Syndecan-4, a transmembrane heparan sulfate
proteoglycan, is a coreceptor with integrins in cell adhesion. It has
been suggested to form a ternary signaling complex with protein kinase
C
and phosphatidylinositol 4,5-bisphosphate
(PIP2). Syndecans each have a unique, central, and
variable (V) region in their cytoplasmic domains, and that of
syndecan-4 is critical to its interaction with protein kinase C and
PIP2. Two oligopeptides corresponding to the variable
region (4V) and whole domain (4L) of syndecan-4 cytoplasmic domain were
synthesized for nuclear magnetic resonance (NMR) studies. Data from NMR
and circular dichroism indicate that the cytoplasmic domain undergoes a
conformational transition and forms a symmetric dimer in the presence
of phospholipid activator PIP2. The solution conformations
of both free and PIP2-complexed 4V have been determined by
two-dimensional NMR spectroscopy and dynamical simulated annealing
calculations. The 4V peptide in the presence of PIP2 formed
a compact dimer with two twisted strands packed parallel to each other
and the exposed surface of the dimer consisted of highly charged and
polar residues. The overall three-dimensional structure in solution
exhibits a twisted clamp shape having a cavity in the center of dimeric
interface. In addition, it has been observed that the syndecan-4V
strongly interacts not only with fatty acyl groups but also the anionic
head group of PIP2. These findings reveal that
PIP2 promotes oligomerization of syndecan-4 cytoplasmic
domain for transmembrane signaling and cell-matrix adhesion.
 |
INTRODUCTION |
Cell adhesion mediated by cell surface receptors triggers signal
transduction cascades. Integrins, which are well characterized transmembrane receptors, are known to be involved in several cell regulatory mechanism including cell proliferation, morphology, and
motility. During cell-matrix interaction, integrin clustering activates
tyrosine kinases (1-3). Adhesion of several cell types to
extracellular matrix molecules such as fibronectin involves both
integrin- and heparin-binding domains, where assays were performed in
the absence of serum and protein synthesis (3-11).
The specific interactions of the cytoplasmic domain of each
integrin member are critical to trigger the downstream signal cascades. Although the functions of these interactions are not clear,
some kinases such as focal adhesion kinase and integrin-linked kinase,
and structural proteins such as
-actinin, talin, and filamin, are
known to interact with the cytoplasmic domain of
1
integrin subunit. The four mammalian syndecans are type I glycoproteins that form dimers or higher order multimers. Oligomerization apparently involves their highly homologous transmembrane domains as well as
regions of their cytoplasmic domains (12-14). The central region of
each syndecan cytoplasmic domain is unique, and has been termed the
variable (V)1 region, flanked
N- and C-terminally by constant (C1 and C2) regions (12, 13).
Syndecan-4 is the only family member that is a widespread focal
adhesion component, potentially functioning as a coreceptor in
integrin-mediated adhesion. It readily forms oligomers that may be
required for its function (12, 13).
Phosphatidylinositol 4,5-bisphosphate (PIP2) plays
important roles in signal transduction, since PIP2 is
hydrolyzed by phospholipase C
to generate two
intracellular messengers: inositol 1,4,5-triphosphate, which mobilizes
Ca2+, and diacylglycerol, which is a physiological
activator of protein kinase C (PKC) (15-19). However, in addition to
its function as precursor for other second messenger, PIP2
may also act as a second messenger by directly or indirectly regulating
several proteins including PKC, phospholipase C
,
phospholipase C
, G protein-coupled receptor kinase, and
phosphatidylinositol 3-kinase. PIP2 binds the cytoskeletal
proteins gelsolin, profilin, and vinculin and regulates their
interaction with actin (20-23). Recently, some
1
integrin cytoplasmic domains have been demonstrated to affect phosphatidylinositol 4,5-kinase, possibly regulating PIP2
synthesis in vivo (24-25). Indeed, it is known that
PIP2 is accumulated in the cytoplasmic face of plasma
membrane during cell adhesion and it may be regulated by G-proteins
such as Rac and Rho, which may activate phosphatidylinositol 4,5-kinase
(26, 27). PIP2 is also known to be involved in focal
adhesion and stress fiber formation (3), probably through its
interactions with focal adhesion components such as vinculin,
-actinin, and syndecan-4.
