FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2024422510> ?p ?o ?g. }

• W2024422510 endingPage "10629" @default.
• W2024422510 startingPage "10624" @default.
• W2024422510 abstract "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. 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 (1Schwartz M.A. Trends Cell Biol. 1992; 2: 304-308Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 2Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1466) Google Scholar, 3Parsons J.T. Curr. Opin. Cell Biol. 1996; 8: 146-152Crossref PubMed Scopus (277) Google Scholar, 4Yamada K.M. Miyamoto S. Curr. Opin. Cell Biol. 1995; 7: 681-688Crossref PubMed Scopus (586) Google Scholar). Phosphatidylinositol 4,5-bisphosphate (PIP2) 1The 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. 1The 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. 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 (5Machesky L.M. Pollard T.D. Trends Cell Biol. 1993; 3: 381-385Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 6Schafer D.A. Cooper J.A. Annu. Rev. Cell Dev. Biol. 1995; 11: 497-518Crossref PubMed Scopus (172) Google Scholar). PIP2 may also interact with α-actinin and vinculin (7Fukami K. Endo T. Imamura M. Takenawa T. J. Biol. Chem. 1994; 269: 1518-1522Abstract Full Text PDF PubMed Google Scholar) and regulate their association with the cytoskeleton (8Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (454) Google Scholar). The level of PIP2 decreases upon detachment of cells from the substratum and increases upon reattachment to fibronectin (1Schwartz M.A. Trends Cell Biol. 1992; 2: 304-308Abstract Full Text PDF PubMed Scopus (189) Google Scholar). 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 (1Schwartz M.A. Trends Cell Biol. 1992; 2: 304-308Abstract Full Text PDF PubMed Scopus (189) Google Scholar), 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 (9Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3809) Google Scholar, 10Zhang J. King W.G. Dillon S. Hall A. Feig L. Rittenhouse S.E. J. Biol. Chem. 1993; 268: 22251-22254Abstract Full Text PDF PubMed Google Scholar, 11Chong L.D. Kaplan A.T. Bokoch G.M. Schwartz M.A. Cell. 1994; 79: 507-513Abstract Full Text PDF PubMed Scopus (592) Google Scholar, 12Ren X. Bokoch G.M. Traynor-Kaplan A. Jenkins G.H. Anderson R.A. Schwartz M.A. Mol. Biol. Cell. 1996; 7: 435-442Crossref PubMed Scopus (199) Google Scholar, 13Tolias K.F. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 17656-17659Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). PIP2 may enter several different pathways in signal transduction. It can be hydrolyzed by phospholipase Cγ 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 (14Wennstrom S. Hawkins P.T. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L.R. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar) and vesicle trafficking (15Shepherd P.S. Reaves B.J. Davidson H.W. Trends Cell Biol. 1996; 6: 92-97Abstract Full Text PDF PubMed Scopus (119) Google Scholar). PIP2 can also be dephosphorylated via the 5-phosphatase to phosphatidylinositol 4-phosphate (16Divecha N. Irvine R.F. Cell. 1995; 80: 269-278Abstract Full Text PDF PubMed Scopus (589) Google Scholar). PIP2 may also directly activate several proteins including PKC. PIP2 is a potent activator of conventional PKC isotypes (α, βI, βII, and γ) in the presence of phosphatidylserine (PS) and calcium (17Chauhan V.S.P. FEBS Lett. 1990; 272: 99-102Crossref PubMed Scopus (18) Google Scholar, 18Chauhan A. Brockerhoff H. Winsniewski H.M. Chauhan V.S.P. Arch. Biochem. Biophys. 1991; 287: 283-287Crossref PubMed Scopus (24) Google Scholar, 19Lee M. Bell R.M. Biochemistry. 1991; 30: 1041-1049Crossref PubMed Scopus (71) Google Scholar). Indeed, PIP2 is more potent than diacylglycerol in stimulating PKCin vitro (20Pap E.H.W. Bastiaens P.I.H. Borst J.W. van den Berg P.A.W. van Hoek A. Snoek G.T. Wirtz K.W.A. Visser A.J.W.G. Biochemistry. 1993; 32: 13310-13317Crossref PubMed Scopus (40) Google Scholar), and it stimulates the translocation of conventional PKC from the soluble to the particulate fraction (18Chauhan A. Brockerhoff H. Winsniewski H.M. Chauhan V.S.P. Arch. Biochem. Biophys. 