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• W1974604649 abstract "Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains a central region unlike that of any other vertebrate or invertebrate syndecan core protein with a cationic motif that binds inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)2) and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. Affinity was much reduced for 3-phosphorylated inositides while no binding of diacylglycerol was detected. Syndecan-2 cytoplasmic domain had negligible affinity for any lipid examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P2. Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down-regulator of syndecan-4 signaling. Similarly, phosphorylation of serine 183 in syndecan-4 cytoplasmic domain reduced PtdIns(4,5)P2 binding affinity by over 100-fold, although interaction could still be detected by nuclear magnetic resonance spectroscopy. Only protein kinase Cα was up-regulated in activity by the combination of syndecan-4 and PtdIns(4,5)P2, with all other isoforms tested showing minimal response. This is consistent with the codistribution of syndecan-4 with the α isoform of protein kinase C in focal adhesions. Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains a central region unlike that of any other vertebrate or invertebrate syndecan core protein with a cationic motif that binds inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)2) and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. Affinity was much reduced for 3-phosphorylated inositides while no binding of diacylglycerol was detected. Syndecan-2 cytoplasmic domain had negligible affinity for any lipid examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P2. Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down-regulator of syndecan-4 signaling. Similarly, phosphorylation of serine 183 in syndecan-4 cytoplasmic domain reduced PtdIns(4,5)P2 binding affinity by over 100-fold, although interaction could still be detected by nuclear magnetic resonance spectroscopy. Only protein kinase Cα was up-regulated in activity by the combination of syndecan-4 and PtdIns(4,5)P2, with all other isoforms tested showing minimal response. This is consistent with the codistribution of syndecan-4 with the α isoform of protein kinase C in focal adhesions. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 1The abbreviations used are: PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; InsP3, inositol 1,4,5-trisphosphate; InsP4, inositol 1,3,4,5-tetrakisphosphate; InsP6, inositol hexakisphosphate; InsS6, inositol hexasulfate; PKC, protein kinase C; BZDC, benzoyldihydrocinnamide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine1The abbreviations used are: PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; InsP3, inositol 1,4,5-trisphosphate; InsP4, inositol 1,3,4,5-tetrakisphosphate; InsP6, inositol hexakisphosphate; InsS6, inositol hexasulfate; PKC, protein kinase C; BZDC, benzoyldihydrocinnamide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinehas multiple roles in cell signaling and the regulation of cell adhesion, morphology, and trafficking (1Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (244) Google Scholar, 2Hurley J.H. Misra S. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 49-79Crossref PubMed Scopus (225) Google Scholar, 3Martin T.F.J. Curr. Opin. Cell Biol. 2001; 13: 493-499Crossref PubMed Scopus (328) Google Scholar, 4Cockcroft S. De Matteis M.A. J. Membrane Biol. 2001; 180: 187-194Crossref PubMed Scopus (69) Google Scholar). It can be cleaved by phospholipases to generate diacylglycerol and inositol trisphosphate (InsP3). These are second messengers that activate some serine/threonine kinases, including conventional and novel protein kinase C (PKC) isoforms (5Newton A.C. Parker P.J. Dekker L.V. Protein Kinase C. R. G. Landes Company, Austin, TX1997: 25-43Google Scholar, 6Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Crossref PubMed Scopus (4244) Google Scholar) and trigger calcium release from intracellular stores (6Berridge M.J. Irvine R.F. Nature. 