1 Purification of heat-stable microtubule-binding
fragment of MAP-2 and tau. Microtubule-protein was digested
with thrombin as described under "Methods". The coomassie
blue stain of a 12% (w/v) polyacrylamide gel shows the
purification during its various stages. Lane 1 is before
cleavage; 2, after cleavage; 3, supernatant after
centrifugation; 4, heat-stable protein; 5, after DEAE-
Sephadex chromatography; 6, a larger amount of sample 5. M
corresponds to molecular weight markers.

53
kDa. Many different ion-exchange resins were tried to
separate these bands such as strong and weak cation
exchangers, and hydroxyapatite chromatography, but all were
unsuccessful. A two-dimensional gel using the NEPHGE
technique in the first dimension was run and all four
fragments were found to be very basic but each fragment was
slightly less basic with increasing molecular weight. This
indicated that the use of ion-exchange resins to separate
the fragments was probably futile and another means of
separation was necessary. The microtubule-binding fragments
in lane 5 of Fig. 3-1 can be readily separated by reverse-
phase HPLC as shown in Fig. 3-2 where SDS gel
electrophoresis revealed that peak C corresponded to the 28
kDa component. This lane also revealed that there is only
minor contamination by a faster migrating component.
Fraction C was subjected to acid-catalyzed hydrolysis and
amino acid analysis, and these results are listed in Table
3-1. I had previously reported this fragment had an
unusually high isoelectric point in comparison with intact
MAP-2 (Flynn et al. 1987), and the amino acid analysis
confirmed this observation. The fragment is comprised of
nearly 14 mole percent in lysyl and arginyl residues.
Curiously it contains a higher than usual proline content.
The analysis also confirmed the fact that this fragment was
low in aromatic amino acids as it contained only two
tyrosine residues and three phenylalanine residues. This
low content of aromatic residues makes it very difficult to
monitor its purification at 280 nm as is usually done for

Absorbance, 220 nm
54
Time after Injection, min
Fig. 3-2 HPLC purification of the microtubule-binding
fragment of MAP-2 digested with microtubules present. The
MAP-fragments seen in lane 5 of Fig. 3-1 were separated by
reverse phase HPLC and the fractions resolved by a 12% (w/v)
polyacrylamide gel (see inset) which is coomassie stained.

55
TABLE 3-1
Amino acid composition of the 28 kDa MAP-2 fragment
Amino Acid
24 h Hydrolysate
(mol%)
Estimated
Residues/mol
Asx
in
•
CO
21
Thr
5.2
14
Ser
9.7
24
Glx
9.2
22
Pro
6.9
18
Gly
9.0
24
Ala
7.5
18
Val
5.5
15
Met
0.2
0
He
4.7
12
Leu
8.1
20
Tyr
0.8
2
Phe
1.4
3
Lys
9.6
25
His
2.9
7
Arg
4.3
11

56
proteins. Also the analysis was rich in its glutamate +
glutamine content as well as its aspartate + asparagine
content even though it has already been established the
fragment was very basic. This meant that most of these
amino acids were in the amide form rather than the
carboxylic acid form.
Amino-terminal sequence of the microtubule-binding fragment
Microsequencing techniques were employed with the 28
kDa fragment electroblotted from SDS-polyacrylamide gels to
a derivatized nylon screen (Immobilon). This allowed
further reduction in any contamination by other MAP
fragments such as that observed in the HPLC preparation.
The amino terminal sequence obtained was, Thr-Pro-His-Thr-
Pro-Gly-Thr-Pro-Lys-Ser-Ala-Ile-Leu-Val-Pro-Ser-Glu-Lys-
Lys, based on the results listed in Table 3-2. In the
absence of thrombin treatment, identical sequence
experiments with either electroblotted MAP-2, as well as
MAP-2 in solution, did not yield any phenylthiohydantoin
derivatized amino acids at detectable levels. Likewise,
experiments with the immobilized 240 kDa projection-arm
fragment yielded no sequence data. Protein samples failing
to yield detectable levels of amino acid derivatives were
subjected to acid-catalyzed hydrolysis and amino acid
analysis to assure that sufficient levels of protein for
sequencing had been employed. In all cases adequate levels
of protein were present above the detection limits for

57
TABLE 3-2
Amino-terminal sequence analysis of the 28 kDa
MAP-2 fragment
Cycle No.
Residue
(pmol)a
Cycle No.
Residue
(pmol)
1
Thr
74
11
Ala
103
2
Pro
131
12
He
74
3
His
15
13
Leu
144
4
Thr
42
14
Val
82
5
Pro
122
15
Pro
102
6
Gly
84
16
Ser
65
7
Thr
54
17
Glu
39
8
Pro
105
18
Lys
105
9
Lys
141
19
Lys
78
10
Ser
85
a Values reported correspond to the sum of the major,
leading, and trailing cycle yields for each amino acid.

Fig. 3-3 Comparison of proteolytic fragmentation patterns and the amino-terminal sequences
of the microtubule-binding fragments of MAP-2 and tau protein. The polypeptide chains and
cleavage patterns for MAP-2 and tau are represented as heavy lines. The closed circles
represent the blocked MAP-2 N-terminus, and the underlined amino acid residues represent
identical and/or conserved amino acid residues common to both MAP-2 and tau proteins. The
tau protein scheme is based on the data of Aizawa et al. (1988) and Lewis et al. (1988).
Arrowheads denote octadecapeptide imperfect repeats.

MAP-2:
240 kDa 28 kDa
:— JT —
r 'i
Thr-Pro-His-Thr-Pro-GIv-Thr-Pro-Lvs-Ser-Ala-lle-Leu-Val-Pro-Ser-Glu-Lvs-Lvs
Tau Proteins:
. j i— i * i
Ser-Ser-Pro-Gly-Ser-Pro-GIv-Thf-Pro-GIv-Ser-Ara-Ser-Afq-Thf-Pro-Ser-Leu-Pro

60
sequencing. These findings suggested that the amino-
terminus of MAP-2 is blocked and that the 240 kDa fragment
is derived from the amino-terminus whereas the 28 kDa
fragment resided at the carboxyl end. These observations
are in accord with the findings by Kosik et al. (1988) who
reported that the N-terminus of MAP-2 appears to be blocked;
moreover, Lewis et al. (1988) reported the entire derived
amino acid sequence using murine MAP-2 cDNA clones. The
primary sequence data with bovine brain MAP-2 correspond to
the murine sequence spanning residues 1626 to 1644 with only
three exceptions. As shown in Fig. 3-3, there is a similar
protease-accessible sequence in the microtubule-binding
fragment of bovine tau protein. In that case, however,
fragments were generated by chymotryptic cleavage (Aizawa et
al., 1988). Both of these cleavage-site sequences reside
approximately thirty-to-forty residues toward the N-terminal
side of the first of three nonidentical octadecapeptide
repeats (indicated schematically by the arrowheads) found in
both MAP-2 and tau (Lewis et al., 1988; Lee et al., 1988).
Amino-terminal sequence of the microtubule-binding fragment
after digestion in the absence of microtubules
My initial experiments to determine if the microtubule¬
binding fragment of MAP-2 also bound to neurofilaments,
digested purified MAP-2 alone in solution. In the preceding
section the amino terminal sequence of the binding fragment
was determined but the fragment was generated by digestion
of microtubule-protein at 37°C in the presence of 1 mM
guanosine triphosphate. In this regard, MAP-2 was digested
in the presence of microtubules, a condition that might have

61
influenced the cleavage point of thrombin were the preferred
site of digestion hindered by interaction with the
microtubule lattice. To determine whether the cleavage
point is the same for the microtubule-binding fragment under
the two different conditions, the amino terminal sequence
was checked once again but now in the absence of
microtubules during the digestion. Purified MAP-2 was
cleaved alone in solution, and passed over a DEAE ion-
exchange column to remove the projection domain. The
breakthrough fractions containing the microtubule-binding
domain were pooled, electrophoresed through a polyacrylamide
gel, transferred to a polyvinylidenedifluoride membrane, and
sequenced by automated Edman chemistry. Ten cycles were
performed yielding ten residues that were exactly the same
as the first ten residues in Table 3-2 and Fig. 3-3. This
indicated that thrombin cleaved MAP-2 at the same argininyl
residue in the presence and absence of microtubules.
Additionally, the breakthrough fractions from the DEAE-
Sephadex column were subjected to reverse phase HPLC similar
to the fragments generated in the presence of microtubules.
When loaded on a C-18 column and eluted with the same
gradient as mentioned in the preceding section, one major
peak corresponding to the 28 kDa fragment was seen (see Fig.
3-4) and one minor peak corresponding to peak A in Fig. 3-2
was seen. The elution time for the 28 kDa fragment was
unchanged from Fig. 3-2 and corresponded to peak C from this
figure.