1991; 287: 283-287Crossref PubMed Scopus (24) Google Scholar). Thus, PIP2 may itself be a primary activator of PKCin vivo, both activating it and inducing its association with the plasma membrane (19Lee M. Bell R.M. Biochemistry. 1991; 30: 1041-1049Crossref PubMed Scopus (71) Google Scholar, 21Bell R.M. Burns D.J. J. Biol. Chem. 1991; 266: 4661-4664Abstract Full Text PDF PubMed Google Scholar). PKC activity is needed for matrix-induced cell spreading (22Vuori K. Ruoslahti E. J. Biol. Chem. 1993; 268: 21459-21462Abstract Full Text PDF PubMed Google Scholar) and for the later stage of focal adhesion assembly (23Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar). Cell surface heparan sulfate proteoglycans have critical role(s) in PKC signaling in focal adhesion and actin stress fiber formation (23Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar, 24Woods A. Couchman J.R. Johansson S. Höök M. EMBO J. 1986; 5: 665-670Crossref PubMed Scopus (322) Google Scholar, 25Woods A. Couchman J.R. Adv. Exp. Med. Biol. 1992; 313: 87-96Crossref PubMed Google Scholar, 26Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar). Cell attachment and spreading can be promoted through integrin interactions with the cell binding domain of fibronectin (23Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar). 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 (23Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar, 24Woods A. Couchman J.R. Johansson S. Höök M. EMBO J. 1986; 5: 665-670Crossref PubMed Scopus (322) Google Scholar, 25Woods A. Couchman J.R. Adv. Exp. Med. Biol. 1992; 313: 87-96Crossref PubMed Google Scholar, 26Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar). 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 (23Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar). 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 (27Woods A. Couchman J.R. Mol. Biol. Cell. 1994; 5: 183-192Crossref PubMed Scopus (277) Google Scholar). 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 PKCα activity in vitro, and PKC interacts with its core protein in vivo and in vitro, and with synthetic peptides of the LGKKPIYKK sequence (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). The interactions between PIP2 and several PIP2-binding proteins may be through their pleckstrin homology domains (20Pap E.H.W. Bastiaens P.I.H. Borst J.W. van den Berg P.A.W. van Hoek A. Snoek G.T. Wirtz K.W.A. Visser A.J.W.G. Biochemistry. 1993; 32: 13310-13317Crossref PubMed Scopus (40) Google Scholar, 29Janmey P.A. Chem. Biol. (Lond.). 1995; 2: 61-65Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 30Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (672) Google Scholar, 31Harlan J.E. Yoon H.S. Hajduk P.J. Fesik S.W. Biochemistry. 1995; 34: 9859-9864Crossref PubMed Scopus (105) Google Scholar, 32Sohn R.H. Chen J. Koblan K.S. Bray P.F. Goldschmidt-Clermont P.J. J. Biol. Chem. 1995; 270: 21114-21120Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), where two lysine residues, which end a β1 strand at the turn, interact with the 4- and 5-phosphates of the inositol head group of PIP2 (31Harlan J.E. Yoon H.S. Hajduk P.J. Fesik S.W. Biochemistry. 1995; 34: 9859-9864Crossref PubMed Scopus (105) Google Scholar). 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). 2J. R. Couchman, A. Woods, E.-S. Oh, G. Prestwich, and A. W. Theibert, unpublished observations. 2J. R. Couchman, A. Woods, E.-S. Oh, G. Prestwich, and A. W. Theibert, unpublished observations. 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. 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). PKCαβγ purified from rabbit brain and recombinant PKCα were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). An alternate source of recombinant PKCα was Life Technologies, Inc., and similar results were obtained for both. [γ-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).Table IThe amino acid sequences of peptides derived from the cytoplasmic domain of syndecan-4, -2, and -1PeptideSequence4LRMKKKDEGSYDLGKKPIYKKAPTNEFYA4VLGKKPIYKK4PALGKKAIYKKScrKYKGKIPLK2VCGERKPSSAAYQK1VCANGGAYQ Open table in a new tab The standard reaction mixture (total 20 μl) contained 50 mm HEPES (pH 7.3), 3 mmmagnesium acetate, PKCαβγ (3 ng) or PKCα (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 [γ-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 (33Hunter T. Ling N. Cooper J.A. Nature. 