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J. Biol. Chem., Vol. 277, Issue 23, 20367-20371, June 7, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/23/20367    most recent M202501200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Murakami, M. Articles by Simons, M. Search for Related Content PubMed PubMed Citation Articles by Murakami, M. Articles by Simons, M. Social Bookmarking                      What's this?

Protein Kinase C (PKC)  Regulates PKC Activity in a Syndecan-4-dependent Manner^*

Masahiro Murakami, Arie Horowitz, Shaoqing Tang^§, J. Anthony Ware^§, and Michael Simons^¶

From the  Angiogenesis Research Center and Section of Cardiology, Dartmouth-Hitchcock Medical Center, Dartmouth Medical School, Lebanon, New Hampshire 03756 and ^§ Eli Lilly and Co., Indianapolis, Indiana 46285

Received for publication, March 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The phosphorylation state of Ser¹⁸³ in the cytoplasmic tail of syndecan-4 determines the binding affinity of the cytoplasmic tail to phosphatidylinositol 4,5-bisphosphate (PIP₂), the capacity of the tail to multimerize, and its ability to activate protein kinase C (PKC) . We sought to identify the kinase responsible for this phosphorylation and to determine its downstream effects on PKC activity and on endothelial cell function. Among several PKC isoenzymes tested, only PKC and - were able to specifically phosphorylate Ser¹⁸³ in vitro. However, studies in cultured endothelial cells showed that the phosphorylation level of syndecan-4 was significantly reduced in endothelial cells expressing a dominant negative (DN) PKC but not a DN PKC mutant. Syndecan-4/PIP₂-dependent PKC activity was significantly increased in PKC DN cells, while PKC overexpression was accompanied by decreased PKC activity. PKC-overexpressing cells exhibited a significantly lower proliferation rate and an impaired tube formation in response to FGF2, which were mirrored by similar observations in PKC DN endothelial cells. These findings suggest that PKC is the kinase responsible for syndecan-4 phosphorylation, which, in turn, attenuates the cellular response to FGF2 by reducing PKC activity. The reduced PKC activity then leads to impaired endothelial cell function. We conclude that PKC regulates PKC activity in a syndecan-4-dependent manner.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The protein kinase C (PKC)¹ family of enzymes is one of the most extensively studied group of proteins involved in intracellular signal transduction. However, to date little information is available regarding specific regulation of function and activity of individual PKC isoforms (1-4). Recently syndecan-4 has been shown to be able to activate PKC in the presence of phosphatidylinositol 4,5-bisphosphate (PIP₂) and in the absence of Ca²⁺ (5-7). Syndecan-4 is a member of the syndecan gene family, a group of heparan sulfate-carrying core proteins present in the plasma cell membrane (8). While sharing the ability of other syndecans to interact with heparin-binding proteins including fibroblast growth factors (FGFs), vascular endothelial growth factors, and numerous other partners, syndecan-4 has been specifically implicated in FGF2 signaling (9) and in regulation of cell cytoskeleton, focal adhesions, and migration (10, 11).

FGF2-dependent activation of syndecan-4 signaling requires oligomerization of its cytoplasmic tails (12) that in turn depends on the phosphorylation state of Ser¹⁸³, which regulates PIP₂ binding to the syndecan-4 tail (6). In previous studies we have demonstrated that phosphorylation of Ser¹⁸³ is carried out by a novel PKC (13). This chain of events, therefore, raises the possibility that one PKC isoform controls the activity of another isoform via the regulation of syndecan-4 phosphorylation. The present study was designed to explore that possibility. We found that PKC is the PKC isoform responsible for syndecan-4 phosphorylation and that alterations in PKC activity result in biologically meaningful alterations in PKC function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- PIP₂, phosphatidylserine, and diolein were purchased from Sigma. Recombinant PKCs were synthesized and prepared as described previously (14). PKC1 optimal substrate peptide (FKLKRKGSFKKFA) was purchased from Genemed Synthesis. 28-amino acid-long syndecan-4 cytoplasmic tail peptides, RMKKKDEGSYDLGKKPIYKKAPTNEFYA (wild type (WT)) and RMKKKDEGAYDLGKKPIYKKAPTNEFYA (mutant (SA)), were synthesized by Genemed Synthesis. Syndecan-4 ectoplasmic and cytoplasmic antisera were a gift from Dr. N. W. Shworak (Dartmouth Medical School). c-Myc antibody was purchased from Santa Cruz Biotechnology. PKC, PKC, PKC, PKC, and PKC antibodies were purchased from Santa Cruz Biotechnology and Transduction Laboratories.