Focal adhesion formation is dependent not only on integrins, but
syndecans (most likely syndecan-4), which may act as co-receptors (2,
8, 9). It has been previously shown that syndecan and PKC are involved
in the focal adhesion formation, and syndecan-4 regulates the
distribution and activity of classical PKC (cPKC). In addition, it has
been suggested the interaction of syndecan-4 cytoplasmic domain and
cPKC depends on cPKC activity. Recently, we have reported that
PIP2 interacts specifically with the cytoplasmic domain of
syndecan-4, and together with PIP2, syndecan-4 cytoplasmic domain potentiated PKC activity (28). It was also shown that PIP2 regulates the interaction of the cytoplasmic domain of
syndecan-4 with PKC
and potentiation activity of syndecan-4
cytoplasmic domain on PKC
(28). As we suggested previously,
syndecan-4 and PIP2 together with PKC may form a ternary
complex. PKC is activated through this complex, which may then
phosphorylate cytoplasmic proteins involved in focal adhesion
formation. Here, we present the solution structures of syndecan-4
cytoplasmic core domain both with and without activator
PIP2 by two-dimensional nuclear magnetic resonance
spectroscopy and dynamical simulated annealing calculations.
 |
EXPERIMENTAL PROCEDURES |
Sample Preparations--
Peptides of both the whole domain with
RMKKKDEGSYDLGKKPIYKKAPTNEFYA sequence corresponding to residues
170-197 (4L) and the variable region with LGKKPIYKKA corresponding to
residues 181-190 (4V) of the chicken syndecan-4 cytoplasmic domain
were synthesized using an improved version of the solid phase method on
a model 431A peptide synthesizer (Applied Biosystems Inc.). The peptide sequences are identical to those of human and rat syndecan-4
cytoplasmic domain. Peptides were purified by reversed-phase liquid
chromatography using a Vydac 218TP152050 C18 column on a Waters Delta
Prep 4000 system. Purification was achieved by equilibrating the column with 0.1% trifluoroacetic acid in water and developing with a linear
gradient of acetonitrile.
The free peptide samples for NMR measurements were prepared by
dissolving in 90% H2O, 10% D2O or 99.9%
D2O solution at pH value of 7.4 with 50 mM
sodium phosphate buffer. The final peptide concentration was 2-4
mM in 0.5 ml of buffer solution. PIP2 was purchased from Sigma. For NMR measurements of PIP2-peptide
complex, 10-50 µl of concentrated PIP2 solution was
titrated to free peptide up to a maximum ratio of 1:1
(PIP2:peptide). The final pH value of 7.0 was adjusted by
adding 0.1 N HCl solution before NMR experiments.
Circular Dichroism--
CD data were collected on a Jasco J-715
spectropolarimeter using 1.0-mm path length cells with scan speed of
500 mM/min, 1-nm bandwidth, and 32 accumulations. Both free
and PIP2-complexed samples of 4L and 4V peptides were
prepared as volumes of 8 µM in H2O solution,
and CD data were collected for temperatures ranges of 20-70 °C. A
standard noise reduction procedure was used to prepare final
spectrum.
NMR Spectroscopy--
All NMR experiments were performed on a
Bruker DMX600 spectrometer in quadrature detection mode equipped with
an SGI INDY computer. All data were collected at either 10 °C or
20 °C, and the strong solvent resonance was suppressed by
water-gated pulse sequence combined with pulsed-field gradient pulses.
The temperature was calibrated using a methanol standard (29). A series
of one-dimensional NMR spectra were recorded for both 4V and 4L peptide
with concentration ranges of 0-2 mM PIP2. All
of the two-dimensional NMR measurements were performed on both free and
peptide-PIP2 (2:1) samples based on results from
PIP2 titration analysis. Mixing times of 100, 200, 300, 400, 500, and 800 ms were used in collecting NOE spectra for both free
and PIP2 complex samples. Total correlation spectroscopy (TOCSY) data were also recorded in both H2O and
D2O solutions with a mixing time of 78 ms using MLEV17 spin
lock pulses(30). Double quantum-filtered (DQF) COSY spectra (31) were
collected in H2O solution. All data were recorded in the
phase-sensitive mode using the time proportional phase incrementation
method (32) with 2048 data points in the acquisition domain and 512 or
256 in the time domain. Two-dimensional NOESY (33) experiments were also performed to identify slowly exchanging amide hydrogens on a
freshly prepared D2O solution after lyophilization of an
H2O sample. Small flexible linear peptides such as 4V have
relatively short correlation times compared with globular proteins. As
a result, longer mixing times or rotating frame Overhauser effect spin
lock times are typically needed before NOE or rotating frame Overhauser
effect cross-peaks begin to build up in the spectrum. From the NOESY
growth curve, we have ascertained that spin-diffusion effects are not
significant even for 600-800-ms mixing times. The interesting
cross-peaks observed in the NOESY spectra are due to direct effects
since no other cross-peaks from protons that could mediate the
intensity through spin diffusion were observed in the spectra. Long
mixing times in the range of 400-800 ms have typically been used for
small peptides.