1984; 311: 480-483Crossref PubMed Scopus (421) Google Scholar), 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). Reaction mixtures prepared as described above with 50 μm PIP2or 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. We first investigated whether phosphoinositides could elevate the activity of a mixture of PKCαβγ in vitro. In the absence of PS and DL, phosphoinositides increased the activity of PKCαβγ to phosphorylate histone III-S (Fig. 1 A) or myelin basic protein (data not shown). PIP2 addition resulted in the highest level of PKCαβγ 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 PKCαβγ by PIP2 was approximately 60% of the maximal activity by conventional stimulation (refer to Fig. 6 A) 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 PKCαβγ to phosphorylate histone III-S (data not shown). The effect of IP6 on the phosphorylation of histone III-S by PKCαβγ was dose-dependent and maximal at 50 μm IP6 (Fig. 1 B). Stimulation of PKCαβγ by PIP2 was also dose-dependent, with half maximal stimulation at 30 μm and a maximum at 50 μm PIP2 (Fig. 1 C).Figure 6Effect of syndecan-4 peptides and calcium on PIP2-induced activation of PKCαβγ and recombinant PKCα. A, autoradiographs show the basal level of phosphorylation by PKCαβγ 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 PKCαβγ 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 PKCα 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 PKCα ± 25 μmcalcium (compare lane 1 with 2 and 3). PIP2 activation of PKCα requires the presence of 25 μm calcium (compare lanes 4 and 5) and is not increased in the presence of peptide 4PA (lanes 6and 7). Maximal phosphorylation is seen in the presence of PS/DL (PL) and 750 μm calcium (lane 8).D, the activation of PKCαβγ by PIP2 and 4L or 4V is calcium-independent, since 1 mm EGTA has little effect (compare lanes 3 and 6 with 2and 5), and high calcium (100 μm) does not increase activation (lanes 4 and 7).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since PKCαβγ are known as calcium-dependent enzymes and PIP2 interacts with PKC through its regulatory domain (18Chauhan A. Brockerhoff H. Winsniewski H.M. Chauhan V.S.P. Arch. Biochem. Biophys. 1991; 287: 283-287Crossref PubMed Scopus (24) Google Scholar, 34Chauhan A. Chauhan V.S.P. Deshmukh D.S. Brockerhoff H. Biochemistry. 1991; 28: 4952-4959Crossref Scopus (35) Google Scholar), we investigated whether calcium affected the increased activity of PKCαβγ 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. 35Schwartz M.A. J. Cell Biol. 1993; 120: 1003-1010Crossref PubMed Scopus (219) Google Scholar, 36Takahashi Y. Yoshida T. Takashima S. J. Gerontol. 1992; 47: 65-70Crossref Scopus (22) Google Scholar, 37Banyard M.R. Tellam R.M. Br. J. Cancer. 1985; 51: 761-766Crossref PubMed Scopus (18) Google Scholar) on the activation of PKCαβγ 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 PKCβ1, -ε, and -ζ in mixed micelles (38Palmer R.H. Dekker L.V. Woscholski R. Le Good J.A Gigg R. Parker P.J. J. Biol. Chem. 1995; 270: 22412-22416Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In contrast, PhosphorImager analysis of autoradiographs with recombinant PKCα 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). 