Absorbance, 220 nm
62
Time after Injection,min
Fig. 3-4 HPLC purification of the microtubule-binding
fragment of MAP-2 digested initially without microtubules.
The inset shows a 12% (w/v) polyacrylamide gel before and
after the HPLC purification: lane 1, digest before
purification; lane 2, fraction 22; lane 3, fraction 23; lane
4, fraction 30; lane 5, fraction 31. Fractions were
collected every two minutes and were 1 ml in volume.

63
Discussion
The experiments described in this chapter were
designed to gain further insight about the microtubule¬
binding fragment of MAP-2. There is now general agreement
that initial proteolytic cleavage of MAP-2 yields two
fragments (Vallee 1980; Flynn et al. 1987). With thrombin,
these initial cleavage products corresponding to values of
240 kDa and 28 kDa based on electrophoresis, are guite
stable with regard to further degradation. All efforts to
seguence Immobilon-1inked MAP-2 and the similarly
immobilized 240 kDa projection arm fragment consistently
failed to yield any detectable levels of PTH-amino acids.
Nevertheless, acid hydrolysis and subseguent amino acid
analysis of these immobilized proteins demonstrated that
sufficient levels were clearly present for detection in the
gas-phase sequencer. This observation led me to believe
that intact MAP-2 showed no evidence of a free N-terminus,
and another group recently reported the same difficulty in
attempts to sequence MAP-2 (Kosik et al. 1988). These
observations suggest that the MAP-2 amino acid sequence, as
derived from nucleotide sequence data (Lewis et al. 1988),
does not provide a complete account of the MAP-2 primary
structure, and further work will be required to establish
the nature of the N-terminal modification blocking Edman
degradation.
A striking common structural feature in MAP-2 and tau
emerges from the combined findings of Aizawa et al. (1988)
and these studies. The former found that chymotryptic

64
cleavage of the bovine tau proteins yielded a microtubule-
binding fragment with the N-terminal sequence shown in Fig.
3-3, and I have now demonstrated that thrombin attacks at a
similarly accessible region in bovine MAP-2 (See also Fig.
3-3). It should be noted that both of these cytomatrix
proteins have four proline residues in exact registration,
and with the exception of the occurrence of a val-pro in the
MAP-2 sequence, each of the prolines in both cleavage sites
was preceded by a hydroxy-amino acid. Efforts to survey
other known sequences in the GenBank database have indicated
the uniqueness of these protease-accessible regions in tau
and MAP-2; however, Earnshaw et al. (1987) described a
centromere-binding protein containing three prolines in
exactly corresponding positions with little other structural
relatedness to tau and MAP-2. Also it should be noted that
the NF-M sequence in chicken contains a proline at every
third residue for 102 residues in the repeated sequence
(EXPXSP)17 (Zopf et al., 1987). The circular dichroism
spectral data of Hernandez et al. (1986) indicates that
uncleaved MAP-2 contains little, if any, alpha helical or
pleated-sheet secondary structure; yet, the preferential
action of the endoprotease thrombin at a single site
suggests that MAP-2 may display some "hinge-point" behavior
akin to the protease-accessible region of myosin. This
region may permit the projection arm to swing away from the
microtubule surface. Certainly, the observed sedimentation
coefficient of 4.5 (Hernandez et al. 1986) also suggests
that MAP-2 has extended a flexible structure. The roughly

65
spherical hemoglobin molecule, itself only one-third the
molecular weight of MAP-2, has an almost identical
sedimentation coefficient (Sanders et al. 1981).
Chymotryptic cleavage between Tyr-128 and Ser-129 in the tau
proteins may reflect a corresponding protease-accessible
site of structural discontinuity between microtubule-binding
and projection domains. These hinge point regions may be
very important in their presentation of the microtubule¬
binding sequences that actually interact with tubulin.

CHAPTER 4
THE MICROTUBULE-BINDING FRAGMENT OF MAP-2:
IDENTIFICATION OF AN ASSEMBLY-PROMOTING PEPTIDE
AND DISPLACEMENT OF HIGH-MOLECULAR-WEIGHT MAPs
Introduction
Microtubule-associated proteins (MAPs) exhibit one of
several properties: the ability to copolymerize with
tubulin during microtubule assembly, the capacity to utilize
tubulin or another MAP as substrates for enzyme-catalyzed
modification, or the use of microtubules as the
architectural framework for motility (Olmsted, 1986; Purich
and Kristofferson, 1984). The first property is shared by
the high-molecular-weight proteins (MAP-1 and MAP-2) as well
as the tau proteins, and these proteins remain associated
with reassembled microtubules during the course of
microtubule-protein purification. Recently, the cDNA-
derived amino acid sequences of the murine MAP-2 (Lewis et
al., 1988) and the murine tau (Lee et al., 1988) proteins
have been defined, and these proteins were both found to
contain a related triad of imperfectly repeated
octadecapeptide sequences in their tubule-binding regions.
Oligopeptide analogues of the repeated sequences in murine
tau and a 190 kD bovine adrenal gland MAP can promote
microtubule assembly as monitored by light scattering
techniques (Ennulat et al., 1989; Aizawa et al., 1989). I
wished to investigate whether the triad of imperfect
66

67
octadecapepti.de repeats of murine MAP-2 and a MAP-2 sequence
from the protease-accessible hinge region could promote
microtubule polymerization and mimic the action of MAPs.
While several peptides corresponding to sequences in
fibrous MAPs can stimulate microtubule assembly, very little
is known about whether these synthetic peptides constitute
the entire site necessary for the MAP binding to
microtubules. At the time little information on MAP-2
sequences reponsible for binding to microtubules or
promotion of tubulin polymerization was available. The
report of Lewis et al. (1988) showed a 100 residue
polypeptide consisting of the first two imperfect repeats
plus flanking sequences could cosediment with MAP-stabilized
microtubules. No information on the binding of small
peptides or promotion of tubulin polymerization was known.
The experiments described in this chapter attempt to define
an "active site" of MAP-2 by testing for sequences promoting
tubulin polymerization. The most likely candidates were the
repeated sequences since two of them were in the 100 residue
polypeptide of Lewis et al. (1988). These peptides were
chemically synthesized along with a hinge-region sequence
and tested for stimulation of tubulin polymerization.
If the repeated sequences are indeed the primary sites
of interaction, then those promoting tubule assembly in the
absence of MAPs may also displace MAPs from microtubules or
block their binding to microtubules. Moreover, I was
motivated to learn whether a particular peptide and MAP
display competitive binding behavior that would indicate the

68
peptide(s) binding to the same site as MAPs on the
microtubule. The effectiveness of all three MAP-2 repeated
peptide analogues in terms of MAP displacement from
microtubules was also checked. These experiments show that
peptides corresponding to the second repeated sequence of
MAP-2 can promote microtubule polymerization and displace
MAP-1 and MAP-2 from recycled microtubule-protein.
Materials and Methods
Materials
[32P]ATP (7000 Ci/mmol) and [3H]GTP (18 Ci/mmol) were
purchased from ICN along with ultrapure grades of ammonium
sulfate, sodium dodecyl sulfate (SDS), acrylamide, and bis-
acrylamide. Liquid scintillation cocktail 3a70 was obtained
from Research Products International. Acetate kinase was a
Boehringer Mannheim product, while phosphocellulose resin
and GF/F glass fiber filters were from Whatman. Anti-B
tubulin antibody was purchased from Amersham. DEAE-Sephadex
A-50 was purchased from Pharmacia; and bovine thrombin,
trifluoroacetic acid, Mes, Pipes, and Tris buffers,
dithiothreitol, guanosine triphosphate, bovine serum
albumin, EGTA, Triton X-100, phenylmethanesulfonylfluoride,
catalytic subunit of cAMP-dependent protein kinase, and goat
anti-murine IgG Texas red conjugate were from Sigma. t-BOC
amino acids and the phenylacetamidomethyl resin were from
Applied Biosystems International. Taxol was a gift supplied
by Dr. Matthew Suffness at the National Cancer Institute,
Bethesda Md.

