From the Department of Cell Biology, Cell Adhesion and Matrix
Research Center and
Department of Neurobiology,
University of Alabama, Birmingham, Alabama 35294
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INTRODUCTION |
The control of cellular adhesion status is complex, involving
several signaling mechanisms (1-4). Phosphatidylinositol
4,5-bisphosphate (PIP2)1 plays
important roles in the organization of the actin cytoskeleton. PIP2 may control actin polymerization by regulating the
binding of actin-binding proteins such as profilin and gelsolin to
actin (5, 6). PIP2 may also interact with
-actinin and
vinculin (7) and regulate their association with the cytoskeleton (8). The level of PIP2 decreases upon detachment of cells from
the substratum and increases upon reattachment to fibronectin (1). The
difference in the levels of PIP2 is probably due to
different rates of phosphorylation of phosphatidyl 4-phosphate to
PIP2 by phosphatidylinositol 4-phosphate 5-kinase.
Phosphatidylinositol 4-phosphate 5-kinase is stimulated 3-4-fold by
adhesion of cells to fibronectin (1), probably through interactions
with the small GTP-binding proteins Rac and Rho, the latter of which
has also been implicated in the regulation of assembly of actin stress fibers and focal adhesions (9-13).
PIP2 may enter several different pathways in signal
transduction. It can be 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). It can be further
phosphorylated by phosphatidylinositol 3-kinase to generate
phosphatidylinositol 3,4,5-triphosphate (PIP3), which has
been proposed to regulate numerous activities including cytoskeletal
organization (14) and vesicle trafficking (15). PIP2 can
also be dephosphorylated via the 5-phosphatase to phosphatidylinositol 4-phosphate (16). PIP2 may also directly activate several
proteins including PKC. PIP2 is a potent activator of
conventional PKC isotypes (
,
I,
II, and
) in the presence
of phosphatidylserine (PS) and calcium (17-19). Indeed,
PIP2 is more potent than diacylglycerol in stimulating PKC
in vitro (20), and it stimulates the translocation of
conventional PKC from the soluble to the particulate fraction (18).
Thus, PIP2 may itself be a primary activator of PKC
in vivo, both activating it and inducing its association
with the plasma membrane (19, 21).
PKC activity is needed for matrix-induced cell spreading (22) and for
the later stage of focal adhesion assembly (23). Cell surface heparan
sulfate proteoglycans have critical role(s) in PKC signaling in focal
adhesion and actin stress fiber formation (23-26). Cell attachment and
spreading can be promoted through integrin interactions with the cell
binding domain of fibronectin (23). However, normal
anchorage-dependent fibroblasts require an additional
signal(s) to form focal adhesions, which occur after binding of a
heparin binding domain of fibronectin or a peptide from this domain to
a cell surface heparan sulfate proteoglycan (23-26). These
interactions may stimulate PKC activity, since PKC inhibitors prevent
focal adhesion formation, and pharmacological activation of PKC can
substitute for stimulation through heparin binding moieties (23).
Syndecan-4 is one of four mammalian transmembrane heparan sulfate
proteoglycans that share a high degree of similarity, and it is
selectively concentrated in focal adhesions in numerous cell types
(27). It may transduce the signal(s) generated on binding of heparin
binding moieties to cells. A unique region of its cytoplasmic domain
(LGKKPIYKK) can potentiate PKC
activity in vitro, and PKC
interacts with its core protein in vivo and in
vitro, and with synthetic peptides of the LGKKPIYKK sequence (28).
The interactions between PIP2 and several
PIP2-binding proteins may be through their pleckstrin
homology domains (20, 29-32), where two lysine residues, which end a
1 strand at the turn, interact with the 4- and 5-phosphates of the
inositol head group of PIP2 (31). The cytoplasmic sequence
of syndecan-4 bears some similarity to pleckstrin homology domains, and
the LGKKPIYKK peptide from the cytoplasmic domain of syndecan-4 can
interact with the phosphoinositides PIP2 and inositol
hexaphosphate
(IP6).2 Since
syndecan-4 can bind PIP2 and activate PKC, we investigated whether PIP2 and syndecan-4 act synergistically to activate
PKC, representing an alternative pathway to those previously
described.
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EXPERIMENTAL PROCEDURES |
Materials--
Synthetic peptides corresponding to the whole
cytoplasmic domain of syndecan-4 (4L) and to the central, unique region
of syndecan-4 (4V), -2 (2V), or -1 (1V), a peptide having the scrambled
sequence of 4V (Scr), and one where the proline was substituted with
alanine (4VPA) were synthesized and sequenced by the University of
Alabama at Birmingham Comprehensive Cancer Center Peptide Synthesis and Analysis Shared Facility (Table I).
PKC