Construction of Rat Fat Pad Endothelial Cell (RFPEC)-derived Cell Lines-- The dominant negative (DN) PKC construct (15, 16) was a generous gift from Dr. Dan Rosson (Lankenau Medical Research Center), and the dominant negative PKC construct was a gift from Dr. I. Bernard Weinstein (Colombia University). These expression plasmids were generated by replacing the conserved lysine in the ATP binding domain with arginine. PKC cDNA containing a c-Myc tag sequence was subcloned into pRc/CMV vector (Invitrogen) between HindIII and XbaI sites. Dominant negative PKC constructs were created by in vitro mutagenesis replacing the conserved lysine in the ATP binding domain in position 376 with tryptophan. These constructs were stably transfected into RFPECs using LipofectAMINE Plus and the protocol provided by the manufacturer (Invitrogen). Following neomycin (Geneticin, Invitrogen) selection, a number of clones were isolated and expanded. Expression of constructs was verified by Western blotting. At least two different clones were used for each construct-related experiment.

Western Blot Analysis-- Cells were lysed with RIPA buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 50 mM NaF, 20 mM sodium pyrophosphate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄), separated on a 10% SDS-polyacrylamide gel, and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore). The blots were probed with appropriate antibodies as described previously (5).

PKC in Vitro Assays-- In vitro PKC assays were carried out as described previously (5, 14). The reaction mixture (30 µl) contained 50 µM ATP and 5 µCi of [-³²P]ATP (PerkinElmer Life Sciences), 1 mM dithiothreitol, 5 mM MgCl₂, 25 mM Tris-HCl (pH 7.5), 20 µM phosphatidylserine, 10 µM diolein, and 0.2 mM CaCl₂ (for PKC assay), 0.5 mM EGTA (for PKC, -, -, -, and - assays), and PKC1 optimal peptide substrate (100 µM) (for PKC, -, -, -, and - assays), PKC peptide substrate (100 µM) (Calbiochem) (for PKC assay), or syndecan-4 cytoplasmic tail peptide (50 µM) in 25 mM Tris-HCl. In syndecan-4-associated PKC assays, the reaction mixture was supplemented with either 50 µM PIP₂ with 0.5 mM EGTA or 20 µM phosphatidylserine and 10 µM diolein with 0.2 mM CaCl₂ as above. Reactions were started by addition of PKC or addition of reaction mixture to the immunoprecipitates and incubated at 30 °C for 10 min. The reaction was stopped by spotting onto P81 phosphocellulose paper or by boiling in Laemmli buffer.

Syndecan-4 Phosphorylation Stoichiometry-- Confluent RFPECs were incubated for 24 h in methionine-, sulfate-, and phosphate-free minimum Eagle's medium prepared from the MEM SELECT-AMINE kit (Invitrogen) with 1% bovine serum albumin (Invitrogen) and radiolabeled for 2 h with 2 mCi/ml [³⁵S]methionine (EasyTag Express, PerkinElmer Life Sciences) and 1 mCi/ml [³²P]orthophosphoric acid (PerkinElmer Life Sciences). Syndecan-4 was immunoprecipitated and gel-resolved, and the ratio between its ³²P and ³⁵S incorporation was measured by scintillation counting as described previously (13).

Proliferation Assay-- 2,000 cells were plated in 96-well tissue culture plates and incubated overnight in 10% FBS, M199 medium. After that cells were starved with 0.5% FBS for 24 h and then treated with 0, 5, or 25 ng/ml FGF2. For measurement of proliferation, 20 µl of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) was added to the wells and incubated for 2 h. Absorbance at 490 nm was measured using a 96-well plate reader both before FGF2 application and 72 h later.