69
Preparation of proteins
Isotonic bovine brain microtubule-protein was isolated
according to the method of Karr et al. (1979) and stored at
-80°C after two cycles of assembly-disassembly. Hypotonic
bovine brain microtubule-protein was isolated by the method
of Shelanski et al. (1973). Tubulin was prepared according
to the method of Kristofferson et al. (1986). [32P]MAP-2
was purified by the procedure of Herzog and Weber (1978) as
modified by Flynn et al. (1987) except the purified protein
was concentrated by ammonium sulfate precipitation after gel
filtration chromatography. Unlabeled MAP-2 was prepared
identically except the phosphorylation reaction was omitted
prior to the gel filtration column.
Preparation of synthetic peptides
All peptides were made with an Applied Biosystems
synthesizer model 430A according to the method of Erickson
and Merrifield (1976) with t-BOC protected amino acids and
starting with a phenylacetamidomethyl resin. Peptides were
cleaved and deprotected using a mixture of hydrogen
fluoride, anisóle, and dimethyl sulfide in a 9:1:0.3 ratio
(v/v) at -10"C for 2.5 hours. After evaporation the resin
was washed with cold diethyl ether and extracted into 1 M
acetic acid and then freeze dried. Purity was tested by
HPLC profile or by gas phase microsequencing. The peptides
were stored at -20°C as a lyophilized powder. All synthetic
peptides except the mN peptide were made by Dr. Jan Pohl of
the microchemical facility at Emory University. The mN

70
peptide was synthesized by the Protein chemistry core
facility at the University of Florida.
Microtubule assembly with synthetic peptides
All assembly experiments were done with a GTP-
regenerating system (MacNeal et al., 1977) consisting of 2
units/ml of acetate kinase, 10 mM acetyl phosphate, and 0.1
mM [3H]GTP (20 Ci/ml). All assay mixes also contained 1 mM
dithiothreitol to maintain reduced sulfhydryls in the
peptides. The peptides were weighed out just before use and
dissolved in 100 mM Pipes, pH 6.8, 1 mM EGTA, 1 mM magnesium
sulfate with 1 mM dithiothreitol. Varying concentrations of
each peptide were added to 1.6 mg/ml pure tubulin and 0.4
mg/ml three-cycle microtubule-protein and incubated at 30°C
for 30 minutes. The extent of microtubule assembly was
monitored by the rapid filtration assay of Maccioni and
Seeds (1978) as modified by Wilson et al. (1982).
Microtubules were diluted 20X into 100 mM Pipes, pH 6.8, 1
mM EGTA, 1 mM magnesium sulfate, 1% (v/v) glutaraldehyde,
10% (v/v) dimethylsulfoxide, 25% (v/v) glycerol, and 1 mM
ATP and kept at 30°C until ready to assay. The diluted
mixture was then applied to Whatman GF/F filters on a vacuum
filtration device presoaked in the same buffer except no
glutaraldehyde was used. Each filter was then washed with
15 ml of the same buffer and the radioactivity was
solubilized in 1.5 ml 0.1 N NaOH for 30 minutes followed by
addition of scintillation cocktail.

71
Preparation of microtubule seeds and elongation assay
Seeds were prepared according to Kristofferson et al.
(1986). Tubulin at 5 mg/ml was assembled with 1 mM GTP in
80 mM Pipes, pH 6.8, 10 mM magnesium chloride, 1 mM EGTA, in
30% (v/v) glycerol at 37°C for 30 minutes. The microtubules
were then sheared with 3 passes through a 22 gauge needle to
produce microtubule seeds. The seeds were diluted 100X into
0.5 mg/ml tubulin and varying concentrations of peptides.
After 30 minutes at 37°C the samples were handled as
described in the preceding section for measuring tritiated
guanine nucleotides.
Electron microscopy
Microtubules were diluted 10X into a warmed solution of
1% (v/v) glutaraldehyde in microtubule-assembly buffer and
fixed for one minute at room temperature. The fixed samples
were placed on a carbon coated Formvar 400 mesh grid and
negatively stained with 1% (w/v) uranyl acetate. Grids were
observed on a JEOL 100 CX microscope at 50,000X
magnification. Samples with and without peptide were
processed identically.
Fluorescence microscopy
Microtubules formed in the presence of synthetic
peptides were diluted 10X into a warmed solution of 1% (v/v)
glutaraldehyde and 0.1% (v/v) Triton X-100 in microtubule-
assembly buffer and fixed for 2 minutes at room temperature.
The microtubules were diluted to 50,000 times their original
concentration and an aliquot of 100 /xl was centrifuged on to
a glass coverslip at 30 psi in a Beckman airfuge for 20

72
minutes. The coverslip was then fixed in -20°C methanol for
4 minutes and blocked with 10 mg/ml bovine serum albumin in
phosphate buffered saline, pH 7.3, with 0.1% (v/v) Triton X-
100 for 10 minutes. The coverslip was then stained with a
murine anti-tubulin antibody at a dilution of 1:200 for 30
minutes followed by washing in the same buffer. A goat
anti-murine IgG secondary antibody conjugated with Texas red
fluorochrome was used at a dilution of 1:80 followed by
washing in phosphate buffered saline, pH 7.3, with 0.1%
(v/v) Triton X-100. The coverslips were mounted in 20 mM
Tris, pH 7.9, with 90% (w/v) glycerol and viewed with a
Zeiss epifluorescence microscope at 1000X power.
Isotonic microtubule experiments
Before use the isotonic bovine brain microtubule-
protein was carried through a third cycle of
assembly/disassembly, and the concentration of protein was
determined by the method of Bradford (1976). Synthetic
peptides were weighed out just prior to use and dissolved in
PEM buffer (100 mM Pipes, pH 6.8, ImM EGTA, 1 mM MgS04)
containing 1 mM dithiothreitol. Peptides were added at the
indicated concentrations to 0.8 mg/ml isotonic microtubule-
protein with 0.5 mM GTP and 1 mM dithiothreitol and
incubated at 37°C for 20 minutes. The microtubules were
subsequently stabilized with 10 /¿M taxol for 10 minutes at
37°C. The samples, 250/ul final volume, were then
centrifuged for 8 minutes at 300,000 x g, 37°C in a Beckman
TL 100.2 rotor. The pellets were dissolved in 8 M urea and
analyzed by gel electrophoresis.

73
Competition with radiolabeled MAP-2
All radiolabeled MAP-2 experiments were performed with
polypropionate airfuge tubes which were coated with 10 mg/ml
bovine serum albumin for 5 minutes and rinsed with PEM
buffer just prior to use. This treatment reduces
nonspecific binding of proteins to the walls of the
centrifuge tubes. The radiolabeled MAP-2 was clarified
prior to use for 20 minutes at 130,000 x g in a Beckman
airfuge to remove any aggregated or denatured protein.
Phosphocellulose-purified tubulin was incubated at 5 mg/ml,
37°C with 1 mM GTP for 20 minutes and subsequently
stabilized with 50 /xM taxol for an additional 10 minutes.
The microtubules were then diluted twenty-fold into a
solution containing 3 /xM radiolabeled MAP-2 with either
unlabeled MAP-2 or synthetic peptides for 20 minutes at
37 °C. The solution also contained 10 /xM taxol and 1 mM GTP
to maintain microtubule stability. The samples, final
volume of 100/xl, were then carefully loaded into coated
airfuge tubes with the aid of a microcapillary pipetter onto
a 50 /xl layer of 20% (w/v) sucrose in PEM buffer warmed to
37°C. The samples were centrifuged for 30 minutes at
130,000 x g and the supernatants removed and discarded. The
pellets were washed with 100 /xl of 10 mg/ml bovine serum
albumin in phosphate buffered saline, pH 7.3, containing
0.1% Triton X-100 and resuspended in 100 /xl 8 M urea.
Aliguots of 25 /xl were taken for liquid scintillation
counting.