1984; 312: 315-321Crossref PubMed Scopus (4244) Google Scholar), respectively. InsP3 can also be the target of kinases that sequentially convert it through InsP4 and InsP5 to InsP6 (inositol hexaphosphate) that has been proposed to have various regulatory functions in phosphatase inhibition, trafficking, calcium influx, and cell growth (7Larsson O. Barker C.J. Sjöholm Å. Carlqvist H.C. Michell R.H. Bertorello A. Nilsson T. Honkanen R.E. Mayr G.W. Zwiller J. Berggren P.-O. Science. 1997; 278: 471-474Crossref PubMed Scopus (122) Google Scholar, 8Llinas R. Sugimori M. Lang E.J. Morita M. Fukuda M. Niinobe M. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1994; 26: 12990-12993Crossref Scopus (88) Google Scholar, 9Irvine R.F. Schell M.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 327-338Crossref PubMed Scopus (534) Google Scholar). PtdIns(4,5)P2 can also be converted to PtdIns(3,4,5)P3 by PI 3-kinases that have also been implicated in regulation of protein trafficking, cell growth and survival, and cytoskeletal organization (10Leevers S.J. Vanhaesebroeck B. Waterfield M.D. Curr. Opin. Cell Biol. 1999; 11: 219-225Crossref PubMed Scopus (572) Google Scholar, 11Insall R.H. Weiner O.D. Dev. Cell. 2001; 1: 743-747Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In addition, PtdIns(4,5)P2 may have roles itself, such as binding and regulation of the actin-associated proteins vinculin, α-actinin, and gelsolin (2Hurley J.H. Misra S. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 49-79Crossref PubMed Scopus (225) Google Scholar). Binding of PtdIns(4,5)P2 to specific sites on these proteins influences their interactive properties with, for example, actin (12Fukami K. Endo T. Imamura M. Takenawa T. J. Biol. Chem. 1994; 269: 1518-1522Abstract Full Text PDF PubMed Google Scholar, 13Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (455) Google Scholar, 14Steimle P.A. Hoffert J.D. Adey N.B. Craig S.W. J. Biol. Chem. 1999; 274: 18414-18420Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Many proteins interact with this phospholipid through defined motifs including pleckstrin homology and epsin N-terminal homology domains (15Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (672) Google Scholar, 16Cullen P.J. Cozier G.E. Banting G. Mellor H. Curr. Biol. 2001; 11: R882-R893Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 17Itoh T. Koshiba S. Kigawa T. Kikuchi A. Yokoyama S. Takenawa T. Science. 2001; 291: 993-994Crossref PubMed Scopus (386) Google Scholar). One cell surface heparan sulfate proteoglycan, syndecan-4, also binds PtdIns(4,5)P2 through a motif in the central portion of its cytoplasmic domain, known as the V (variable) region. The V region of syndecan-4 is unlike that of any other family member and has the sequence LGKKPIYKKA (18Oh E.-S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 19Couchman J.R. Chen L. Woods A. Int. Rev. Cytol. 2001; 207: 113-150Crossref PubMed Scopus (122) Google Scholar). The two pairs of lysine residues appear to be critical for this activity, and the motif KKXXXKK is known to bind inositol polyphosphate based on previous studies, for example of synaptotagmin II (20Mehrotra B. Elliott J.T. Chen J. Olszewski J.D. Profit A.A. Chaudhary A. Fukuda M. Mikoshiba K. Prestwich G.D. J. Biol. Chem. 1997; 272: 4237-4244Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The functional consequences of inositol phospholipid binding at this site appears to be the stabilization of a dimer of syndecan-4 cytoplasmic domain in an unusual twisted clamp motif, determined by nuclear magnetic resonance (NMR) spectroscopy (21Lee D. Oh E.-S. Woods A. Couchman J.R. Lee W. J. Biol. Chem. 1998; 273: 13022-13029Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 22Shin J. Lee W. Lee D. Koo B.-K. Han I. Lim Y. Woods A. Couchman J.R. Oh E.-S. Biochemistry. 2001; 40: 8471-8478Crossref PubMed Scopus (43) Google Scholar). Dimers, or more probably, higher order oligomers (23Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), then can bind protein kinase Cα (PKCα), and cause it to be strongly activated (18Oh E.