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

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 PKCα 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 PKCαβγ show that in the absence of phosphatidylserine and diolein, PIP2 increased the extent of autophosphorylation of PKCαβγ 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 PKCαβγ 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 PKCα, localize them to forming focal adhesions, and potentiate PKCα activity there.

The control of cellular adhesion status is complex, involving several signaling mechanisms (1)(2)(3)(4). Phosphatidylinositol 4,5bisphosphate (PIP 2 ) 1 plays important roles in the organization of the actin cytoskeleton. PIP 2 may control actin polymerization by regulating the binding of actin-binding proteins such as profilin and gelsolin to actin (5,6). PIP 2 may also interact with ␣-actinin and vinculin (7) and regulate their association with the cytoskeleton (8). The level of PIP 2 decreases upon detachment of cells from the substratum and increases upon reattachment to fibronectin (1). The difference in the levels of PIP 2 is probably due to different rates of phosphorylation of phosphatidyl 4-phosphate to PIP 2 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). PIP 2 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 Ca 2ϩ , 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 (PIP 3 ), which has been proposed to regulate numerous activities including cytoskeletal organization (14) and vesicle trafficking (15). PIP 2 can also be dephosphorylated via the 5-phosphatase to phosphatidylinositol 4-phosphate (16). PIP 2 may also directly activate several proteins including PKC. PIP 2 is a potent activator of conventional PKC isotypes (␣, ␤I, ␤II, and ␥) in the presence of phosphatidylserine (PS) and calcium (17)(18)(19). Indeed, PIP 2 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, PIP 2 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)(24)(25)(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)(24)(25)(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 PIP 2 and several PIP 2 -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 PIP 2 (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 PIP 2 and inositol hexaphosphate (IP 6 ). 2 Since syndecan-4 can bind PIP 2 and activate PKC, we investigated whether PIP 2 and syndecan-4 act synergistically to activate PKC, representing an alternative pathway to those previously described.

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. [␥-32 P]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 PIP 2 , IP 6 , and inositol tetraphosphate (IP 4 ), histone III-S, myelin basic protein, and other chemicals were purchased from Sigma. PIP 3 was synthesized by Dr. Roy Gigg (National Institute of Medical Research, London, UK).
In Vitro PKC Assay-The standard reaction mixture (total 20 l) contained 50 mM HEPES (pH 7.3), 3 mM magnesium acetate, PKC␣␤␥ (3 ng) or PKC␣ (1 ng), and 4 g 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. CaCl 2 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 M ATP (0.5 mCi of [␥-32 P]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 ϫ 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 M PIP 2 or IP 6 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. 6 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). PIP 2 addition resulted in the highest level of PKC␣␤␥ activity (approximately 4-fold over control levels; compare lanes 1 and 2). The same concentrations of PIP 3 and IP 6 (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 PIP 2 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, PIP 2 , PIP 3 , IP 4 , and IP 6 had no significant effect on the ability of PKC␣␤␥ to phosphorylate histone III-S (data not shown). The effect of IP 6 on the phosphorylation of histone III-S by PKC␣␤␥ was dose-dependent and maximal at 50 M IP 6 ( Fig.  1B). Stimulation of PKC␣␤␥ by PIP 2 was also dose-dependent, with half maximal stimulation at 30 M and a maximum at 50 M PIP 2 (Fig. 1C).