74
Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis was done by the
method of Bloom et al. (1985) omitting sodium dodecyl
sulfate in the separating and stacking gels and adding 2 M
urea to the separating gel. Gels were stained with
coomassie Brilliant Blue R-250 and scanned with an LKB
ultrascan densitometer.
Results
Peptide interactions with tubulin and microtubule-protein
To further analyze sequence(s) responsible for MAP-2
binding to tubulin within the 28 kDa fragment, four peptides
were synthesized. The first (mN = TPHTPGTPK) corresponded
to the N-terminus of the 28 kDa fragment from bovine MAP-2
that was determined previously by microsequencing (see Table
3-2). The others corresponded to the three octadecapeptide
repeats (m1 = VKSKIGSTDNIKYQPKGG, m2 = VTSKCGSLKNIRHRPGGG,
m3 = AQAKVGSLDNAHHVPGGG). Peptide mM was based on the
bovine sequence data while the murine MAP-2 sequence data
was used for m.,, m2, and m3, because no such data are yet
available for the bovine MAP-2. The high state of purity of
each peptide was confirmed on the basis of HPLC elution
profile analysis or gas-phase microsequencing.
First, I sought to determine whether any of these
peptides would influence the assembly of microtubule-protein
that contained both tubulin and MAPs. I worked with
recycled microtubule-protein to which sufficient pure
tubulin was added to lower the content of MAPs to about one-
fifth their normal level. This final composition was

75
approximately 5% MAPs and 95% tubulin by weight. This ratio
was chosen to accentuate any stimulatory effects of the
peptides on the assembly process, and no microtubule
polymerization occurred at the protein concentrations used
without peptide addition. To assay the extent of
microtubule assembly at different levels of peptides mN, m1,
m2, and m3, [3H]guanine nucleotide uptake was measured with
the glass fiber filter assay of Maccioni and Seeds (1978) as
modified by Wilson et al. (1982). Only peptide m2,
corresponding to the second repeat in MAP-2, stimulated
microtubule-assembly as evidenced by the data shown in Fig.
4-1. The level of m2 peptide reguired for polymerization
was very high. Concentrations greater than 250 /¿M were
needed to stimulate microtubule polymerization. When
peptides mN, m1, or m3 were employed individually, no
incorporation of guanine nucleotide was observed above
background levels. Moreover, in companion experiments, I
found that none of these peptides mixed individually with
peptide m2 altered the stimulation of microtubule assembly
by peptide m2. A range of concentrations from 0-1 mM was
tried for peptides m, and m3 with 0.5 mM or 0.75 mM m2
peptide but none showed any effect on the extent of
microtubule polymerization.
I also found that assembly of pure tubulin could be
stimulated by m2 only. Indeed, assembly with tubulin and m2
exhibits a typical time-course for the polymerization
process as shown in Fig. 4-2. An initial lag phase
indicative of subunit nucleation was seen followed by a

Incorporation, 10 cpm
77
Minutes
Fig. 4-2 Time course of peptide induced assembly.
Phosphocellulose-purified tubulin (1.0 mg/ml) was incubated
with m2 peptide (1.0 mM) for 30 min. at 37°C. At the times
indicated, the amount of GTP incorporation was determined as
described in "methods1'.

78
rapid polymerization phase that plateaued around 30 minutes.
Without addition of m2 peptide, no tritium label is retained
on the glass fiber filters. I verified that the observed
polymerization resulted in microtubules by using electron
microscopy (see Fig. 4-3) and immunofluorescence microscopy
(see Fig. 4-4). The electron micrographs revealed a
morphology typical of microtubules composed solely of
tubulin. When tubulin (1 mg/ml) was incubated with and
without peptide m2 (1 mM) , microtubules were observed only
in those micrographs of samples to which this peptide had
been added. The same was seen for the immunofluorescence
micrographs in Fig. 4-4 where panel A had the same
concentrations of protein and peptide as the electron
micrograph in Fig. 4-3 and panel B was without added
peptide. Panel A shows typical in vitro microtubules
stained with an anti-tubulin antibody. The concentration of
tubulin used was 1 mg/ml because it was clearly above the
critical concentration for peptide m2 induced assembly while
for tubulin alone it was just at the lower limit for
polymerization (see Fig. 4-5). Any molecule that shifts the
x-intercept to the left is a microtubule-stabilizer and any
molecule that shifts it to the right is a microtubule-
destabilizer. Clearly, m2 is a stabilizer of microtubule
polymerization. I also tested the action of several common
inhibitors of microtubule assembly to learn whether peptide
m2 induced assembly in a manner akin to normal assembly of
brain microtubules. Inclusion of colchicine (0.1 mM),

79
Fig. 4-3 Electron micrograph of peptide induced assembly.
Tubulin (1.0 mg/ml) and m2 peptide (1.0 mM) were incubated
for 30 min. at 37"C and then diluted into 1% glutaraldehyde
in microtubule assembly buffer warmed to 37"C. After
fixation for one minute, the sample was processed for
electron microscopy. An identical sample without m2 peptide
was also done but showed no microtubules. Magnification is
50,000X and the bar equals 0.25 Jim. Formvar coated grids,
uranyl acetate, and photographs were supplied by the
Electron Microscopy core facility

80
Fig. 4-4 Immunofluorescence of microtubules polymerized with
and without m2 peptide. Panel A shows the same tubulin and
peptide concentrations as Fig. 4-3 and panel B shows just
tubulin with no peptide addition. Both samples were stained
with anti-beta-tubulin followed by an anti-mouse IgG
conjugated with Texas red fluorochrome.

Incorporation, 10 cpm
81
Fig. 4-5 Critical concentration plot of peptide induced
tubulin polymerization. Varying concentrations of tubulin
plus microtubule seeds were mixed with or without m2 peptide
(1.0 mM) and assayed for GTP incorporation after 30 min. at
37 ° C.

82
calcium ion (2 mM) , or podophyllotoxin (0.1 mM) resulted in
complete inhibition of peptide m2-induced assembly.
These observations indicate that only the peptide m2,
with a sequence corresponding to the second repeated region
of the microtubule-binding fragment MAP-2 could stimulate
tubulin assembly. Nonetheless, the possibility remained
that the other peptides could still promote elongation, but
not nucleation, of microtubule assembly. To investigate
this possibility, I added pre-formed microtubule seeds to
tubulin (0.5 mg/ml) and [3H]GTP in the presence or absence
of the peptides. Without any peptide additions, only a
minimal increase in guanine nucleotide incorporation was
observed; however, upon addition of peptide m2, significant
assembly was again observed. By contrast, peptides m1 and
m3 failed to cause any significant increase of labeled
guanine nucleotide incorporation into microtubules beyond
background levels (see Fig. 4-6) . Thus, m2 is the only
peptide that can stimulate nucleation and elongation.
Displacement of MAPs from recycled microtubule-protein by
MAP-2 repeated sequence peptides
While only peptide-m2 promoted microtubule self-
assembly, I was interested in determining whether m1 and m3
might also bind to assembled tubules and displace MAP-2.
Therefore, the ability of the MAP-2 repeated sequence
peptides to displace high-molecular-weight MAPs from
assembled microtubules was assessed. For this purpose,
microtubule-protein isolated by the isotonic extraction
method (Karr et al., 1979) was used because such protein as

83
Fig. 4-6 Seeded assembly of tubulin with synthetic peptides.
Microtubule seeds were added to a solution containing 0.5
mg/ml tubulin (a level below the critical concentration).
Varying amounts of m1# m2, and m3, were added and
polymerization initiated by warming to 37°C. The plotted
values correspond to radiolabel incorporation over a 30
minute period.