All NMR data were processed using nmrPipe (Biosym/Molecular Simulations
Inc.) or Bruker XWIN-NMR (Bruker Instruments) software on SGI
Indigo2 workstation. Prior to Fourier transformation in the
t1 dimension (34), the first row was
half-weighted to suppress t1 ridges. The
DQF-COSY data were processed to 8192 × 1024 data matrices to
obtain a maximum digital resolution for coupling constant measurements. The proton chemical shifts were referenced with internal sodium 4,4-dimethyl-4-silapentane 1-sulfonate.
Molecular Modeling Calculations--
All calculations were
performed for syndecan-4V using XPLOR 3.1 (Biosym/Molecular
Simulations, Inc) on an SGI Indigo2 workstation using the
topology and parameter files of topallhdg.pro and parallhdg.pro.
Monomer 4V structures were generated with random backbone dihedral
angles and used as starting structures for simulated annealing (SA)
calculations (35-38). The procedure by Nilges (35) was also used for
symmetric dimer generations with minor modifications described by Lee
et al. (39) as follows. Symmetric dimers were generated by
duplications of the random coordinates. The combined use of XPLOR
noncrystallographic symmetry and symmetry pseudo-NOE term were served
to satisfy monomer symmetry as described by Nilges (35). The modeling
protocol used by us consists of two separate stages: (i) initial and
extensive regulation and (ii) simulated annealing and refinement.
Regulation procedures were cycled twice to satisfy both experimental
constraints and noncrystallographic symmetry parameters, followed by
simulated annealing and refinement. The potential energy function
consisted of covalent, repulsion, NOE, and torsional angle terms. The
target function forms of NOE and torsional angles are the same as used
by Driscoll et al. (40). A total of 116 distance restraints
and 16 torsional angle constraints were used for structural
calculations. All NOEs were classified and converted to distance
constraints as strong (1.8-2.7 Å), medium (1.8-3.3 Å), and weak
(1.8-5.0 Å) based on their intensities of NOESY spectrum. Corrections
for pseudoatom representations were used for non-stereo specifically
assigned methylene, methyl group, and tyrosine ring protons (32).
Dihedral angle restraints were derived from measured
3JHN-H
coupling constants in DQF-COSY
spectra in H2O solution (41, 42). The structure of
4V·PIP2 complex was generated with Insight II
(Biosym/Molecular Simulation Inc.) based on intermolecular NOE data of
two-dimensional NOESY spectrum.
 |
RESULTS AND DISCUSSION |
CD of Free and PIP2-complexed Peptide--
The CD
spectra of the 4V peptide (sequence LGKKPIYKKA) at pH 7.4 and 25 °C
were consistent with a random coil characteristics (43). However, the
CD spectra obtained from 4V peptide complexed with PIP2
were different from that of peptide alone, having a small positive mean
residue ellipticity at 222 nm. The same spectral changes of 4L peptide
were observed in the presence of PIP2. The CD data
suggested that the structural transitions of both peptides were
occurred in the presence of PIP2. Fig.