PIP 2 and IP
Calcium Dependence of Activation-Since PKC␣␤␥ are known as calcium-dependent enzymes and PIP 2 interacts with PKC through its regulatory domain (18,34), we investigated whether calcium affected the increased activity of PKC␣␤␥ in the presence of PIP 2 and IP 6 ( 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 PIP 2 or IP 6 , indicating calciumindependence. Minor increases in phosphorylation were seen with PIP 2 and IP 6 at 1-30 M calcium, but at concentrations above 30 M, calcium significantly inhibited the activity. This is consistent with previous reports demonstrating the inhibition by calcium of PIP 2 -induced potentiation of the activity of PKC␤1, -⑀, and -in mixed micelles (38). In contrast, Phosphor Imager analysis of autoradiographs with recombinant PKC␣ indicated calcium dependence, with 25 M causing a 2.7-and LGKKPIYKK 4PA LGKKAIYKK 3.5-fold increase in phosphorylation of histone III-S in the presence of PIP 2 and IP 6 , respectively (not shown, but see Fig. 6).
PIP 2 , but Not IP 6 , Directly Activates PKC␣␤␥-Phosphoinositides such as PIP 2 and IP 6 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 PIP 2 or IP 6 could increase autophosphorylation of PKC␣␤␥ in the absence of PS/DL (Fig. 3). PIP 2 increased autophosphorylation of PKC␣␤␥ over that seen in the absence of PIP 2 (compare lanes 1 and 2). However, autophosphorylation of PKC␣␤␥ in the presence of IP 6 was not increased (compares lanes 3 and 4). Thus, IP 6 may increase PKC␣␤␥ phosphorylation of basic substrates by charge interactions that increase substrate accessibility. In contrast, PIP 2 may directly affect PKC␣␤␥. To substantiate this hypothesis, PKC␣␤␥ assays were performed in the presence of PIP 2 or IP 6 using a peptide substrate from the EGF receptor (Fig. 4). PIP 2 increased PKC␣␤␥ phosphorylation of this substrate approximately 3-fold (compare lanes 1 and 3), whereas no increase was seen with IP 6 (compare lanes 1 and 9). Although this activation was less than that seen using histone III-S as substrate, it was statistically significant (p Ͻ 0.001). As seen with histone III-S phosphorylation, PIP 3 , but not IP 4 , also increased the phosphorylation of the EGF receptor peptide approximately 2.5-fold (compare lanes 1 and 5).
Syndecan-4 Further Potentiates PKC␣␤␥ Activity Induced by PIP 2 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 PIP 2 -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 PIP 2 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 PIP 3 (compare lanes 5 and 6), IP 4 (compare lanes 7 and 8), or IP 6 (compare lanes 9 and 10). Similar results were obtained monitoring histone III-S phosphorylation (Fig. 5A). PIP 2 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 PIP 2 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 IP 6 (Fig. 5B, compare lanes 3 and 4), again suggesting that IP 6 and PIP 2 act through different mechanisms.
To investigate whether the potentiation of PIP 2 -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 PIP 2 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 M calcium normally induced maximal phosphorylation (lane 2), as seen in our assays (28) and by others (39). In the presence of 50 M PIP 2 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 PIP 2 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 PIP 2 (lane 3) did, and a further increase was seen in the presence of both 4L and PIP 2 (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 PIP 2 -mediated activation (Fig. 6C) 4 and 5). Again, 4PA peptide did not increase the activity seen with PIP 2 alone Ϯ 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 M calcium. Activation of recombinant PKC␣ by PIP 2 appears to be dependent on at least 25 M 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 PIP 2 -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 M (lanes 3 and 6) or 100 M (lanes 4 and 7) calcium or even 1 mM EGTA (lanes 2 and 5).
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 PIP 2 -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 PIP 2 . Synthetic peptides 4L (lane 1) and 4V (lane 2) potentiated PIP 2 -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 PIP 2 .

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/Ca 2ϩ produces a large en-  6 and 7). Maximal phosphorylation is seen in the presence of PS/DL (PL) and 750 M calcium (lane 8). D, the activation of PKC␣␤␥ by PIP 2 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 M) does not increase activation (lanes 4 and 7). hancement of the PS/DL/Ca 2ϩ -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), PIP 2 , 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 M phosphatidylserine and 40 M phosphatidylethanolamine, most phosphoinositides, including PIP 2 , did not significantly activate PKC␣. They also failed to detect any significant activation of PKC␣ by 10 M PIP 2 in the absence of phospholipid (48). Our experiments show that PKC␣␤␥ requires 50 M PIP 2 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 PIP 2 may be as high as 140 -240 M (49), supporting physiological activation of PKC by PIP 2 .
In contrast to published reports, we report here that there is little or no calcium dependence for PIP 2 stimulation of PKC␣␤␥ in the presence of 50 M PIP 2 and absence of PS/DL, although activation of recombinant PKC␣ is dependent on low levels of calcium (25 M). 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 PIP 2 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 crosslinking 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 PIP 2 , 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 PIP 2 , in conjunction with the PKCbinding protein syndecan-4, can regulate PKC␣␤␥ activity through a novel calcium-independent pathway and that PKC␣ activation requires only low levels (25 M). 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, PIP 2 levels increase, and this may be an important regulatory factor for actin polymerization and stress fiber and focal adhesion formation (11,12). In addition, PIP 2 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 PIP 2 2 , and this promotes oligomerization of the syndecan-4 cytoplasmic domain (53). The fact that PIP 2 in the presence of syndecan-4 can together give rise to high PKC activity suggests that ternary interactions between PIP 2 , 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, PIP 2 , 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 PIP 2 probably binds the regulatory domain of PKC␣ (19, 24) even more strongly than diacylglycerol (20).
Both PKC␣ and PIP 2 appear to interact with the same region of syndecan-4, namely the central V region (LGKKPIYKK). The binding of PIP 2 and PKC to this region is not mutually exclusive. Although PIP 2 or 4V alone modestly up-regulate PKCmediated 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 IP 6 can also interact with syndecan-4. However, in contrast to PIP 2 , IP 6 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 IP 6 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 PIP 2 rather than IP 6 is the active participant in a signaling complex. Although IP 6 can also bind the syndecan-4 peptide, PIP 2 , but not IP 6 , 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).