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 PKCαβγ was due to either increased PKCαβγ activity or increased accessibility of the substrate to PKCαβγ. We therefore investigated whether PIP2 or IP6 could increase autophosphorylation of PKCαβγ in the absence of PS/DL (Fig. 3). PIP2 increased autophosphorylation of PKCαβγ over that seen in the absence of PIP2 (compare lanes 1 and 2). However, autophosphorylation of PKCαβγ in the presence of IP6 was not increased (compares lanes 3 and 4). Thus, IP6 may increase PKCαβγ phosphorylation of basic substrates by charge interactions that increase substrate accessibility. In contrast, PIP2 may directly affect PKCαβγ. To substantiate this hypothesis, PKCαβγ assays were performed in the presence of PIP2or IP6 using a peptide substrate from the EGF receptor (Fig. 4). PIP2 increased PKCαβγ 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).Figure 4The effects of phosphoinositides and syndecan-4 peptide on the ability of PKCαβγ 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our previous studies showed that syndecan-4 could directly activate recombinant PKCα and potentiate its activation by phospholipid through a defined region of the syndecan-4 cytoplasmic domain (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Further experiments determined whether syndecan-4 could also affect the PIP2-induced activation of PKCαβγ 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 PKCαβγ to phosphorylate the EGF receptor peptide in the presence of PIP2 from approximately 3-fold to 7-fold (Fig. 4, compare lanes 3 and 4with 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. 5 A). PIP2 alone increased the activity of PKCαβγ to phosphorylate histone III-S approximately 5-fold (Fig. 5 A, compare lanes 1and 3). Peptide 4V in the absence of inositol lipid or phospholipid showed a direct activation, as seen previously (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), but to a smaller (approximately 1.5-fold) extent (lane 2). The presence of both PIP2 and 4V potentiated the activation of PKCαβγ to approximately 11 times that of control levels (Fig. 5 A, compare lanes 3 and 4 with 1). However, 4V did not further increase phosphorylation of histone III-S by PKCαβγ in the presence of IP6 (Fig. 5 B, compare lanes 3 and 4), again suggesting that IP6 and PIP2 act through different mechanisms. To investigate whether the potentiation of PIP2-induced PKC activity by syndecan-4 could be significant in vivo, we compared the maximal activity of PKCαβγ or PKCα 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 (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), basal levels of phosphorylation were detected in the absence of phospholipid and calcium (Fig. 6 A, lane 1). PS/DL in the presence of 750 μm calcium normally induced maximal phosphorylation (lane 2), as seen in our assays (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) and by others (39Kikkawa U. Takai Y. Minakuchi R. Inohara S. Nishizuka Y. J. Biol. Chem. 1982; 257: 13341-13348Abstract Full Text PDF PubMed Google Scholar). In the presence of 50 μm PIP2and the syndecan-4 peptide 4L or 4V, there was even greater activity of PKCαβγ, even in the absence of PL and calcium (Fig. 6 A, compare lanes 3 and 4 with 2). Again PIP2 alone induced some activation of PKCαβγ in the absence of PS/DL, peptide, or calcium (lane 5). With recombinant PKCα (Fig. 6 B), similar results were seen, although low levels of calcium were required. Calcium alone did not activate PKCα (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 PKCα activity (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), also had no effect on PIP2-mediated activation (Fig. 6 C). Neither 25 μm calcium (lane 1) nor the 4PA peptide ± calcium (lanes 2 and 3) activated PKCα. PIP2 activation of PKCα 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 6and 7 with lanes 4 and 5). Lane 8 shows the maximal activity of PKCα in the presence of PS/DL and 750 μm calcium. Activation of recombinant PKCα by PIP2 appears to be dependent on at least 25 μm calcium (Fig. 6 C, lanes 4 and 5), whereas that of purified PKCαβγ is not (Fig. 6 A, lane 5). This was confirmed (Fig. 6 D) by the fact that potentiation of PIP2-induced PKCαβγ 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). Since all syndecans have high homology in 2 regions of the cytoplasmic domain with intervening variable sequences (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), 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 PKCαβγ in the presence of PIP2. Synthetic peptides 