84
isolated from the gray matter of the brain is rich in both
MAP-1 and MAP-2. The isotonic microtubule-protein was
stabilized with taxol after 20 minutes of 1 mM GTP at 37°C.
This method reduced the amount of abnormal microtubule
structures common with taxol-induced polymerization where
taxol is added at the start of polymerization. In this
experiment, taxol was added after 20 minutes of microtubule
assembly. After a 10 minute incubation, the peptides were
added and the samples kept at 37"C for an additional 20
minutes. In addition to the three repeats of MAP-2, two
other peptides were tested. The first (m^) was a glycine
substitution for a lysine in the m, peptide converting the
carboxyl terminal sequence to that of all the other repeats.
The second (m2') was the same as m2 plus the next three
residues present in the MAP-2 sequence. These additional
residues were RVK which made the peptide more electro¬
positive as well as possibly adding more structural
conformation. As seen in Fig. 4-7, SDS gel
electropherograms of the microtubule after assembly and
centrifugation, indicate that MAP-2 was only susceptible to
displacement by a 21-amino acid peptide m2' corresponding to
the m2 sequence above plus residues RVK at the C-terminus.
Interestingly, MAP-lb was selectively displaced by peptide
m2, and all high-molecular-weight MAPs were removed from
microtubules in the presence of peptide m2' . Densitometry
tracings of lanes 2, 5, and 6 indicate the profiles of MAPs
with m2, m2', and no peptide, respectively (Fig. 4-8).
Because the 21-amino acid peptide was more effective in

85
MAP-1 MAP-2 a,b
rri! m2 m3 m; vn'2 none
Fig. 4-7 Effects of MAP-2 peptides on MAP binding to
microtubules. Coomassie Blue staining of proteins in
pelleted microtubule fractions after electrophoresis on a 4%
polyacrylamide gel: (Lane 1) m1; (lane 2) m?; (lane 3) m3;
(lane 4) m1 • ; (lane 5) m2'; (lane 6) no peptide. All
peptides were added to a concentration of 2.0 mM.

86
Fig. 4-8 Densitometry of the coomassie blue stained gel.
Upper trace represents lane 6, middle trace lane 2, and the
lower trace is lane 5 of the gel depicted in Fig. 4-7.

87
displacing MAPs, it was compared to the m2-peptide with
regard to the promotion of microtubule assembly using
tritiated GTP incorporation as a measure of polymerized
protein (see Fig. 4-9). I found that tubulin polymerization
was considerably more effective in the presence of m2',
especially at lower peptide concentrations. At the greatest
concentration of peptide, the level of tritiated GTP
incorporation was the same for both m2 and m2' . Together,
these observations suggest that only peptides corresponding
to the second repeated sequence can displace MAPs from
assembled microtubules. The data also indicate that both
MAP-1 and MAP-2 can be displaced by peptide-m2' , suggesting
further that this peptide may bind to common, or closely
overlapping, sites on microtubules. Peptides m,, m2, and m3
were otherwise without effect, as was an analogue of m,
containing a gly in place of lys toward the C-terminus.
This m1 analogue was synthesized because the lysine residue
was disrupting a possible beta turn structure. In m2 and m3
as well as all three repeats of tau, the carboxyl termini
are proline-(glycine)3. This structure was hypothesized to
be important for microtubule-assembly since both the first
two tau repeats contained this carboxyl terminal tail and
could promote tubule polymerization. The first repeat of
MAP-2 was very similar to the first repeat of tau except for
the glycine difference; however, the m, analogue failed to
promote tubulin polymerization and also failed to displace
high-molecular-weight MAPs.

88
PEPTIDE CONCENTRATION (mM)
Fig. 4-9 Comparison of the stimulation of tubulin
polymerization by peptides m2 and m2'. The incorporation of
tritiated GTP into microtubule-polymer was measured to
examine the assembly promoting activity of m? and the
extended analogue m2' . Phosphocellulose-purified tubulin
was used at 1 mg/ml. The open circles represent m2 and the
closed circles m2' .

89
A gradient of m2' peptide was used to determine the
effective concentration range of displacing the MAPs. The
results shown in Fig. 4-10 demonstrate that the extended
second repeated sequence peptide m2' removes MAP-1 and MAP-
2 from microtubules in a concentration-dependent manner.
Again, densitometry was used to gauge the extent of MAP
depletion in the assembled tubule fraction, and the
concentration of peptide-m2' that displaces 50% of MAP-2 was
about 1.5-2.0 mM (data not shown). This level of peptide-
m2' is about four times the concentration needed to promote
tubulin polymerization in the absence of MAPs or
microtubule-stabilizing agents.
Radiolabeled MAP-2 binding to microtubules
While the findings presented in Figs. 4-7 and 4-10
provide clear evidence of MAP displacement, a quantitative
displacement/binding assay was developed to more accurately
measure the MAP displacement. MAP-2 was incubated with
3',51-cyclic-AMP-stimulated protein kinase and [c-32P]ATP
under conditions that have been found to result in the
incorporation of about 1-1.5 phosphoryl groups per MAP-2
molecule (Flynn et al., 1987). First I determined the
concentrations of tubulin and MAP-2 necessary for saturation
of binding to microtubules. The level of tubulin used was
0.25 mg/ml which was taxol-stabilized like the isotonic
microtubule-protein described in the previous section. At
this low level of tubulin, 1-5 ¡jlM of 32P-MAP-2 was seen to
saturate the available binding sites on the microtubules.
This is depicted in Fig. 4-11.

90
MAP-1 MAP-2a,b
1
M 0
**
0 0.5 1.0 1.5 2.0 2.5
Fig. 4-10 Effects of increasing the m2' concentration on
high-molecular-weight MAP binding to microtubules. Peptide
m2' was added to isotonic microtubule-protein to the final
mM concentration indicated at the bottom of each lane.
After centrifugation, the pellet fractions were analyzed by
gel electrophoresis. The coomassie Blue staining of a 4%
polyacrylamide gel is shown.

91
Because extensive MAP-2 phosphorylation can alter the
affinity of MAP-2 to microtubules (Murthy and Flavin, 1983;
Hoshi et al., 1988), I wanted to compare the binding
behavior of the trace phosphorylated MAP-2 with unmodified
MAP-2 isolated by the standard recycling preparation
protocol (Shelanski et al., 1973; Herzog and Weber, 1978).
Accordingly, taxol-stabilized microtubules were incubated
with the phosphorylated MAP-2 in the presence of several
concentrations of unphosphorylated MAP-2 (Fig. 4-12). The
data points show that the amount of radiolabeled MAP-2 bound
to microtubules decreases at increasing concentrations of
the unlabeled MAP-2; the solid line is the theoretical curve
calculated on the basis of isotopic dilution, using the
ratio R = [MAP-2*]/([MAP-2] + [MAP-2*]), where labeled and
unlabeled protein are MAP-2* and MAP-2, respectively. The
data normalized with respect to R (Fig. 4-12 inset) indicate
that the relative affinities of both MAP-2 species are the
same within experimental error.
Displacement of labeled MAP-2 by a second repeated seguence
peptide
To gain a more quantitative view of MAP-2 displacement,
the amount of microtubule-bound [32P]MAP-2 as a function of
the concentration of peptide-m2' was examined. As shown in
Fig. 4-13, MAP-2 is displaced by this peptide, but the
desorption process is not described by a typical hyperbolic
dissociation curve. The basis of the slight stimulation of
MAP-2 binding at 0.5 mM peptide-m2* is unclear, but careful
inspection of the MAP-2 band in lane 2 of Fig. 4-10 revealed

92
cone. MAP-2 (mg/ml)
Fig. 4-11 32P-MAP-2 binding to taxol-stabilized
microtubules. Taxol-stabilized microtubules were diluted to
0.25 mg/ml in increasing concentrations of radiolabeled MAP-
2. After incubation at 37°C for 20 minutes and
centrifugation, the level of MAP-2 binding was determined by
liquid scintillation counting.

10 CPM
93
Fig. 4-12 Displacement of trace phosphorylated MAP-2 from
taxol-stabilized microtubules by unlabeled MAP-2. See
"Competition with Radiolabeled MAP-2" under the "Methods"
section. The molarity of MAP-2 was calculated using a
molecular weight of 200,000 daltons.

