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J Biol Chem, Vol. 273, Issue 17, 10624-10629, April 24, 1998

Syndecan-4 Proteoglycan Cytoplasmic Domain and Phosphatidylinositol 4,5-Bisphosphate Coordinately Regulate Protein Kinase C Activity*

Eok-Soo Oh, Anne Woods, Ssang-Taek Lim, Anne W. TheibertDagger , and John R. Couchman§

From the Department of Cell Biology, Cell Adhesion and Matrix Research Center and Dagger  Department of Neurobiology, University of Alabama, Birmingham, Alabama 35294

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in the organization of the actin cytoskeleton by regulating actin-associated proteins. The transmembrane heparan sulfate proteoglycan syndecan-4 also plays a critical role in protein kinase C (PKC) signaling in the formation of focal adhesions and actin stress fibers. The cytoplasmic domain of syndecan-4 core protein directly interacts with and potentiates PKCalpha activity, and it can directly interact with the phos- phoinositide PIP2. We, therefore, investigated whether the interaction of inositol phosphates and inositol phospholipids with syndecan-4 could regulate PKC activity. Data from in vitro kinase assays using purified PKCalpha beta gamma show that in the absence of phosphatidylserine and diolein, PIP2 increased the extent of autophosphorylation of PKCalpha beta gamma and partially activated it to phosphorylate both histone III-S and an epidermal growth factor receptor peptide. This activity was dose-dependent, and its calcium dependence varied with PKC isotype/source. Addition of the cytoplasmic syndecan-4 peptide, but not equivalent syndecan-1 or syndecan-2 peptides, potentiated the partial activation of PKCalpha beta gamma by PIP2, resulting in activity greater than that observed with phosphatidylserine, diolein, and calcium. This study indicates that syndecan-4 cytoplasmic domain may bind both PIP2 and PKCalpha , localize them to forming focal adhesions, and potentiate PKCalpha activity there.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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 Cgamma 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 (alpha , beta I, beta II, and gamma ) 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 PKCalpha 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 beta 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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). PKCalpha beta gamma purified from rabbit brain and recombinant PKCalpha were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). An alternate source of recombinant PKCalpha was Life Technologies, Inc., and similar results were obtained for both. [gamma -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).

                              
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Table I
The amino acid sequences of peptides derived from the cytoplasmic domain of syndecan-4, -2, and -1 

In Vitro PKC Assay-- The standard reaction mixture (total 20 µl) contained 50 mM HEPES (pH 7.3), 3 mM magnesium acetate, PKCalpha beta gamma (3 ng) or PKCalpha (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 [gamma -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 alpha beta gamma -- 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.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

PIP2 and IP6 Can Partially Activate PKCalpha beta gamma -- We first investigated whether phosphoinositides could elevate the activity of a mixture of PKCalpha beta gamma in vitro. In the absence of PS and DL, phosphoinositides increased the activity of PKCalpha beta gamma to phosphorylate histone III-S (Fig. 1A) or myelin basic protein (data not shown). PIP2 addition resulted in the highest level of PKCalpha beta gamma 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 PKCalpha beta gamma 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 PKCalpha beta gamma to phosphorylate histone III-S (data not shown). The effect of IP6 on the phosphorylation of histone III-S by PKCalpha beta gamma was dose-dependent and maximal at 50 µM IP6 (Fig. 1B). Stimulation of PKCalpha beta gamma 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 PKCalpha beta gamma 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).

Calcium Dependence of Activation-- Since PKCalpha beta gamma 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 PKCalpha beta gamma 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 PKCalpha beta gamma 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 PKCbeta 1, -epsilon , and -zeta in mixed micelles (38). In contrast, PhosphorImager analysis of autoradiographs with recombinant PKCalpha 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 PKCalpha beta gamma 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.

PIP2, but Not IP6, Directly Activates PKCalpha beta gamma -- 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 PKCalpha beta gamma was due to either increased PKCalpha beta gamma activity or increased accessibility of the substrate to PKCalpha beta gamma . We therefore investigated whether PIP2 or IP6 could increase autophosphorylation of PKCalpha beta gamma in the absence of PS/DL (Fig. 3). PIP2 increased autophosphorylation of PKCalpha beta gamma over that seen in the absence of PIP2 (compare lanes 1 and 2). However, autophosphorylation of PKCalpha beta gamma in the presence of IP6 was not increased (compares lanes 3 and 4). Thus, IP6 may increase PKCalpha beta gamma phosphorylation of basic substrates by charge interactions that increase substrate accessibility. In contrast, PIP2 may directly affect PKCalpha beta gamma . To substantiate this hypothesis, PKCalpha beta gamma assays were performed in the presence of PIP2 or IP6 using a peptide substrate from the EGF receptor (Fig. 4). PIP2 increased PKCalpha beta gamma 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 PKCalpha beta gamma autophosphorylation by PIP2 but not IP6. PIP2 or IP6 was added at 50 µM as under "Experimental Procedures," and autophosphorylation of PKCalpha beta gamma 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 PKCalpha beta gamma 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.