4L (lane 1) and 4V (lane 2) potentiated PIP2-induced activity of PKCαβγ, 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 PKCαβγ activation by PIP2. A variety of evidence implicates PKC activity (22Vuori K. Ruoslahti E. J. Biol. Chem. 1993; 268: 21459-21462Abstract Full Text PDF PubMed Google Scholar, 40Lewis J.M. Cheresh D.A. Schwartz M.A. J. Cell Biol. 1996; 134: 1323-1332Crossref PubMed Scopus (176) Google Scholar, 41Chun J.S. Jacobson B.S. Mol. Biol. Cell. 1993; 4: 271-281Crossref PubMed Scopus (67) Google Scholar, 42Somers C.E. Mosher D.F. J. Biol. Chem. 1993; 268: 22277-22280Abstract Full Text PDF PubMed Google Scholar, 43Ginsberg M.H. Du X. Plow E.F. Curr. Opin. Cell Biol. 1992; 4: 766-771Crossref PubMed Scopus (388) Google Scholar, 44Herbert J.M. Biochem. Pharmacol. 1993; 45: 527-537Crossref PubMed Scopus (37) Google Scholar) in cell-cell and cell-matrix interactions. In most cases, the isoform of PKC is unknown, though a role for PKCα emerges from its presence in focal adhesions of normal, but not transformed, cells (45Hyatt S.L. Klauck T. Jaken S. Mol. Carcinog. 1990; 3: 45-53Crossref PubMed Scopus (50) Google Scholar, 46Jaken S. Leach K. Klauck T. J. Cell Biol. 1989; 109: 697-704Crossref PubMed Scopus (253) Google Scholar). PKCαβγ 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 PKCα. 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 (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Similar to published reports, the present studies show that a phosphoinositide previously implicated in transmembrane signaling (16Divecha N. Irvine R.F. Cell. 1995; 80: 269-278Abstract Full Text PDF PubMed Scopus (589) Google Scholar, 47Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (348) Google Scholar), 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. (48Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar) have investigated the activation of PKC isotypes by phosphoinositides. In the presence of 10 μm phosphatidylserine and 40 μmphosphatidylethanolamine, most phosphoinositides, including PIP2, did not significantly activate PKCα. They also failed to detect any significant activation of PKCα by 10 μm PIP2 in the absence of phospholipid (48Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar). Our experiments show that PKCαβγ requires 50 μmPIP2 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 (49Machesky L. Goldschmidt-Clermont P.J. Pollard T. Cell Regul. 1990; 1: 937-950Crossref PubMed Scopus (92) Google Scholar), 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 PKCαβγ in the presence of 50 μm PIP2and absence of PS/DL, although activation of recombinant PKCα is dependent on low levels of calcium (25 μm). This may be due to differences in preparation of PKCαβγ and recombinant PKCα, leading to varying degrees of phosphorylation (50Bornancin F. Parker P.J. J. Biol. Chem. 1997; 272: 3544-3549Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). 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 Cγ after ligand binding to receptors (8Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (454) Google Scholar). 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 (1Schwartz M.A. Trends Cell Biol. 1992; 2: 304-308Abstract Full Text PDF PubMed Scopus (189) Google Scholar), others only show this response during adhesion through a subset of integrins (35Schwartz M.A. J. Cell Biol. 1993; 120: 1003-1010Crossref PubMed Scopus (219) Google Scholar, 51Leavesley D.I. Schwartz M.A. Rosenfeld M. Cheresh D.A. J. Cell Biol. 1993; 121: 163-170Crossref PubMed Scopus (347) Google Scholar). The PKCαβγ activity in vitroinduced 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 PKCαβγ activity through a novel calcium-independent pathway and that PKCα 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 PKCα to the plasma membrane at the sites of focal adhesion formation (45Hyatt S.L. Klauck T. Jaken S. Mol. Carcinog. 1990; 3: 45-53Crossref PubMed Scopus (50) Google Scholar, 46Jaken S. Leach K. Klauck T. J. Cell Biol. 1989; 109: 697-704Crossref PubMed Scopus (253) Google Scholar). 