Matrigel Assay-- Matrigel (Becton Dickinson) plates were prepared by adding 0.5 ml of thawed Matrigel to a 12-well tissue culture plate. The gel was allowed to solidify for 1 h at 37 °C. 100,000 cells were plated in each well with 25 ng/ml FGF-2 in 0.5% FBS. Cells were imaged after a 24-h incubation at 37 °C in a humidified chamber with 5% CO₂. Analysis of Matrigel results was carried out as described previously (9).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

To define the PKC isoform responsible for phosphorylation of Ser¹⁸³ in the syndecan-4 cytoplasmic domain, we assayed the ability of all PKC isoforms expressed in RFPECs to phosphorylate Ser¹⁸³ in vitro. To determine the PKC isozyme preferentially phosphorylating Ser¹⁸³ over other potentially phosphorylatable residues in the syndecan-4 tail, we synthesized a peptide corresponding to the syndecan-4 cytoplasmic domain with Ser¹⁸³ replaced by Ala (SA peptide). While all PKC isoforms phosphorylated both the wild type and mutant peptides with similar efficiencies, only PKC and PKC preferentially phosphorylated the Ser¹⁸³ site (i.e. preferential phosphorylation of the wild type peptide compared with the SA peptide, Fig. 1A). Since our previously published PKC inhibitor studies (13) strongly argue against PKC as a biologically relevant PKC isoform phosphorylating syndecan-4, we compared the extent of syndecan-4 cytoplasmic domain phosphorylation in growth-arrested wild type RFPECs and in an RFPEC-derived cell line stably expressing dominant negative PKC, -, or - constructs (Fig. 1B). While PKC dominant negative expression resulted in a 2.5-fold reduction in the stoichiometry of syndecan-4 cytoplasmic tail phosphorylation, the expression of another non-calcium-dependent PKC (PKC) or calcium-dependent PKC had no effect on syndecan-4 phosphorylation stoichiometry. At the same time, overexpression of PKC increased the extent of Ser¹⁸³ site phosphorylation (Fig. 1B). Taken together with previously published observations (13), these results identify PKC as the PKC isoenzyme responsible for syndecan-4 phosphorylation.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Syndecan-4 cytoplasmic tail is phosphorylated by PKC. A, phosphorylation of syndecan-4 cytoplasmic tail peptides by PKC isoenzymes. Phosphorylation levels of RMKKKDEGSYDLGKKPIYKKAPTNEFYA (WT) and RMKKKDEGAYDLGKKPIYKKAPTNEFYA (SA) peptides were measured in vitro with six different recombinant PKC isoenzymes (0.002-0.005 units/sample). The graph shows the ratio between the phosphorylation levels of WT and SA peptides (mean ± S.D., n = 3; * indicates here and elsewhere p < 0.05). B, in vivo phosphorylation stoichiometries of the syndecan-4 cytoplasmic tail Ser183 site in wild type, PKC, PKC dominant negative (PKC DN) and PKC dominant negative (PKC DN) RFPEC-derived cell lines (mol of phosphate/mol of protein, mean ± S.D., n = 4). * indicates p < 0.05 versus wild type RFPECs.

Since syndecan-4 phosphorylation affects its ability to activate PKC in a PIP₂-dependent manner, we reasoned that PKC may inhibit PKC activity by phosphorylating the cytoplasmic tail of syndecan-4. To examine the role of PKC in syndecan-4-dependent regulation of PKC activity, we studied RFPEC-derived cell lines expressing PKC and PKC dominant negative constructs as well as a wild type PKC construct. Since expression of dominant negative isoform-specific PKC constructs can potentially influence activities of PKCs other then the intended target (17), we assayed the activity of all PKC isoforms present in RFPECs expressing PKC or PKC dominant negative constructs. In both cases the dominant negative construct expression resulted in a significant inhibition of the intended target (Fig. 2A) while not affecting activities of other present PKCs (Fig. 2B). At the same time, overexpression of PKC resulted in a significant increase in PKC activity in cells (Fig. 2C).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Expression and activities of PKC constructs in RFPECs. A, in vitro PKC and PKC activity assays in RFPECs expressing PKC and PKC dominant negative constructs. Total cell lysates from vector-transfected, PKC DN, and PKC DN RFPECs were immunoprecipitated with, respectively, anti-PKC and -PKC antibodies, and in vitro PKC activity assays were performed as described under "Experimental Procedures." PKC isozyme activities were measured in triplicate in each experiment. Graphs summarize the results of three independent experiments (mean ± S.D.). * indicates p < 0.05 versus vector-transfected cells. Insets, PKC isozymes levels in total cell lysates (top) and immunoprecipitates (bottom). Immunoblots were done with the same isozyme antibodies as used for immunoprecipitation (IP). B, in vitro PKC, -, and - activity assays in PKC and PKC DN RFPECs. Total cell lysate from vector-, PKC DN-, and PKC DN-transfected RFPECs were immunoprecipitated with PKC, -, and - antibodies, and in vitro PKC assays were performed. PKC isozyme activities were measured in triplicate in each experiment. Graphs summarize the results of three independent experiments (mean ± S.D.). * indicates p < 0.05 versus control vector-transfected cells. Insets, PKC isozymes levels in total cell lysates (top) and immunoprecipitates (bottom). Immunoblots were done with the same isozyme antibodies as used for immunoprecipitation. C, in vitro PKC assay in PKC-overexpressing (o/e) RFPECs. Total cell lysate from vector-transfected and PKC-overexpressing RFPECs were immunoprecipitated with anti-c-Myc antibody, and an in vitro PKC assay was performed as described under "Experimental Procedures" (mean ± S.D., n = 3). * indicates p < 0.05. Inset, expression of the PKC construct detected by the anti-c-Myc tag antibody. AU, absorbance unit.