Syndecan-4 Further Potentiates PKCalpha beta gamma Activity Induced by PIP2 but Not by Other Phosphoinositides-- Our previous studies showed that syndecan-4 could directly activate recombinant PKCalpha 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 PKCalpha beta gamma 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 PKCalpha beta gamma 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 PKCalpha beta gamma 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 PKCalpha beta gamma 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 PKCalpha beta gamma 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 PKCalpha beta gamma 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.

To investigate whether the potentiation of PIP2-induced PKC activity by syndecan-4 could be significant in vivo, we compared the maximal activity of PKCalpha beta gamma or PKCalpha 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 PKCalpha beta gamma , even in the absence of PL and calcium (Fig. 6A, compare lanes 3 and 4 with 2). Again PIP2 alone induced some activation of PKCalpha beta gamma in the absence of PS/DL, peptide, or calcium (lane 5). With recombinant PKCalpha (Fig. 6B), similar results were seen, although low levels of calcium were required. Calcium alone did not activate PKCalpha (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 PKCalpha 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 PKCalpha . PIP2 activation of PKCalpha 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 PKCalpha in the presence of PS/DL and 750 µM calcium. Activation of recombinant PKCalpha by PIP2 appears to be dependent on at least 25 µM calcium (Fig. 6C, lanes 4 and 5), whereas that of purified PKCalpha beta gamma is not (Fig. 6A, lane 5). This was confirmed (Fig. 6D) by the fact that potentiation of PIP2-induced PKCalpha beta gamma 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 PKCalpha beta gamma and recombinant PKCalpha . A, autoradiographs show the basal level of phosphorylation by PKCalpha beta gamma 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 PKCalpha beta gamma 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 PKCalpha 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 PKCalpha  ± 25 µM calcium (compare lane 1 with 2 and 3). PIP2 activation of PKCalpha 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 PKCalpha beta gamma 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).

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 PKCalpha beta gamma in the presence of PIP2. Synthetic peptides 4L (lane 1) and 4V (lane 2) potentiated PIP2-induced activity of PKCalpha beta gamma , 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 PKCalpha beta gamma activation by PIP2.


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  		Fig. 7.   Effect of syndecan peptides on PIP2-induced PKCalpha beta gamma 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).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 PKCalpha emerges from its presence in focal adhesions of normal, but not transformed, cells (45, 46). PKCalpha beta gamma 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 PKCalpha . 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 PKCalpha . They also failed to detect any significant activation of PKCalpha by 10 µM PIP2 in the absence of phospholipid (48). Our experiments show that PKCalpha beta gamma 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 PKCalpha beta gamma in the presence of 50 µM PIP2 and absence of PS/DL, although activation of recombinant PKCalpha is dependent on low levels of calcium (25 µM). This may be due to differences in preparation of PKCalpha beta gamma and recombinant PKCalpha , 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 Cgamma 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 PKCalpha beta gamma 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 PKCalpha beta gamma activity through a novel calcium-independent pathway and that PKCalpha 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 PKCalpha 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 PKCalpha copatches when syndecan-4 is patched by the addition of ectodomain antibodies to spreading fibroblasts, and they can be coimmunoprecipitated. Moreover, PKCalpha , 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 PKCalpha 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 PKCalpha may be the most relevant activation of PKCalpha 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 Cgamma -dependent calcium fluxes or diacylglycerol production. However, it is not yet known whether interactions of two of the three components, syndecan-4, PIP2, and PKCalpha , 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 PKCalpha (28), whereas PIP2 probably binds the regulatory domain of PKCalpha (19, 24) even more strongly than diacylglycerol (20).

Both PKCalpha 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 PKCalpha beta gamma only when phosphorylating histone III-S as a substrate, not when using the EGF receptor peptide as a substrate. Experiments examining the autophosphorylation of PKCalpha beta gamma 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 PKCalpha beta gamma by the oligomeric peptide (53).

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM50194 (to J. R. C.) and MH50102 and HD32901 (to A. W. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Cell Biology, University of Alabama, VH 201C, University Blvd., Birmingham, AL 35294-0019. Tel.: 205-934-2626; Fax: 205-975-9956

1 The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate; PKC, protein kinase C; PS, phosphatidylserine; IP6, inositol hexaphosphate; IP4, inositol tetraphosphate; DL, diolein; EGF, epidermal growth factor; PL, phospholipid.

2 J. R. Couchman, A. Woods, E.-S. Oh, G. Prestwich, and A. W. Theibert, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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