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 (11Chong L.D. Kaplan A.T. Bokoch G.M. Schwartz M.A. Cell. 1994; 79: 507-513Abstract Full Text PDF PubMed Scopus (592) Google Scholar, 12Ren X. Bokoch G.M. Traynor-Kaplan A. Jenkins G.H. Anderson R.A. Schwartz M.A. Mol. Biol. Cell. 1996; 7: 435-442Crossref PubMed Scopus (199) Google Scholar). In addition, PIP2 and PKC activation are both required for focal adhesion and stress fiber formation (24Woods A. Couchman J.R. Johansson S. Höök M. EMBO J. 1986; 5: 665-670Crossref PubMed Scopus (322) Google Scholar, 52Baciu P.C. Goetinck P.F. Mol. Biol. Cell. 1995; 6: 1503-1513Crossref PubMed Scopus (120) Google Scholar). We have previously shown (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) that PKCα copatches when syndecan-4 is patched by the addition of ectodomain antibodies to spreading fibroblasts, and they can be coimmunoprecipitated. Moreover, PKCα, 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 PKCα activity (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). A synthetic peptide of the same sequence also interacts with PIP22, and this promotes oligomerization of the syndecan-4 cytoplasmic domain (53Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). 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 PKCα may be the most relevant activation of PKCα 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 Cγ-dependent calcium fluxes or diacylglycerol production. However, it is not yet known whether interactions of two of the three components, syndecan-4, PIP2, and PKCα, 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 PKCα (28Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), whereas PIP2 probably binds the regulatory domain of PKCα (19Lee M. Bell R.M. Biochemistry. 1991; 30: 1041-1049Crossref PubMed Scopus (71) Google Scholar, 24Woods A. Couchman J.R. Johansson S. Höök M. EMBO J. 1986; 5: 665-670Crossref PubMed Scopus (322) Google Scholar) even more strongly than diacylglycerol (20Pap E.H.W. Bastiaens P.I.H. Borst J.W. van den Berg P.A.W. van Hoek A. Snoek G.T. Wirtz K.W.A. Visser A.J.W.G. Biochemistry. 1993; 32: 13310-13317Crossref PubMed Scopus (40) Google Scholar). Both PKCα 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 (53Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). 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 (27Woods A. Couchman J.R. Mol. Biol. Cell. 1994; 5: 183-192Crossref PubMed Scopus (277) Google Scholar, 52Baciu P.C. Goetinck P.F. Mol. Biol. Cell. 1995; 6: 1503-1513Crossref PubMed Scopus (120) Google Scholar). 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. 54Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (962) Google Scholar). Our binding data indicates that IP6 can also interact with syndecan-4. However, in contrast to PIP2, IP6could activate PKCαβγ only when phosphorylating histone III-S as a substrate, not when using the EGF receptor peptide as a substrate. Experiments examining the autophosphorylation of PKCαβγ 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 PKCαβγ by the oligomeric peptide (53Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar)." @default.
• W2024422510 created "2016-06-24" @default.
• W2024422510 creator A5035485431 @default.
• W2024422510 creator A5046605351 @default.
• W2024422510 creator A5051382096 @default.
• W2024422510 creator A5064957549 @default.
• W2024422510 creator A5080806595 @default.
• W2024422510 date "1998-04-01" @default.
• W2024422510 modified "2023-10-01" @default.
• W2024422510 title "Syndecan-4 Proteoglycan Cytoplasmic Domain and Phosphatidylinositol 4,5-Bisphosphate Coordinately Regulate Protein Kinase C Activity" @default.
• W2024422510 cites W1487858339 @default.
• W2024422510 cites W1500148089 @default.
• W2024422510 cites W1539113719 @default.
• W2024422510 cites W1569108584 @default.
• W2024422510 cites W1587369907 @default.
• W2024422510 cites W1600280692 @default.
• W2024422510 cites W1643628872 @default.
• W2024422510 cites W1895065514 @default.
• W2024422510 cites W189749484 @default.
• W2024422510 cites W1931472297 @default.
• W2024422510 cites W1966067563 @default.