-S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 23Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 24Horowitz A. Murakami M. Gao Y. Simons M. Biochemistry. 1999; 38: 15871-15877Crossref PubMed Scopus (78) Google Scholar). This may explain why syndecan-4 and PKCα are both focal adhesion components in many cell types (25Couchman J.R. Woods A. J. Cell Sci. 1999; 112: 3415-3420Crossref PubMed Google Scholar, 26Baciu P.C. Goetinck P.F. Mol. Biol. Cell. 1995; 6: 1503-1513Crossref PubMed Scopus (120) Google Scholar). Our current hypothesis is that syndecan-4, when clustered, signals through the kinase at nascent focal adhesions and contributes to focal adhesion assembly, with possible involvement of the G protein RhoA (19Couchman J.R. Chen L. Woods A. Int. Rev. Cytol. 2001; 207: 113-150Crossref PubMed Scopus (122) Google Scholar,23Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 24Horowitz A. Murakami M. Gao Y. Simons M. Biochemistry. 1999; 38: 15871-15877Crossref PubMed Scopus (78) Google Scholar, 25Couchman J.R. Woods A. J. Cell Sci. 1999; 112: 3415-3420Crossref PubMed Google Scholar, 27Saoncella S. Echtenmeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (331) Google Scholar). One unresolved issue is the regulation of the syndecan-4, PtdIns(4,5)P2, and PKCα signaling complex. There are several possibilities. First, all syndecans can interact with PDZ domain proteins through a C-terminal FYA sequence (19Couchman J.R. Chen L. Woods A. Int. Rev. Cytol. 2001; 207: 113-150Crossref PubMed Scopus (122) Google Scholar, 28Zimmerman P. David G. FASEB J. 1999; 13: S91-S100Crossref PubMed Google Scholar). Such interactions could lead to complex disassembly or internalization or to stabilization. This has not been examined. Second, Horowitz et al. (24Horowitz A. Murakami M. Gao Y. Simons M. Biochemistry. 1999; 38: 15871-15877Crossref PubMed Scopus (78) Google Scholar, 29Horowitz A. Simons M. J. Biol. Chem. 1998; 273: 25548-25551Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 30Horowitz A. Simons M. J. Biol. Chem. 1998; 273: 10914-10918Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) showed that phosphorylation of the single serine residue at the boundary of the membrane proximal C1 region and V region of syndecan-4 cytoplasmic domain (Ser183) can lead to decreased signaling through PKCα. In part this may be due to decreased affinity of PtdIns(4,5)P2 for the phosphorylated syndecan-4 cytoplasmic domain (24Horowitz A. Murakami M. Gao Y. Simons M. Biochemistry. 1999; 38: 15871-15877Crossref PubMed Scopus (78) Google Scholar). A third, alternate, hypothesis is that PtdIns(4,5)P2 may be displaced by another compound, yielding a form of syndecan-4 cytoplasmic domain unable to bind or activate PKCα. One possibility is InsP6. The KKXXXKK motif within syndecan-4 V region provides a potential site for InsP6 binding (31Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar), although we showed previously that this inositol phosphate neither promotes syndecan-4 cytoplasmic domain oligomerization nor activates PKCα (18Oh E.-S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Here we have examined these hypotheses by analyzing the specificity of phospholipid and inositol phosphate binding to syndecan-4 cytoplasmic domain peptides, whether InsP6 can compete with PtdIns(4,5)P2 for binding to the cytoplasmic domain of syndecan-4 core protein and the role of Ser183phosphorylation. Both InsP6 and phosphorylation may down-regulate syndecan-4-mediated signaling, since both strongly affect PtdIns(4,5)P2 interactions. In addition, InsP6diminished microfilament bundle formation in fibroblasts, under experimental conditions where syndecan-4 and PKCα are involved. Syndecan-2 and −4 peptides corresponding to the entire cytoplasmic domains of human syndecan-4 (4L) and syndecan-2 (2L) were synthesized by SynPep (Dublin, CA), and their sequences confirmed by mass spectroscopy. These sequences were (C)RMKKKDEGSYDLGKKPIYKKAPTNEFYA