Since the extent of syndecan-4 phosphorylation can affect PKC activity, we measured Ca²⁺- and PIP₂-activated PKC activity in wild type RFPECs and in clones expressing wild type or dominant negative PKC. While the expression of both constructs had no effect on Ca²⁺-activated activity, overexpression of PKC significantly decreased syndecan-4/PIP₂-activated PKC activity, while expression of PKC dominant negative significantly increased syndecan-4/PIP₂-activated PKC activity (Fig. 3).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Syndecan-4-associated PKC activities. Wild type, PKC DN, and PKC-overexpressing (o/e) RFPECs were starved in 0.5% FBS for 24 h and then incubated for another 20 min with 25 ng/ml FGF2. Total cell lysates were immunoprecipitated with antiserum to the ectoplasmic domain of syndecan-4 and then divided into two equal fractions. In vitro PKC assays were performed in two conditions: in the presence of PIP2 or Ca2+, phosphatidylserine (PS), and diolein (DAG). Data are mean ± S.D., n = 4 for all experiments. * indicates p < 0.05. Inset, syndecan-4-associated PKC levels in wild type, PKC DN, and PKC-overexpressing RFPECs. Total cell lysates were immunoprecipitated using anti-syndecan-4 ectoplasmic antibody, and PKC was detected with a specific antibody.

To study whether these changes in PIP₂-activated PKC activity translate into functionally relevant changes in cell behavior, we measured the ability of FGF2 to induce proliferation of wild type RFPECs or RFPECs expressing either PKC and PKC dominant negative constructs or unmodified PKC. In accord with previously published results (18), expression of the PKC dominant negative construct significantly inhibited cell growth. PKC-overexpressing cells also had a significantly lower proliferation rate compared with vector-transfected RFPECs (Fig. 4A). In fact, the proliferation rate of PKC overexpressors was close to that of cells expressing the PKC dominant negative construct. At the same time, cells expressing the PKC dominant negative construct demonstrated enhanced proliferation compared with vector-transfected cells. One interesting finding was a high rate of growth of cells expressing the PKC dominant negative construct even in the absence of FGF2 presumably because FGF2-activated syndecan-4 phosphatase, in the absence of syndecan-4 phosphorylation, was no longer needed to activate PKC (Fig. 4A). Similar results were obtained in an in vitro Matrigel angiogenesis assay with PKC overexpressors and cells expressing the PKC dominant negative construct demonstrating reduced vascular structure formation compared with vector-transfected RFPECs (Fig. 4B).

View larger version (73K): [in this window] [in a new window]   Fig. 4.   Effects of the overexpression of WT PKC, PKC DN, and PKC DN constructs on endothelial cell function. A, proliferation assays with RFPECs. Vector-transfected, PKC-overexpressing (o/e), PKC DN, and PKC DN RFPECs were starved in 0.5% FBS for 24 h and then treated with either 0, 5, or 25 ng/ml FGF2 for 72 h. The results are presented as -fold increase in cell number after the 72-h incubation relative to the initial cell number (mean ± S.D., n = 4; each cell line was assayed in duplicate). B, Matrigel assays. Vector-transfected, PKC DN, PKC DN, and PKC-overexpressing RFPECs were plated on Matrigel with 25 ng/ml FGF2 in 0.5% FBS. Cells were imaged after a 24-h incubation. Each cell line was assayed in duplicate.