94
a similar behavior. Once again the effective concentration
range for displacement was in the low millimolar range
comparable to the SDS gels shown in Fig. 4-10. Thus, the
same displacement behavior is observed whether or not MAP-2
is trace phosphorylated by the 3',5'-cyclic AMP-stimulated
protein kinase.
Competitive binding of peptide-m-,1 and MAP-2
I was particularly interested in the mode of inhibition
of MAP-2 binding to microtubules by the extended second
repeated sequence peptide-m2' . Therefore a series of
radiolabeled MAP-2 binding measurements were conducted over
the concentration range of MAP-2 shown in Fig. 4-14. These
experiments were carried out in the absence or presence of
1.5 mM peptide-m2'. The results indicated that peptide-m2'
did indeed act as a competitive inhibitor of MAP-2 binding
as binding of MAP-2 to microtubules was reduced in the
presence of the peptide as opposed to without the peptide.
When the data was transformed into a double reciprocal plot,
the lines intersected at the y-axis (see Fig. 4-15)
suggesting the peptide was a competitive inhibitor of MAP-2
binding to microtubules. The data presented here also
indicated that the MAP-2 interaction with microtubules was
defined by a single class of binding sites. The maximal
extent of MAP-2 binding was found to correspond to one
molecule of MAP-2 per four molecules of polymerized tubulin
dimers. The dissociation constant for MAP-2 binding to
microtubules was 3.4 ¿iM in the absence of peptide-m2* and 14
MM in the presence of 1.5 mM of this peptide. From the

MAP-2 Bound, 10 CPM
95
Peptide m£ (mM)
Fig. 4-13 Displacement of trace phosphorylated MAP-2 from
taxol-stabilized microtubules by peptide m2'. See
"Competition with Radiolabeled MAP-2" in the "Methods"
section.

10 CPM
96
Fig. 4-14 Radiolabeled MAP-2 binding to taxol-stabilized
microtubules in the presence and absence of 1.5 mM m2'
peptide. Plot of bound MAP-2 vesus total MAP-2 in absence
(closed circles) and presence (open circles) of m2'. In
this experiment the microtubules were diluted into a
solution containing radiolabeled MAP-2 with or without 1.5
mM peptide.

97
1/[MAP-2]
Fig. 4-15 Double reciprocal plot of MAP-2 binding. The data
from Fig. 4-14 were transformed into reciprocals and
plotted. With peptide (open circles), without peptide
(closed circles).

98
change in slope, the iu2' inhibition constant was estimated
to be 0.5 mM. Thus, the dissociation constant for MAP-2 is
about 100 times less than the corresponding constant for a
peptide-m2.
Discussion
The experiments described in this chapter were designed
to gain further insight about the microtubule-binding
fragment of MAP-2 and determined what sequences were
responsible for MAP-2 binding to microtubules. I have
demonstrated that a single octadecapeptide corresponding to
the second repeated sequence (from Val-1705 through Gly-
1722 in murine MAP-2) promoted microtubule nucleation and
elongation. To my knowledge, these are the first
observations that a sequence amounting to less than 1% of
the overall MAP-2 molecule is sufficient to interact with
tubulin, but some additional considerations of MAP-2
structure seem appropriate.
That the second repeated sequence in murine MAP-2 can
promote microtubule assembly is indeed interesting. Lewis
et al. (1988) had studied microtubule binding of an in vitro
translation product spanning amino acids 1621 through 1722
(including the first and second repeated sequences). While
they did not attempt to demonstrate promotion of microtubule
assembly, Lewis et al. (1988) succeeded in showing that
their radioactively labeled 100-residue polypeptide
copurified through two cycles of assembly/disassembly with
MAP-containing microtubule protein. I found that a single
octadecapeptide can not only copurify with tubulin, but can

99
promote tubulin polymerization at concentrations below the
critical concentration of pure tubulin in vitro, even in the
absence of MAPs.
While the possibility of multiple binding of peptide m2
to tubulin cannot be discounted, establishing the
stoichiometry of ligand binding to a protein can be
particularly challenging for ligands that bind in the
millimolar concentration range. This is due to the fact
that most techniques are not sensitive enough to accurately
measure the protein-ligand complex versus the free ligand
concentration with dissociation constants in the millimolar
range. It should also be emphasized that approximately 0.5
mM of the peptide m2 is needed to promote assembly, but MAP-
2 is effective in the 1-5 /¿M range. This may mean that the
several repeats in MAP-2 reinforce each other in promoting
assembly or that the octadecapeptide cannot readily assume
the assembly-promoting conformation. Obviously, the
conformational latitude of the second repeated sequence
could be greatly influenced by the other residues in the
entire microtubule-binding domain of MAP-2. At this point,
however, the combined findings of Lewis et al. (1988) and
these observations attest to the importance of the second
octadecapeptide in MAP-2 interactions with tubulin.
It was interesting to note that just three additional
amino acids at the carboxyl terminus of m2, yielding m2',
promoted tubulin polymerization at lower peptide
concentrations. This could have been due to the increased
positive charge by adding a lysine and arginine interacting

100
with the anionic carboxyl termini of alpha and beta tubulin,
or it may have been due to a more restricted conformation at
the tail of the peptide that promoted tubulin
polymerization.
The peptide corresponding to the second repeated
sequence has an overall isoelectric point that is more basic
than the first and third octadecapeptides fe.a.. m1, m2 and
m3 have calculated pi values of 10.5, 11.6, and 8.0). In
the pH 6.8 assembly buffer, m1, m2 and m3 have overall
charges of +3, +4, and +0.4, respectively. There are other
indications that ionic interactions are important in MAP-2
binding to tubulin and/or microtubules. Flynn et al.
(1987), for example, showed that the 28 kDa tubule-binding
fragment of MAP-2 had an isoelectric point of about 10.5
whereas the larger projection arm fragment was considerably
more anionic (pi about 4.8). Earlier findings suggested
that MAP-2 interacts with microtubules largely through ionic
forces (Vallee, 1982). Aside from the ability of
intermediate salt concentrations (e.g., 0.4 - 0.6 M NaCl) to
block MAP binding to tubules, the inhibitory action of
polyanions and polycations should also be noted. Among the
polyanionic inhibitors of assembly are RNA and polyglutamate
(Bryan, 1976; Bryan et al., 1975), phosphatidyl inositol
(Yamauchi and Purich, 1987), and estramustine phosphate
(Wallin et al,. , 1985) . These agents are thought to bind to
the microtubule-binding region of MAP-2. Polycations also
bind to microtubules and displace MAPs (Purich and
Kristofferson, 1984). MAP binding is thought to occur at

101
the glutamate-rich C-termini of the tubulin a and & chains,
and the polycations presumably block these MAP-binding sites
on tubulin and/or microtubules. Indeed, subtilisin
treatment of tubulin results in loss of a 4 kD fragment
containing the C-terminus, and tubulin proteolyzed in this
manner readily assembles but fails to bind MAPs (Serrano et
al.. 1984). Nonetheless, I cannot conclude that ionic
interactions alone determine the effectiveness of peptides
in promoting assembly. An interesting case was m1 and its
analogue utilized in this chapter. The original repeat
contained a lysine toward the carboxyl terminus and the
analogue contained a glycine, but neither promoted
microtubule polymerization. Obviously something else
besides charge interactions was involved in proper binding
to the microtubule-lattice, possibly a hydrophobic effect.
The observation that an octadecapeptide can promote
assembly suggests a route for preparing low-molecular-weight
modulators of microtubule assembly. As more information on
the binding of MAP-2 to tubulin is developed, it may be
possible to improve the binding efficiency of the
oligopeptides. Already it has been shown that a slightly
longer analogue, m2', was a more efficient stimulator of
microtubule polymerization. Moreover, the availability of
these peptides should also permit additional studies of tau
interactions with tubulin and MAP-2 interactions with other
cytoskeletal elements such as the neurofilament proteins.
That MAP-1 and MAP-2 were both displaced from
microtubules in the presence of peptides based on the second

102
repeated sequence suggested that peptides m2 and m21 can
bind at or near the site(s) of MAPs interaction with
microtubules. Until a quantitative binding assay for MAP-1
is developed, I cannot discern whether these peptides act as
competitive inhibitors, as was found to be the case for MAP-
2. At the same time, I can be confident that peptides m2
and m2' will be found to competitively inhibit MAP-2c
binding to microtubules. This assumption is based on the
interesting finding of Papandrikopoulo et al. (1989) that
the embryonic MAP-2c has an identical amino acid sequence in
the C-terminal microtubule-binding motif as the adult forms,
MAP-2a and MAP-2b. The embryonic protein lacks 1372 amino
acid residues spanning positions 147 to 1519 of adult rat
MAP-2 as a result of an alternative splicing event.
That peptides corresponding to the second repeated
sequence of MAP-2 can both promote assembly and displace
MAPs opens the way for developing high affinity peptide
analogues. I now have developed functional assays for the
assembly-promoting and MAP-displacing characteristics of
such peptides, and the latter can be extended by the
electrophoresis experiments to test for specificity of MAP
displacement (i.e., preferential desorption of a particular
MAP). Again it has already been found that the extended
second sequence peptide-m2' was more effective than peptide-
m2 in displacing MAPs.
Finally, there is mounting evidence that a group of
microtubule-associated proteins achieve binding to
microtubules by way of a triad of nonidentical repeated

103
octadecapeptide sequences (Lewis et al., 1988). The
stimulation of tubulin polymerization by synthetic peptides
corresponding to several repeated sequences is consistent
with, but does not prove, this hypothesis. On the other
hand, the findings presented here indicate that peptides
resembling the second repeated sequence of MAP-2 can
displace MAP-2 from microtubules and can competitively
inhibit MAP-2 binding. Because only these peptides promote
tubulin polymerization, I am drawn to the conclusion that
these peptides do indeed mimic MAP binding to microtubules.

CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS
The experiments designed in this project focused on the
microtubule-binding domain of MAP-2 with the aim of
determining the sequences and/or regions responsible for
several known functions and properties of MAP-2. During the
course of these studies, I gained information on the
structural features of MAP-2, its association with
neurofilaments, and sequences responsible for promotion of
both tubulin polymerization and MAP displacement.
Interactions with Neurofilaments
When MAP-2 is incubated with neurofilaments, the
protein binds to the filaments and cosediments with them in
a manner analogous to MAP association, or binding, with
microtubules. The purpose of this research was to determine
if the same region of MAP-2 that bound to microtubules was
responsible for binding to neurofilaments. In part, this
effort also addressed the question of whether a single MAP-
2 molecule could cross-link a microtubule and neurofilament
at the same time. If this was the case, MAP-2 could be
responsible for many physiologically relevant connections
microscopically observed within the neuronal cytoskeleton.
Through the use of protease digests and ultracentrifugations
of polymerized protein, the 28 kDa fragment of MAP-2 was
shown to bind to both types of structures. This result
104

105
implied that a single MAP-2 molecule does not cross-link a
microtubule and neurofilament at the same time; however,
this does not exclude the possibility that MAP-2 is involved
in connections between the two structures. If a higher
order of MAP-2 structure such as a dimer exists, then two
molecules of MAP-2 could cross-link the structures. To date
no such dimer has ever been reported; only the 280 kDa
monomer and a very large-molecular-weight aggregate composed
of many MAP-2 molecules has been observed (Hernandez et al.,
1986). Another possibility is that in vivo MAP-2 cross-
linking is lost upon in vitro purification, perhaps the
consequence of structural damage to the protein or the loss
of another component necessary for cross-linking. While
beyond the scope of this work, assays for cross-linking
ability using falling-ball viscometry could be helpful. The
addition of MAPs to microtubules, composed solely of
tubulin, and neurofilaments causes a rise in the viscosity
of the solution, but the addition of heat-treated MAPs or
pure MAP-2 does not (Flynn and Purich, 1987). If MAP-2 is
involved in the cross-linking of microtubules with
neurofilaments, one could fractionate phosphocellulose-
purified MAPs and add back various fractions to MAP-2 in the
presence of microtubules and neurofilaments. The viscosity
of the resulting solution can be monitored to check for
cross-linking. With further fractionation it may be
possible to identify key proteins in a complex that cross¬
links the two structures.

106
The identification of the 28 kDa microtubule-binding
domain possessing a neurofilament binding site was never
further analyzed for specific sequence interactions as was
the case for microtubules. This was partly due to the lack
of a functional assay for neurofilaments. Tubulin
polymerizes in the presence of MAP-2 peptides but
neurofilaments are already polymers and remain rather static
in vitro. The peptides used in my work are probably too
short to cross-link neurofilaments in a falling-ball
viscometry assay. If the peptides were radiolabeled during
synthesis (by using tritiated amino acids), one might be
able to assay for cosedimentation of peptides with
neurofilaments using liquid scintillation counting of the
pellet and supernatant fractions. Alternatively, the
peptides could be labeled after synthesis by acetylation of
amino residues with 14C-labeled acetate. A major pitfall of
this technique is that the covalent modification of the
peptide could alter its properties for binding to
neurofilaments.
The binding of neurofilaments to MAP-2 was examined in
a preliminary manner from a different approach to learn what
part of NF-L binds to MAP-2. It had already been
established that the L subunit of neurofilaments interacted
with MAP-2 (Heimann et al., 1986). The L subunit sequence
was known to contain one tryptophan residue in the middle of
the rod forming domain common to all intermediate filament
proteins. This tryptophan can be cleaved with BNPS-skatole
to yield a 40 kDa carboxyl terminal fragment and a 30 kDa

107
amino terminal fragment. These fragments no longer form
filaments; therefore cosedimentation is not a possible
assay. Instead, the fragments were separated by SDS
electrophoresis and electroblotted to nitrocellulose. The
membrane was incubated with 32P-labeled MAP-2 and exposed to
X-ray film to determine which bands interacted with the MAP-
2. Care must be taken in blocking the membrane after
electroblotting and before incubating with the labeled
protein. One has to include 2.5% (v/v) Triton X-100 with 10
mg/ml BSA in the blocking step. The BSA bound to any
nitrocellulose not used in the electroblotting process and
the added detergent displaced SDS from the blotted proteins.
If SDS was not displaced, then the labeled MAP-2 bound
everywhere on the blot and the autoradiogram appeared
completely black. Removing the SDS allowed for a clear
exposure. When 32P-MAP-2 was incubated with a blot of
skatole-cleaved NF-L, the labeled protein bound only to the
30 kDa amino terminus and intact, uncleaved NF-L. No
interaction with the 40 kDa carboxyl terminal fragment was
seen. An interesting note about use of this technigue
referred to specificity. While no interaction with the 40
kDa fragment was seen, several protein molecular weight
markers showed weak interacions that could never be
selectively removed with various salts or detergents. The
most notable marker showing such an interaction was carbonic
anhydrase. This line of research could be advanced with
further cleavages of the 30 kDa fragment, but the non¬
specific binding to molecular weight markers should be

108
characterized and taken into consideration when analyzing
binding to fragments of NF-L.
Structure of MAP-2
Valuable information on the overall organization of the
molecule was gained through the use of protease digests and
microsequencing techniques. The microtubule-binding
fragment was derived from the carboxyl portion of the
molecule and the projection domain from the amino portion.
This was later confirmed by the cDNA work of Lewis et al.
(1988). For such a large structure to yield only two
distinct fragments after a thrombin digest would indicate
some order of secondary structure; yet previous studies of
MAP-2 by circular dichroic measurements indicated that MAP-
2 possessed little organized secondary structure (Hernandez
et al., 1986). Also, the cDNA-derived amino acid sequence
revealed little secondary structure upon computer-assisted
structural analysis. If there was little organized
structure, one would expect far more than the two major
fragments in thrombin digests, especially with eleven
arginine residues present in the 28 kDa binding domain alone
and many more present in the larger projection domain.
When MAP-2 was analyzed by electron microscopy, the
structure most often seen was an elongated rod with a
globular head at one end (Voter and Erickson, 1984). It is
tempting to speculate that the globular head is the
microtubule-binding domain and that the protease-sensitive
hinge region connects the globular head and the elongated
tail. The low level of observed secondary structure in the