and RMRKKDEGSYDLGERKPSSAAYQKAPTKEFYA, respectively. Two modified 4L peptides were also synthesized, one lacking the three C-terminal amino acids (FYA; denoted 4ΔE) and a second incorporating a phosphoserine at position 183 in place of serine (p-4L). Also used were peptides corresponding to the central, variable (V) region of syndecan-4 and −2 with the sequences (C)LGKKPIYKK and (C)LGERKPSSAAYQ, respectively. At least two different batches of each peptide were used. All inositol phospholipids and diacylglycerols were purchased from Biomol (Plymouth Meeting, PA). PtdIns(4,5)P2 was also purchased from Avanti Polar Lipids (Alabaster, AL), as were phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. InsP6, InsP4, InsP3, and inositol hexasulfate (InsS6), HEPES, CHAPS, and one batch of PtdIns(4,5)P2 were from Sigma-Aldrich. Lipids were dissolved in a chloroform/methanol solution at 2 mg/ml as described previously (29Horowitz A. Simons M. J. Biol. Chem. 1998; 273: 25548-25551Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Solubilized lipids were dried under N2 and sonicated for 2 min in ice-cold H2O at a final concentration of 1 mg/ml. Peptides were incubated on ice for 30 min with the indicated concentrations of lipid in 10 mm Tris-HCl (pH 7.5) in a reaction volume of 100 μl. The samples were spun in filter units (Ultrafree-MC, 30,000 NMWL, Millipore, Bedford, MA) at 2000 ×g for 70 s following the method described by Haareret al. (32Haarer B.K. Petzold A.A. Brown S.S. Mol. Cell. Biol. 1993; 10: 7864-7873Crossref Scopus (94) Google Scholar). The filtrates (40 μl of each in 4% SDS, 10% glycerol, 1.5% dithiothreitol, 0.004% bromphenol blue, 50 mm Tris-HCl, pH 6.8) were resolved by SDS-PAGE on 16.5% Tris-Tricine gels, stained with Coomassie Brilliant Blue G-250 (BioRad). The stained peptides were scanned on a BioRad GS670 imaging densitometer and quantitated. All experiments were performed at least in triplicate. Bound and free phospholipid were then calculated and averaged from replicates. The data were used to generate Scatchard plots, from which affinities were estimated. In addition, two forms of displacement experiments were performed. Peptides were first incubated with differing concentrations of inositol phosphate or sulfate for 30 min, then with 50 μm inositol phospholipid for an additional 30 min, or PtdIns(4,5)P2 was incubated first with the peptide, then the inositol phosphate or sulfate was added. The preparation and use of [3H]benzoyldihydrocinnamide (BZDC)-InsP4 and -InsP6 probes has been described previously (33Theibert A.B. Prestwich G.D. Jackson T.R. Hammonds-Odie L.P. Shears S. Signaling by Inositides: A Practical Approach. Oxford University Press, 1997: 117-150Google Scholar). Experiments were performed in 96-well plates. To 21 μl of buffer, consisting of 25 mm Tris-HCl, pH 7.4, 1 mmEDTA, and 1 mm dibasic potassium phosphate, was added 1 μg of peptide in 5 μl of buffer and 3 μl of water, with or without unlabeled InsP4 or InsP6, as appropriate, to a final concentration of 20 μm. After a 10-min incubation on ice, 1 μl of photoactivable probe (0.5 μCi, 20–70 nm) was added. The plate was incubated for 60 min on ice before exposure to UV irradiation. The reaction was stopped by the addition of 4× SDS-PAGE sample buffer. Samples were resolved by 20% SDS-PAGE at ∼30 mA, with prestained low molecular mass standards (Invitrogen) in one or two lanes. The gels were impregnated with Entensify (DuPont), dried and fluorographs exposed for 1 week. In other experiments, increasing concentrations of unlabeled InsP6were included in the assays, in order to ascertain theK i as an approximate measure of affinity of the inositol phosphate for the V region of syndecan-4. Gel filtration procedures were as previously (22Shin J. Lee W. Lee D. Koo B.-K. Han I. Lim Y. Woods A. Couchman J.R. Oh E.-S. Biochemistry. 