purified from rabbit brain and recombinant PKC
were
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). An
alternate source of recombinant PKC
was Life Technologies, Inc., and
similar results were obtained for both. [
-32P]ATP was
obtained from NEN Life Science Products. The peptide representing the
phosphorylation site in the epidermal growth factor (EGF) receptor and
P81 phosphocellulose paper were obtained from Biomol Research (Plymouth
Meeting, PA) and Whatman (Fairfield, NJ), respectively.
Phosphoinositides PIP2, IP6, and inositol
tetraphosphate (IP4), histone III-S, myelin basic protein,
and other chemicals were purchased from Sigma. PIP3 was
synthesized by Dr. Roy Gigg (National Institute of Medical Research,
London, UK).
In Vitro PKC Assay--
The standard reaction mixture (total 20 µl) contained 50 mM HEPES (pH 7.3), 3 mM
magnesium acetate, PKC

(3 ng) or PKC
(1 ng), and 4 µg of
histone III-S or myelin basic protein as a substrate. 0.2 mg/ml PS and
0.02 mg/ml diolein (DL) were added as required, and different amounts
of phosphoinositides were added as detailed in the text.
CaCl2 was added as indicated in the figure legends and
text, and 0.25 mg/ml each of synthetic peptides were present. Reactions
were started by the addition of 200 µM ATP (0.5 mCi of
[
-32P]ATP). After 10 min at room temperature, the
reaction was stopped by adding SDS-polyacrylamide gel electrophoresis
sample buffer and separated by 20% SDS-polyacrylamide gel
electrophoresis, and phosphorylated histone III-S or myelin basic
protein was detected by autoradiography and quantified by Bio-Rad Model
GS-670 imaging densitometer. In assays using 0.1 mg/ml EGF receptor
peptide of the sequence RKRTLRRL as an alternate substrate (33), the
reaction was stopped by spotting the whole reaction mixture onto
phosphocellulose filters (Whatman, p81, 2.1 cm) and dropping these into
75 mM phosphoric acid. Filters were washed 3 × 10 min, immersed in 95% ethanol for 5 min, dried, and counted with 4 ml
of scintillation mixture in a scintillation counter (Wallac Model
1409).
Autophosphorylation of PKC 

--
Reaction mixtures
prepared as described above with 50 µM PIP2
or IP6 in the absence of any activators (PS/DL, calcium) or substrate were incubated at 30 °C for 5 min and stopped by the addition of SDS sample buffer and heating to 95 °C for 5 min. Proteins were separated by 7.5% SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.
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RESULTS |
PIP2 and IP6 Can Partially Activate
PKC

--
We first investigated whether phosphoinositides
could elevate the activity of a mixture of PKC

in
vitro. In the absence of PS and DL, phosphoinositides increased
the activity of PKC

to phosphorylate histone III-S (Fig.
1A) or myelin basic protein (data not shown). PIP2 addition resulted in the highest
level of PKC

activity (approximately 4-fold over control
levels; compare lanes 1 and 2). The same
concentrations of PIP3 and IP6 (lanes
3 and 5, respectively) also increased activity
(approximately 3-fold), whereas the effect of inositol tetraphosphate
(lane 4) was not significant. The activation of PKC

by PIP2 was approximately 60% of the maximal activity by
conventional stimulation (refer to Fig. 6A) by PS/DL (0.2 mg/ml PS and 0.02 mg/ml DL) and calcium. When PS/DL was present,
PIP2, PIP3, IP4, and
IP6 had no significant effect on the ability of
PKC

to phosphorylate histone III-S (data not shown). The
effect of IP6 on the phosphorylation of histone III-S by
PKC

was dose-dependent and maximal at 50 µM IP6 (Fig. 1B). Stimulation of
PKC

by PIP2 was also dose-dependent, with half maximal stimulation at 30 µM and a maximum at
50 µM PIP2 (Fig. 1C).