To further link the effect of PKC on PKC activity to changes in syndecan-4 Ser¹⁸³ site phosphorylation, we determined the stoichiometry of this site in RFPEC-derived cell lines expressing the PKC construct. As expected, PKC overexpression increased base-line syndecan-4 phosphorylation (0.28 ± 0.05 versus 0.19 ± 0.03 mol of phosphate/mol of protein, p < 0.05; PKC overexpressor versus wild type syndecan-4). At the same time, FGF2 treatment had no appreciable effect on syndecan-4 phosphorylation in PKC-overexpressing cells (without FGF2, 0.28 ± 0.05, and with FGF2, 0.27 ± 0.06 mol of phosphate/mol of protein).

The results of this study show that PKC is the kinase responsible for syndecan-4 cytoplasmic domain phosphorylation and that PKC regulates PKC activity via this mechanism. Several observations support these conclusions. The conclusion that PKC is the PKC isoenzyme responsible for Ser¹⁸³ phosphorylation in the syndecan-4 cytoplasmic domain is supported by its ability to preferential phosphorylate the Ser¹⁸³ site in vitro.

While PKC also preferentially phosphorylated this site in vitro, only PKC had this activity in vivo as demonstrated by a decrease in the extent of Ser¹⁸³ phosphorylation in vivo in cells expressing a PKC DN construct and an increase in cells overexpressing PKC. At the same time, expression of the PKC dominant negative construct had no effect on syndecan-4 phosphorylation in vivo. It is not clear why the PKC effect on syndecan-4 phosphorylation is different in in vitro versus in vivo settings. PKC does not directly interact with syndecan-4 but rather binds to the syndecan-4-PIP₂ complex (6). It is quite possible that when such a complex is formed, as would be the case in vivo, the ¹⁸³Ser site is no longer accessible to PKC.

The modulation of the extent of syndecan-4 phosphorylation, achieved by expression of either PKC or PKC dominant negative constructs, affected its ability to activate PKC in the PIP₂-dependent manner. Interestingly the Ca²⁺-dependent PKC activity in cells expressing the wild type PKC or the PKC dominant negative construct was not affected. These alterations in PKC or its dominant negative construct expression (and corresponding changes in syndecan-4 Ser¹⁸³ phosphorylation) resulted in significant changes in cellular function as demonstrated by the proliferation and the in vitro Matrigel angiogenesis assays.

The changes in endothelial cell function induced by PKC overexpression in these experiments, inhibition of endothelial cell growth and angiogenesis, are consistent with prior publications including growth inhibition in smooth muscle cells (19), fibroblasts (20), and capillary endothelial cells (21, 22). Furthermore, the similarity of the functional effects between PKC overexpression and the expression of a PKC dominant negative construct, accompanied by increased PKC activity, is in agreement with the previously reported positive effects of PKC and inhibitory effects of PKC on endothelial cell migration (21). It is also interesting to note that vascular endothelial growth factor-induced increase in endothelial cell migration and proliferation is accompanied by a decrease in PKC activity (23).

The indirect modulation of the activity of one PKC isoform by another by means of regulation of the syndecan-4 phosphorylation state represents a novel mechanism of modulation of PKC activity. It is also interesting to note that changes in PIP₂- but not Ca²⁺-dependent PKC activation correlated with changes in cell function, suggesting that syndecan-4/PIP₂-dependent regulation of PKC activity reflects its physiological function.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants HL62289 and HL63609 (to M. S.) and HL51043 (to J. A. W.) and American Heart Association Scientist Development Grant 9730282N (to A. H.).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: Section of Cardiology, Dartmouth-Hitchcock Medical Center, One Medical Center Dr., Lebanon, NH 03756. Tel.: 603-650-3540; Fax: 603-650-6164; E-mail: michael.simons@dartmouth.edu.

Published, JBC Papers in Press, March 26, 2002, DOI 10.1074/jbc.M202501200

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; DN, dominant negative; PIP₂, phosphatidylinositol 4,5-bisphosphate; FGF, fibroblast growth factor; WT, wild type; SA, mutant in which Ser¹⁸³ replaced by Ala; RFPEC, rat fat pad endothelial cell; FBS, fetal bovine serum.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/23/20367    most recent M202501200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Murakami, M. Articles by Simons, M. Search for Related Content PubMed PubMed Citation Articles by Murakami, M. Articles by Simons, M. Social Bookmarking                      What's this?