109
whole MAP-2 molecule does not exclude the possibility that a
small portion of the protein has organized secondary
structure. Indeed, the microtubule-binding domain is only
one-tenth of the entire protein, and its secondary structure
organization may be masked in circular dichroic measurements
using the whole protein. Accordingly, circular dichroic
measurements of the 28 kDa fragment could be carried out to
check for local areas of secondary structure. The immediate
carboxyl terminus of MAP-2 shares some homology with leucine
zipper DNA-binding proteins implying that some helical
structure is present (Lewis et al., 1989).
Another interesting avenue to pursue regarding MAP-2
structure concerns the rat embryonic form, MAP-2c. This
protein results from alternative mRNA splicing that leads to
a deletion of residues 147 through 1519 (see Fig. 1-4). The
protein contains the thrombin cleavage site of adult MAP-2
as well as the triad of octadecapeptide repeats and the
amino terminal site for the regulatory subunit of cAMP-
dependent protein kinase. It would be of interest to digest
MAP-2c with thrombin to test if the same protease accessible
site still exists. If the conformation in this hinge region
is similar to the adult form, then a stable 28 kDa
microtubule-binding domain could be produced.
MAP-2 Sequence Interactions with Microtubules
That the second octadecapeptide repeat of MAP-2 could
promote tubulin polymerization in vitro was very
interesting. At the time it was the first result indicating
small sequences of MAPs could direct tubule assembly. A few

110
months later, the report of Ennulat et al. (1989) revealed
that both the first and second octadecapeptide repeats of
tau could promote tubulin polymerization. These sequences
are similar to the MAP-2 repeated sequences, but the first
repeat of MAP-2 could not promote tubulin polymerization.
The third repeated sequences of tau and MAP-2 are similar in
structure, and less basic than m1, t1, m2, or t2. Neither m3
nor t3 promotes microtubule formation. Nonetheless, a
sequence that fails to promote tubulin polymerization may
still interact with microtubules. These sequences could
bind weakly to the microtubule lattice, and in MAP-2 they
would be in close proximity to the microtubule wall already
since m2 interacts with tubulin. It is possible that m2
directs the initial MAP-2 interaction with tubulin and that
the m1 and m3 repeats bind afterward.
A test of binding for the m1 and m3 repeats to tubulin
could be accomplished if the peptides are radiolabeled. One
could then use taxol-stabilized microtubules composed solely
of tubulin in a pelleting assay followed by liquid
scintillation counting to check for cosedimentation of the
peptides. An interesting variation of this experiment could
be carried out to compare binding of the peptides to a
microtubule lattice versus tubulin monomer. If the
incubation of peptide with tubulin is done at 4°C and
without taxol, the binding can be monitored by gel
filtration chromatography. If the peptides bind, then a
shift should be seen in their elution time from a gel
filtration column. The peptide would coelute with the

Ill
tubulin monomer at approximately 100 kDa rather than at
approximately 2 kDa without the presence of tubulin.
Alternatively, one could use extrinsic fluorescence as a
tool to monitor peptide interactions with microtubules. The
peptides can be covalently modified with an extrinsic
fluorochrome such as dansyl chloride and the emission
spectrum can be monitored in the presence and absence of
microtubules to observe possible changes. One caveat to
this approach is the possible change in peptide properties
after modification with the flúor. In that case, quenching
or enhancement of the intrinsic tubulin fluorescence might
prove to be useful.
To ensure that the peptides, including m2, bind to the
known site on tubulin, gel filtration again could be
employed. It has been well established that the glutamate-
rich carboxyl termini of both alpha and beta tubulin is the
MAP-binding site (Serrano et al., 1984; Littauer et al.,
1986). It appears that the second repeated sequence of MAP-
2 binds at the same tubulin site as MAP-2 since the binding
of the peptide is competitive with MAP-2 (see Fig. 4-15) .
To confirm this result the carboxyl terminal regions of both
alpha and beta tubulin could be synthesized as was done for
the MAP-2 peptides. Then these tubulin peptides could be
incubated with the MAP-2 peptides to check for binding. If
there is binding of the two sets of peptides, then a shift
to an earlier elution time should be seen with a gel
filtration column.

112
The MAP displacement studies demonstrated that the
second repeated sequence of MAP-2 could displace MAP-la and
MAP-lb as well as MAP-2. This result suggests that MAP-1
binds to the same site or a nearby overlapping site. The
microtubule-binding sequence for MAP-1 is a portion of the
protein containing many KKEE and KKE(I/V) (Noble et al.,
1989), indicating it has a different type of sequence
interacting with microtubules than MAP-2. This sequence may
interact with the carboxyl termini of alpha and beta tubulin
at the same residues as MAP-2 or it could be slightly
displaced. Another high-molecular-weight MAP from HeLa
cells, designated MAP-4, contains 23 tandem repeats of
KDMXLPXETEVALA (J. Olmsted, personal communication). It now
appears there may be several distinct classes of MAP
sequences responsible for microtubule binding and/or tubulin
polymerization. The MAP-2- and tau-like sequences are
listed in Fig. 6-1. Alignment of these repeats reveals
several interesting features, including the conservation of
the four carboxyl terminal residues and the spacing of
positive charges, as discussed in Chapter 4. It is possible
that peptide synthesis can be used to study the effects of
potentially key residues on tubulin polymerization. Also,
chemical modification of important residues may alter the
ability of a peptide to promote tubulin polymerization. For
example, cysteinyl thiol present in both MAP-2 and tau
second repeats could be carboxymethylated to introduce a
negative charge adjacent to the conserved positive charge at
position four. Alternatively, the cysteine could be

113
1 2
3 4 5 6
7 8 9 10 11
( + )
( + )
(")
m1
V -K •
-S -K -I -G
-S -T -D -N -I
( + )
( + )
(-)
bt1
V -R •
-S -K -I -G
-S -T -E -N -L
K
( + )
( + )
(-)
ag
V -R
-S -K -V -G
-S -T -E -N -I
( + )
( + )
HI2
V -T â– 
-S -K -C -G
-S -L -K -N -I
( + )
^2
V -T â– 
-S -K -C -G
-S -L -G -N -I
bt2
Q
K -D
( + ) (-)
( + )
(")
m3
A -Q •
-A -K -V -G
-S -L -D -N -A
( + )
(")
tj
V -Q •
-S -K -I -G
-S -L -D -N -I
bt3
T
C
G
1 2
3 4 5 6
7 8 9 10 11
12 13 14 15 16 17 18
(+) (+)
-K -Y -Q -P -K -G -G
( + ) 5+
-K -H -Q -P -G -G -G
( + ) 5+
-K -H -Q -P -G -G -G
( + ) <5+( + )
-R -H -R -P -G -G -G
6+ 5+ (+)
-H -H -K -P -G -G -G
L V
( + )
8+ 8+
-H -H -V -P -G -G -G
-T -H -V -P -G -G -G
H K
(S+ ( + )
12 13 14 15 16 17 18
Fig. 5-1 The octadecapeptide repeats of murine MAP-2 (m1f
m2, m3) , murine tau (t1f t2, t3) , bovine tau (bt1f bt2, bt3) ,
and a corresponding sequence of the 190 kDa adrenal gland
(ag) are shown. The (+) signs represent full positive
charges at pH 6.8, whereas the delta represents partially
positive imidazolium side-chain groups of histidyl residues.

114
aminoethylated to increase the positive charge in this
region. There is a wealth of potential work in determining
essential residues for tubulin polymerization in the m2
peptide, and converting a non-functional peptide such as m1
to a polymerization promoting peptide.
The exact roles of the various microtubule-associated
proteins in neurons remain to be elucidated. Nonetheless,
the structural findings presented in this dissertation
should be helpful in developing better probes of the in vivo
interactions of MAPs with the neuronal cytoskeleton.

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BIOGRAPHICAL SKETCH
John Charles Joly was born on July 27, 1963, in Long
Branch, New Jersey. He earned his Bachelor of Arts degree
in May 1985, majoring in chemistry and biology from the
University of Virginia in Charlottesville. In August of
1985, he began his graduate education in the Department of
Biochemistry and Molecular Biology at the University of
Florida, Gainesville, working under the direction of Dr.
Daniel L. Purich. He plans to work for Dr. Bill Wickner at
the UCLA Molecular Biology Institute studying protein
translocation across biological membranes.
125

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctotqf Philosophy.
Daniel L. Purich, Chairman
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
La M ÚM <3
.es M. Allen, Jr.
Charles
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Richard P. Boyce
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Thomas W. O'Brien
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
â– Wm/
Gerald P.J. Shaw
Assistant Professor of Neuroscience

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Christopher M. West
Associate Professor of Anatomy
and Cell Biology
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
May, 1990
Dean, College of Medicine
Dean, Graduate School

UNIVERSITY OF FLORIDA



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