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Fig. 1.
Phosphoinositide activation of PKC 
to phosphorylate histone III-S in the absence of phospholipid.
A, phosphoinositides (50 µM) were added to
assays in the absence of both calcium and PL, and autoradiographs of
phosphorylated histone III-S (HIS, inset) were quantified by
densitometer. Values shown are mean ± S.E. (n = 3). Activation was dose-dependent with both IP6
(B) and PIP2 (C).
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Calcium Dependence of Activation--
Since PKC

are known
as calcium-dependent enzymes and PIP2 interacts
with PKC through its regulatory domain (18, 34), we investigated
whether calcium affected the increased activity of PKC

in the
presence of PIP2 and IP6 (Fig.
2). In contrast to that observed with
PS/DL, no effect was seen at physiological intracellular calcium levels
(50-100 nM; Refs. 35-37) on the activation of
PKC

by either PIP2 or IP6, indicating
calcium-independence. Minor increases in phosphorylation were seen with
PIP2 and IP6 at 1-30 µM calcium,
but at concentrations above 30 µM, calcium significantly
inhibited the activity. This is consistent with previous reports
demonstrating the inhibition by calcium of PIP2-induced potentiation of the activity of PKC
1, -
, and -
in mixed
micelles (38). In contrast, PhosphorImager analysis of
autoradiographs with recombinant PKC
indicated calcium dependence,
with 25 µM causing a 2.7- and 3.5-fold increase in
phosphorylation of histone III-S in the presence of
PIP2 and IP6, respectively (not shown, but see
Fig. 6).

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Fig. 2.
The effect of calcium on activation of
PKC  by PIP2 and IP6. PKC activity
was measured in the absence of phospholipid but presence of 50 µM PIP2 (A) or IP6
(B) and with the different concentrations of calcium as
indicated. Representative autoradiograms of phosphorylated histone
III-S are shown.
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PIP2, but Not IP6, Directly Activates
PKC

--
Phosphoinositides such as PIP2 and
IP6 are highly negatively charged, whereas histone III-S
and myelin basic protein are positively charged. It was possible,
therefore, that increased phosphorylation of substrate by PKC

was due to either increased PKC

activity or increased
accessibility of the substrate to PKC

. We therefore investigated whether PIP2 or IP6 could increase
autophosphorylation of PKC

in the absence of PS/DL (Fig.
3). PIP2 increased
autophosphorylation of PKC

over that seen in the absence of
PIP2 (compare lanes 1 and 2).
However, autophosphorylation of PKC

in the presence of
IP6 was not increased (compares lanes 3 and
4). Thus, IP6 may increase PKC

phosphorylation of basic substrates by charge interactions that
increase substrate accessibility. In contrast, PIP2 may
directly affect PKC

. To substantiate this hypothesis, PKC

assays were performed in the presence of PIP2
or IP6 using a peptide substrate from the EGF receptor
(Fig. 4). PIP2 increased PKC

phosphorylation of this substrate approximately 3-fold (compare lanes 1 and 3), whereas no increase was
seen with IP6 (compare lanes 1 and
9). Although this activation was less than that seen using
histone III-S as substrate, it was statistically significant
(p < 0.001). As seen with histone III-S
phosphorylation, PIP3, but not IP4, also
increased the phosphorylation of the EGF receptor peptide approximately
2.5-fold (compare lanes 1 and 5).

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Fig. 3.
Stimulation of PKC 
autophosphorylation by PIP2 but not IP6.
PIP2 or IP6 was added at 50 µM as
under "Experimental Procedures," and autophosphorylation of
PKC  in the absence of calcium was detected by
autoradiography.
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Fig. 4.
The effects of phosphoinositides and
syndecan-4 peptide on the ability of PKC  to phosphorylate the
EGF receptor peptide in the absence of calcium. Phosphoinositides
were added at 50 µM and peptide at 250 µg/ml. Results
are the mean activity relative to phosphorylation in the absence of any
agent (lane 1), quantified by densitometric analysis of
autoradiographs.
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Syndecan-4 Further Potentiates PKC

Activity Induced by
PIP2 but Not by Other Phosphoinositides--
Our previous
studies showed that syndecan-4 could directly activate recombinant
PKC
and potentiate its activation by phospholipid through a defined
region of the syndecan-4 cytoplasmic domain (28). Further experiments
determined whether syndecan-4 could also affect the
PIP2-induced activation of PKC

using EGF receptor peptide (Fig. 4) or histone III-S (Fig.
5) as substrates. The results for both
were similar. Peptide 4V from the cytoplasmic domain of syndecan-4
potentiated the activity of PKC

to phosphorylate the EGF
receptor peptide in the presence of PIP2 from approximately 3-fold to 7-fold (Fig. 4, compare lanes 3 and 4 with 1). It had no effect, however, on activity in the
presence of PIP3 (compare lanes 5 and
6), IP4 (compare lanes 7 and
8), or IP6 (compare lanes 9 and
10). Similar results were obtained monitoring histone III-S phosphorylation (Fig. 5A). PIP2 alone increased
the activity of PKC