1 shows that the positive ellipticity
intensity of 4V·PIP2 complex at 222 nm decreased
gradually at temperature ranges of 20-60 °C, indicating a slow
process of dissociation.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Circular dichroism spectra of
4V·PIP2 complex at temperatures of 20-60 °C, pH 7.0. Closed triangle, 20 °C; open triangle,
25 °C; closed circle, 55 °C; open circle,
60 °C.
|
|
NMR Resonance Assignments and Secondary Structures--
The
one-dimensional proton NMR spectra of both 4V and 4L peptides are shown
in Fig. 2. The spectra have demonstrated
that both peptides produced considerable changes in the whole peptide resonance as well as in linewidths of resonance throughout
PIP2 titration. In particular, 4L peptide experiences a
severe aggregation at ratio of 8:6 (4V:PIP2) (Fig. 2,
B, d). There are only single glycine, tyrosine, and alanine
residues in the 4V peptide sequence. Glycine was easily identified by
the distinctive fingerprint pattern in the H2O COSY
spectra, and the lone alanine residue was identified from
connectivities in TOCSY spectra by its characteristic methyl resonance.
One AMX spin system from the tyrosine residue was easily identified in
TOCSY spectra. These preliminary resonance assignments served as
starting points for the sequence-specific assignment procedure (44).
Sequential resonance assignments were made based on two-dimensional
TOCSY and NOESY spectra in 90% H2O, 10% D2O solution. Sequential assignments for the backbone protons were completed by following d
N connectivities from
NH-C
H COSY cross-peaks of previously identified amino
acids. The side-chain proton chemical shifts were completed by TOCSY
connectivities. The resonances of PIP2 were identified from
the previously published chemical shifts (45) and PIP2
titration procedure (Fig. 2). Some of the PIP2 resonance
signals in 4V·PIP2 complex were assigned from proton
one-dimensional spectrum, followed by PIP2 titration to 4V.
Since most of PIP2 resonances were severely overlapped with
peptide signals, PIP2 chemical shifts were identified from all assigned peptide resonances in two-dimensional TOCSY and NOESY spectra. All these sequential and medium NOE connectivities for both
free and complex are summarized in Fig.
3. Most of the NOEs shown in Fig. 3 were
measured at mixing times of 100-400 ms. For 4L peptide, since the
backbone NMR resonances were severely overlapped due to 7 lysine
residues in the sequence, not all NOEs from residues involved in the
variable domain could be assigned
completely.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
A, proton one-dimensional NMR spectra of
4V peptide with addition of 0 mg (a), 0.5 mg (b),
and 1.0 mg (c) of PIP2, respectively. The molar
ratios of peptide to PIP2 are indicated on the
left side of the spectra. B, proton
one-dimensional NMR spectra of 4L with PIP2 of 0 mg
(a), 0.25 mg (b), 0.33 mg (c), and 0.5 mg (d), respectively.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 3.
Summary of the NOE connectivities identified
in both free (A) and 4V·PIP2 (B)
complex (peptide:PIP2 = 2:1) at 20 °C, pH 7.0 (complex)
and pH 7.4 (free), respectively. Mixing times of 100-400 ms were
used, and observed NOE intensities are classified by the thickness of
the lines.
|
|
The proton chemical shifts for the individual residues in free 4V
peptide are in excellent agreement with the corresponding random coil
values (46). As expected for a highly flexible peptide, NH-C
H coupling constants for all the residues are of the
order of 6.5-8.0 Hz, suggesting retention of significant
conformational flexibility. In addition, the sequential NOE intensities
displayed a standard pattern of the preferred random coil conformation
of the peptide. However, when peptide was titrated with
PIP2, both the chemical shifts and NOE patterns exhibited a
significant change in the entire peptide sequence indicating a
structural transition (Figs. 3 and 4). In
addition, a number of intermolecular NOEs, which are NOEs between
peptide and PIP2 and intersubunit NOEs on the 4V dimer,
were observed. The aliphatic proton resonance region of the
two-dimensional NOESY spectrum of 4V·PIP2 is shown in
Fig. 5, displaying both intersubunit and
intermolecular NOEs in the complex.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 4.
Plot of proton chemical shift differences of
4V calculated from free and 4V·PIP2 complex.
Backbone NH, C H, and C H chemical shifts
are used.
|
|

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 5.