• W2024422510 cites W1966260369 @default.
• W2024422510 cites W1967941200 @default.
• W2024422510 cites W1968013329 @default.
• W2024422510 cites W1968253797 @default.
• W2024422510 cites W1973064894 @default.
• W2024422510 cites W1975166712 @default.
• W2024422510 cites W1976121619 @default.
• W2024422510 cites W1977294762 @default.
• W2024422510 cites W1981744194 @default.
• W2024422510 cites W1984760832 @default.
• W2024422510 cites W1987118848 @default.
• W2024422510 cites W1991708828 @default.
• W2024422510 cites W2006878196 @default.
• W2024422510 cites W2007660746 @default.
• W2024422510 cites W2010265636 @default.
• W2024422510 cites W2016477781 @default.
• W2024422510 cites W2021469314 @default.
• W2024422510 cites W2023800215 @default.
• W2024422510 cites W2026987638 @default.
• W2024422510 cites W2046974658 @default.
• W2024422510 cites W2058240687 @default.
• W2024422510 cites W2060538012 @default.
• W2024422510 cites W2062980512 @default.
• W2024422510 cites W2063761685 @default.
• W2024422510 cites W2072871114 @default.
• W2024422510 cites W2073478575 @default.
• W2024422510 cites W2081450796 @default.
• W2024422510 cites W2086811440 @default.
• W2024422510 cites W2107937825 @default.
• W2024422510 cites W2113221012 @default.
• W2024422510 cites W2113266466 @default.
• W2024422510 cites W2121598303 @default.
• W2024422510 cites W2128511563 @default.
• W2024422510 cites W2135112491 @default.
• W2024422510 cites W2140969448 @default.
• W2024422510 cites W2142714350 @default.
• W2024422510 cites W2144796435 @default.
• W2024422510 cites W2168918180 @default.
• W2024422510 cites W2169819991 @default.
• W2024422510 cites W2170766885 @default.
• W2024422510 cites W2323768455 @default.
• W2024422510 cites W34966227 @default.
• W2024422510 cites W4247231961 @default.
• W2024422510 doi "https://doi.org/10.1074/jbc.273.17.10624" @default.
• W2024422510 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9553124" @default.
• W2024422510 hasPublicationYear "1998" @default.
• W2024422510 type Work @default.
• W2024422510 sameAs 2024422510 @default.
• W2024422510 citedByCount "177" @default.
• W2024422510 countsByYear W20244225102012 @default.
• W2024422510 countsByYear W20244225102013 @default.
• W2024422510 countsByYear W20244225102014 @default.
• W2024422510 countsByYear W20244225102015 @default.
• W2024422510 countsByYear W20244225102016 @default.
• W2024422510 countsByYear W20244225102017 @default.
• W2024422510 countsByYear W20244225102018 @default.
• W2024422510 countsByYear W20244225102019 @default.
• W2024422510 countsByYear W20244225102020 @default.
• W2024422510 countsByYear W20244225102021 @default.
• W2024422510 countsByYear W20244225102022 @default.
• W2024422510 crossrefType "journal-article" @default.
• W2024422510 hasAuthorship W2024422510A5035485431 @default.
• W2024422510 hasAuthorship W2024422510A5046605351 @default.
• W2024422510 hasAuthorship W2024422510A5051382096 @default.
• W2024422510 hasAuthorship W2024422510A5064957549 @default.
• W2024422510 hasAuthorship W2024422510A5080806595 @default.
• W2024422510 hasBestOaLocation W20244225101 @default.
• W2024422510 hasConcept C134306372 @default.
• W2024422510 hasConcept C1491633281 @default.
• W2024422510 hasConcept C184235292 @default.
• W2024422510 hasConcept C185592680 @default.
• W2024422510 hasConcept C189165786 @default.
• W2024422510 hasConcept C190062978 @default.
• W2024422510 hasConcept C2777853560 @default.
• W2024422510 hasConcept C2779335624 @default.
• W2024422510 hasConcept C2780610907 @default.
• W2024422510 hasConcept C33923547 @default.

• next