to phosphorylate histone III-S
approximately 5-fold (Fig. 5A, compare lanes 1 and 3). Peptide 4V in the absence of inositol lipid or
phospholipid showed a direct activation, as seen previously (28), but
to a smaller (approximately 1.5-fold) extent (lane 2). The
presence of both PIP2 and 4V potentiated the activation of
PKC

to approximately 11 times that of control levels (Fig. 5A, compare lanes 3 and 4 with
1). However, 4V did not further increase phosphorylation of
histone III-S by PKC

in the presence of IP6 (Fig.
5B, compare lanes 3 and 4), again
suggesting that IP6 and PIP2 act through
different mechanisms.

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Fig. 5.
The effect of syndecan-4 peptides on the
increased phosphorylation of histone III-S (HIS) by
PKC  in the presence of 50 µM
PIP2 (A) and IP6 (B).
In vitro PKC assays were performed in the absence of PS/DL
and calcium. Results are the mean ± S.E. (n = 3)
of densitometric analysis of autoradiograms, a representative one of
which is inset.
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To investigate whether the potentiation of PIP2-induced PKC
activity by syndecan-4 could be significant in vivo, we
compared the maximal activity of PKC

or PKC
in the presence
of both PIP2 and syndecan-4 peptide with that of PKC
induced by other physiological PKC phospholipid activators (Fig.
6). As seen previously (28), basal levels
of phosphorylation were detected in the absence of phospholipid and
calcium (Fig. 6A, lane 1). PS/DL in the presence of 750 µM calcium normally induced maximal
phosphorylation (lane 2), as seen in our assays (28) and by
others (39). In the presence of 50 µM PIP2
and the syndecan-4 peptide 4L or 4V, there was even greater activity of
PKC

, even in the absence of PL and calcium (Fig.
6A, compare lanes 3 and 4 with
2). Again PIP2 alone induced some activation of
PKC

in the absence of PS/DL, peptide, or calcium (lane
5). With recombinant PKC
(Fig. 6B), similar results
were seen, although low levels of calcium were required. Calcium alone
did not activate PKC
(lane 1) but peptide 4L (lane
2) or PIP2 (lane 3) did, and a further
increase was seen in the presence of both 4L and PIP2
(lane 4). An additional control was that the altered 4V
peptide (proline substituted with alanine), which had no effect in
potentiating PS/DL-mediated PKC
activity (28), also had no effect on
PIP2-mediated activation (Fig. 6C). Neither 25 µM calcium (lane 1) nor the 4PA peptide ± calcium (lanes 2 and 3) activated PKC
.
PIP2 activation of PKC
was dependent on the presence of
25 µM calcium (compare lanes 4 and
5). Again, 4PA peptide did not increase the activity seen
with PIP2 alone ± calcium (compare lanes 6 and 7 with lanes 4 and 5). Lane
8 shows the maximal activity of PKC
in the presence of PS/DL
and 750 µM calcium. Activation of recombinant PKC
by
PIP2 appears to be dependent on at least 25 µM calcium (Fig. 6C, lanes 4 and
5), whereas that of purified PKC

is not (Fig.
6A, lane 5). This was confirmed (Fig.
6D) by the fact that potentiation of
PIP2-induced PKC

phosphorylation of histone III-S
(lane 1) by the syndecan 4L (lanes 2-4) and 4V
(lanes 5-7) peptides was virtually unaffected by the
presence of 10 µM (lanes 3 and 6)
or 100 µM (lanes 4 and 7) calcium
or even 1 mM EGTA (lanes 2 and 5).