600-MHz two-dimensional NOESY proton spectrum
with mixing time of 400 ms in 90% H2O/10% D2O
solution at 20 °C and pH value of 7.0. The spectrum are labeled
with some of NOEs including intersubunit NOEs (marked as *) as well as
intermolecular NOEs between PIP2 and 4V peptide.
|
|
Three-dimensional Structures--
It is of interest to compare the
proton chemical shift differences between free and PIP2
complex 4V. Fig. 4 shows that, whereas proton chemical shifts of the
side chains showed not much changes, those of the backbone NH and
C
H changed dramatically in N-terminal as well as
C-terminal regions. The side-chain chemical shifts, especially
Pro185 and Ile186 residues in the center of
peptide, exhibited significant changes. These interesting results
indicate that the 4V dimer has extensive intermolecular contacts not
only at both peptide terminus but also in the middle of the peptide
(Fig. 4). In addition, these results strongly suggest that, while the
side-chain atoms would have close intersubunit contacts with each other
in the center of peptide, the backbone atoms do so at peptide
termini.
The solution structures for the 4V dimer were generated using the
experimental constraints described above. A total of 90 starting
substructures were used in the initial stage. After two cycles of
regulation protocol, 60 structures that show no constraint violations
greater than 0.5 Å for distances and 5° for torsional angles were
identified. Among 60 structures, the 14 lowest energy structures
(
SA
kr) were selected for detailed analysis (Table I).
The structures are well defined with a root-mean-square deviation between backbone atom coordinates of 1.05 Å for all residues. The
average structure was generated from the geometrical average from 14 structure coordinates and was subjected to restrained energy
minimization to correct bond length and angle distorsions. This average
structure exhibited 0.59-Å root-mean-square deviation for backbone
atoms with respect to 14
SA
kr structures. A best fit
superposition of all final structures and the backbone conformation for
average restrained energy minimized structure
(

kr) are shown in Fig.
6. The atomic average root-mean-square
deviations of the final structures with respect to average restrained
energy minimized structure are shown in Fig.
7. A Ramachandran plot (Fig. 8) indicates that
,
values of all
14 final NMR structures are distributed properly in energetically
acceptable regions (47).

View larger version (103K):
[in this window]
[in a new window]
|
Fig. 6.
A, a stereo view of the backbone
superposition of the energy-minimized average structure
(  kr) over the family of 14 final
simulated annealing structures ( SA kr) with subunit A
(red) and B (green). B, a
ball-and-stick model of the energy-minimized average structure
displaying all atoms. Subunits are colored in red and
green, respectively. C, side view of the
energy-minimized average structure displaying a cavity in the center of
dimeric interface. Backbone conformation is drawn as red
ribbon.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 7.
Distribution of the average atomic
root-mean-square deviations with respect to average structure of the
backbone (A) and side-chain (B) atoms in the 14 final simulated annealing structures for the 4V dimer.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
Distribution of all ( , ) values for the
final 14 simulated annealing structures of the 4V in
4V·PIP2 complex. The glycine residues are
represented by open circles.
|
|
An inspection of the average energy-minimized structure in Fig.
6B clearly shows that major driving forces for dimer
formation could be originated from hydrophobic interactions among
side-chain atoms. 4V peptide in 4V·PIP2 complex forms a
compact symmetric dimer with two extended strands twisted parallel each
other. This compactness explains why 4V dimer dissociates slowly during
the subunit exchange experiment as shown in Fig. 1. The exposed surface of the 4V consisted of very polar residues with basic side chains. Interestingly, the Pro185 residue that resides in the
center of the cytoplasmic core has been shown to play a key role for
twisted dimeric formation. No standard regular secondary structural
elements were observed, as shown in Fig. 1. However, each monomer
conformation exhibits similar to that of extended form with both N and
C termini with kink. Strikingly, the overall three-dimensional
structure of the 4V in solution demonstrates a twisted clamp shape
having a cavity in the center of dimeric twist (Fig.
6C).
Molecular Interaction with PIP2--
A number of NOE
contacts between 4V and PIP2 have been observed, which are
NOEs between Pro185 ring protons of 4V and head group
protons of PIP2, Ile186
C
H3 methyl group of 4V and fatty acyl group
of PIP2, and C
H2 of
Lys188 and olefinic protons of PIP2. These NOEs
strongly suggest that both anionic head group and fatty acyl side chain
of PIP2 have close contacts with center of the peptide.
Table II summarizes NOEs between 4V and
PIP2. Especially, a possible hydrogen bond between
phosphatidylinositol 4-phosphate oxygen and Lys188
NH3+ of 4V was observed from modeling
structure based on NMR data. Fig. 9 shows
the averaged energy-minimized solution structure of 4V complexed with
PIP2. The 4V dimer grips PIP2 having
interactions with both head group and fatty acyl chains of
PIP2. The solution structure clearly reveals that side
chains of cytoplasmic core residues play an important role for
PIP2 binding.

View larger version (92K):
[in this window]
[in a new window]
|
Fig. 9.
A, model of 4V·PIP2
complex generated with Insight II (Biosym/Molecular Simulation Inc.).
PIP2 (space-filling model) and 4V (van der Waals and ribbon
representation) are shown in violet and red,
respectively. B, electrostatic potential surface of 4V and
space-filling model of PIP2 (yellow) in
4V·PIP2 complex. The negative electrostatic potential is
represented in red, the positive in blue, and the
neutral in white. The potential surface was calculated using
the Delphi program (Biosym/Molecular Simulation Inc.).
|
|
It has been suggested that the syndecan core proteins, which are highly
conserved among the syndecan family, exhibit a propensity to form
non-covalently linked dimers and higher order oligomers (12-14). It is
also well known that oligomer formation is essential in protein kinase
C interaction and activation (12). Recently, we have reported that
SDS-resistant multimerization of syndecan-4 core protein correlates
with PKC regulatory activity and variable region unique to syndecan-4
(4V) could potentiate phospholipid-induced activation of PKC

(12, 13). In addition, it has been shown that a central penta-peptide
KPIYK sequence is necessary for multimerization of syndecan-4 core
protein, resulting in both direct activation of PKC

and
phospholipid-induced activation (12, 13). Further, 4V or 4L peptides in
the presence of PIP2 strongly activate PKC
(28). The
data from NMR and gel filtration clearly support the hypothesis that
PIP2 induces dimerization of both 4V and 4L peptide, and
the induced dimer of 4V forms a compact structure bound to PIP2 (Fig. 2). We have recently reported that the peptide
of whole cytoplasmic domain (4L) displayed less tendency to oligomerize than 4V in the absence of PIP2, resulting reduced activity
of 4L. This could be due to highly basic residues of the conserved (C1)
region, which lies between the V region of the cytoplasmic domain and
the membrane. These residues may decrease the self-association of the
4L peptide. However, very recently, we have shown that both 4V and 4L
peptides exhibited essentially the same activity with respect to
PIP2-mediated activation of PKC (28). Therefore, we
concluded that the solution structure of 4L in the presence of
PIP2 should resemble that of PIP2-4V complex;
in other words, the molecular topology of 4L and 4V in the presence of
PIP2 must be similar. Major interaction of 4V peptide with
PIP2 occurs between aliphatic side chains of peptide and
whole area of PIP2. No close contacts of peptide backbone
to PIP2 has been observed. The other finding is that, even
though PIP2 promotes association of 4V initially, further
driving or stabilizing forces could be peptide-peptide interactions.
The NMR structure clearly shows that peptide backbones of each monomer
consists of intertwined circular shape and side chains provide
additional stabilizing forces by hydrophobic interactions from an
outward position. Surprisingly, two extended monomers form a quite
stable twisted dimeric conformation, even if there is no hydrogen bonds
between monomers. Since the calculated 14 structures
(
SA
kr) based on NMR constraints exhibited a good convergence, we conclude that the 4V dimer has a stable and unique conformation in solution state in the presence of PIP2.
This was also confirmed by melting experimental data based on circular dichroism (Fig. 1) and NMR linewidth analysis at temperature ranges of
20-70 °C (data not shown). We have recently demonstrated that 4V
peptides of altered sequence have much reduced ability to regulate PKC
activity (12, 13, 28). It was suggested that substitution of
Lys184, Lys188, Pro185, and
Ile186 compromised both oligomerization and cPKC
activation. The data here support this hypothesis, showing that all
these residues take part in monomer-monomer interactions. Our previous
data also showed that, while replacement of Tyr187 with
phenylalanine had a profound effect on PKC activation, it had the least
effect on dimer formation. This is in very good agreement with solution
structure analysis. Tyr187 appears not to be involved in
oligomerization of 4V, and we hypothesize that since its side chain is
mostly solvent-exposed, the hydroxyl group may be critical for contact
with, and activation of, PKC. Structural studies of the
4V·PIP2·PKC
ternary complex will provide this
important information.
We thank TMSI Korea for the use of the
molecular simulation programs (Molecular Modeling Tools, Molecular
Simulations Inc.).