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Fig. 6.
Effect of syndecan-4 peptides and calcium on
PIP2-induced activation of PKC  and recombinant
PKC . A, autoradiographs show the basal level of
phosphorylation by PKC  of histone III-S in the absence of
PS/DL (PL) and 750 µM calcium (lane 1) and
normally maximal phosphorylation in their presence (lane 2).
Phosphorylation by PKC  is even higher in the presence of
PIP2 and peptide 4L (lane 3) or 4V (lane
4), with lower levels in the presence of PIP2 alone
(lane 5). B, recombinant PKC is not activated
in the presence of 25 µM calcium (lane 1), but
this is sufficient to allow activation by peptide 4L (lane
2) or PIP2 (lane 3) and potentiation of
activity with a combination of PIP2 and 4L peptide
(lane 4). C, peptide 4PA (proline substituted by
alanine) does not activate recombinant PKC ± 25 µM
calcium (compare lane 1 with 2 and 3).
PIP2 activation of PKC requires the presence of 25 µM calcium (compare lanes 4 and 5)
and is not increased in the presence of peptide 4PA (lanes 6 and 7). Maximal phosphorylation is seen in the presence of
PS/DL (PL) and 750 µM calcium (lane 8).
D, the activation of PKC  by PIP2 and 4L
or 4V is calcium-independent, since 1 mM EGTA has little
effect (compare lanes 3 and 6 with 2 and 5), and high calcium (100 µM) does not
increase activation (lanes 4 and 7).
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The Effect on PKC Activity Is Unique to a Syndecan-4 Cytoplasmic
Sequence--
Since all syndecans have high homology in 2 regions of
the cytoplasmic domain with intervening variable sequences (28), we
determined whether the potentiation of PIP2-induced PKC
activity was unique to syndecan-4 (Fig.
7). We used synthetic peptides corresponding to the whole cytoplasmic domain of syndecan-4 (4L), the
unique regions of the cytoplasmic domain of syndecans-4 (4V), -2 (2V)
or -1 (1V), and a peptide where the normal sequence of 4V was scrambled
(Scr) in assays monitoring phosphorylation of histone by PKC

in the presence of PIP2. Synthetic peptides 4L (lane
1) and 4V (lane 2) potentiated PIP2-induced
activity of PKC

, but Scr (lane 4) and 2V
(lane 5) or 1V (lane 6) had no effect. Thus, the
cytoplasmic domain of syndecan-4, but not those of syndecan-1 or
syndecan-2, can potentiate PKC

activation by
PIP2.

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Fig. 7.
Effect of syndecan peptides on
PIP2-induced PKC  activation. In vitro
PKC assays were performed in the presence of PIP2 (50 µM) and different synthetic peptides (250 µg/ml) as
shown but in the absence of both PL and calcium. Autoradiographs of
histone III-S (inset) were quantified by densitometer and
shown as the mean ± S.E. (n = 3).
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DISCUSSION |
A variety of evidence implicates PKC activity (22, 40-44) in
cell-cell and cell-matrix interactions. In most cases, the isoform of
PKC is unknown, though a role for PKC
emerges from its presence in
focal adhesions of normal, but not transformed, cells (45, 46).
PKC

have been characterized as calcium and
phospholipid-dependent isozymes, requiring both cofactors
for activity. We have previously shown that a peptide sequence from the
cytoplasmic domain of syndecan-4 can directly activate PKC
. In the
absence of PS/DL and calcium, a modest increase is observed (1.5-fold),
whereas addition of syndecan-4 peptide in the presence of
PS/DL/Ca2+ produces a large enhancement of the
PS/DL/Ca2+-stimulated activities, leading to an 11-fold
stimulation over basal activity (28). Similar to published reports, the
present studies show that a phosphoinositide previously implicated
in transmembrane signaling (16, 47), PIP2, partially
activates PKC in the absence of PS/DL, and this is increased by the
syndecan-4 peptide.
Previous studies by Toker et al. (48) have investigated the
activation of PKC isotypes by phosphoinositides. In the presence of 10 µM phosphatidylserine and 40 µM
phosphatidylethanolamine, most phosphoinositides, including
PIP2, did not significantly activate PKC
. They also
failed to detect any significant activation of PKC
by 10 µM PIP2 in the absence of phospholipid (48).
Our experiments show that PKC

requires 50 µM
PIP2 for maximum activation in the absence of PS/DL to
phosphorylate three different substrates: histone III-S, myelin basic
protein, and the EGF receptor peptide. In platelets, the concentration
of PIP2 may be as high as 140-240 µM (49),
supporting physiological activation of PKC by PIP2.
In contrast to published reports, we report here that there is little
or no calcium dependence for PIP2 stimulation of
PKC

in the presence of 50 µM PIP2
and absence of PS/DL, although activation of recombinant PKC
is
dependent on low levels of calcium (25 µM). This may be
due to differences in preparation of PKC

and recombinant
PKC
, leading to varying degrees of phosphorylation (50). The
phosphorylation status of intracellular PKC isoforms is not clear. IP3,
which activates intracellular calcium channels, is known to be produced
by the hydrolysis of PIP2 by phospholipase C
after
ligand binding to receptors (8). It is, however, not entirely resolved
whether or not calcium transients accompany integrin ligation and are
required for focal adhesion assembly. Although several cell types
undergo a transient increase of intracellular calcium levels during
integrin-mediated adhesion or integrin cross-linking with antibodies
(1), others only show this response during adhesion through a subset of
integrins (35, 51). The PKC

activity in vitro
induced by the combination of syndecan-4 and PIP2, even in
the absence of calcium, was greater than that maximally induced by the
conventional PKC activators PS and DL in the presence of high calcium
concentrations. It, therefore, appears that PIP2, in
conjunction with the PKC-binding protein syndecan-4, can regulate PKC

activity through a novel calcium-independent pathway and that PKC
activation requires only low levels (25 µM).
Indeed, this pathway has one extremely exciting feature; it overcomes the requirement for the nonphysiologically high calcium levels normally
required in vitro. The transient increase in calcium levels
in response to some integrin stimulation may be more involved in the
translocation of conventional PKC isotypes, especially PKC
to the
plasma membrane at the sites of focal adhesion formation (45, 46).
During focal adhesion formation, when cells adhere to an extracellular
matrix molecule such as fibronectin, PIP2 levels increase, and this may be an important regulatory factor for actin polymerization and stress fiber and focal adhesion formation (11, 12). In addition,
PIP2 and PKC activation are both required for focal adhesion and stress fiber formation (24, 52). We have previously shown
(28) that PKC
copatches when syndecan-4 is patched by the addition
of ectodomain antibodies to spreading fibroblasts, and they can be
coimmunoprecipitated. Moreover, PKC
, once activated by phospholipid
or phorbol esters, can interact in vitro with the
cytoplasmic domain of syndecan-4 through the sequence LGKKPIYKK, and
this potentiates PKC
activity (28). A synthetic peptide of the same
sequence also interacts with PIP22, and this
promotes oligomerization of the syndecan-4 cytoplasmic domain (53). The
fact that PIP2 in the presence of syndecan-4 can together
give rise to high PKC activity suggests that ternary interactions
between PIP2, syndecan-4 cytoplasmic domain, and PKC
may
be the most relevant activation of PKC
in the regulation of focal
adhesion and stress fiber formation. This would not require an
involvement of any other second messenger signaling mechanism such as
phospholipase C
-dependent calcium fluxes or
diacylglycerol production. However, it is not yet known whether
interactions of two of the three components, syndecan-4,
PIP2, and PKC
, influences further binding of the third
to form a ternary complex. Our previous data suggest that syndecan-4
core protein interacts with the catalytic domain of PKC
(28), whereas PIP2 probably binds the regulatory domain of PKC
(19, 24) even more strongly than
diacylglycerol (20).
Both PKC
and PIP2 appear to interact with the same
region of syndecan-4, namely the central V region (LGKKPIYKK). The
binding of PIP2 and PKC to this region is not mutually
exclusive. Although PIP2 or 4V alone modestly up-regulate
PKC-mediated phosphorylation of substrates, the addition of both agents
leads to a synergistic stimulation of kinase activity. In addition,
only oligomeric forms of syndecan-4 stimulate PKC activity (53).
Therefore syndecan-4 has multiple copies of the 4V region present when
interacting with and activating PKC. This activity is unique to
syndecan-4, which is the only syndecan that is widespread in focal
adhesions (27, 52). The three other mammalian syndecan core proteins and the Drosophila homolog all lack the essential V region
sequence, and PKC activity is not regulated by 2V and 1V (3V has a
sequence closely similar to 1V and, therefore, probably also
lacks activity; Ref. 54).
Our binding data indicates that IP6 can also interact with
syndecan-4. However, in contrast to PIP2, IP6
could activate PKC

only when phosphorylating histone III-S as
a substrate, not when using the EGF receptor peptide as a substrate.
Experiments examining the autophosphorylation of PKC

indicate
that IP6 may not directly activate the enzymes but rather
increase the apparent activity by changing substrate accessibility.
Since most experiments investigating PKC activation by
phosphoinositides have used highly basic substrates including myelin
basic protein, any increased phosphorylation seen may be due to either
or both increased activity or substrate accessibility. One further
experiment also supports the hypothesis that PIP2 rather
than IP6 is the active participant in a signaling complex.
Although IP6 can also bind the syndecan-4 peptide,
PIP2, but not IP6, will promote the
oligomerization of full-length syndecan-4 cytoplasmic domain (4L), with
a concomitant stimulation of kinase activity of PKC

by the
oligomeric peptide (53).