2001; 40: 8471-8478Crossref PubMed Scopus (43) Google Scholar). Synthetic peptides or a mixture of synthetic peptide and phosphoinositide were loaded onto a Sephadex G-50 gel filtration column (0.7 × 50 cm) equilibrated with 50 mm HEPES (pH 7.3), 0.1% CHAPS, and 150 mmNaCl. Peptides were eluted with the same buffer at a flow rate of 3 ml/h at room temperature, and 1-ml fractions were assayed on a UV monitor at 280 nm. The column was calibrated with molecular standards containing thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.3 kDa). Primary rat embryo fibroblasts were seeded onto fibronectin-coated glass coverslips in 24-well plates as previously (34Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar) for 30 min. At this time InsP4, InsP6, or InsS6 were added to the serum-free medium at various concentrations for up to an additional 2.5 h. In some cases, after 1.5 h, adherent cells were returned to normal growth medium, as a control to ascertain that adhesion inhibition was reversible. For interference reflection microscopy analysis of focal adhesion formation (35Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar), cultures were fixed in 3% glutaraldehyde in phosphate-buffered saline for 15 min and washed and mounted in serum-free medium. Other cultures were fixed in 4% paraformaldehyde in phosphate-buffered saline, containing 0.1% Triton X-100. Texas Red-conjugated phalloidin (Molecular Probes, Eugene, OR) staining of microfilaments was performed as before (35Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar). The percentage of cells with focal adhesions or microfilament bundles was counted in three separate replicates, with at least 100 cells per coverslip counted in each case. In further studies, cells were seeded on the integrin-binding 110-kDa fragment of plasma fibronectin (19Couchman J.R. Chen L. Woods A. Int. Rev. Cytol. 2001; 207: 113-150Crossref PubMed Scopus (122) Google Scholar, 34Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar, 35Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar) for 1.5 h, then stimulated for 30 min at 37 °C with 10 ng/ml recombinant 31-kDa hepII domain of fibronectin (35Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar) or 25 μg/ml purified monoclonal antibody 150.9 against the N-terminal of syndecan-4 (36Longley R.L. Woods A. Fleetwood A. Cowling G.J. Gallagher J.T. Couchman J.R. J. Cell Sci. 1999; 112: 3421-3431Crossref PubMed Google Scholar) to promote focal adhesion formation (19Couchman J.R. Chen L. Woods A. Int. Rev. Cytol. 2001; 207: 113-150Crossref PubMed Scopus (122) Google Scholar, 27Saoncella S. Echtenmeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (331) Google Scholar). Some cultures were treated with InsP6 or InsS6 for the final 35 min. Cells were fixed and stained for microfilament bundles with Texas Red-conjugated phalloidin as before. Recombinant isoforms of PKC were incubated with, or without, the addition of PtdIns(4,5)P2and syndecan-4L cytoplasmic domain peptide, using identical methods as previously (18Oh E.-S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 23Oh E.-S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Phosphorylated histone IIIS was resolved by 20% SDS-PAGE and autoradiography. Quantitation was by laser scanning densitometry as before (18Oh E.-S. Woods A. Lim S.-T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Phosphoinositide titration experiments were performed on a Bruker DRX500 spectrometer in quadrature detection mode, equipped with a triple resonance probe head with triple-axis gradient coils. A series of one-dimensional NMR spectra were recorded for 4L, p-4L, and 2L peptides with different concentrations of PtdIns(4,5)P2. All data were collected at 25 °C, and the strong solvent resonance was suppressed by water-gated pulse sequence combined with pulsed-field gradient (PFG) pulses. All NMR data were processed using Bruker XWIN-NMR (Bruker Instruments) software on an SGI Indigo 2W. Lee and J. R. Couchman, unpublished data. work station. The proton chemical shifts were referenced to internal sodium 4,4-dimethyl-4-silapentane 1-sulfonate (DSS). Fig.1 shows the sequences of syndecans-2 and −4 with their constant (C1 and C2) and variable (V) regions denoted. Lipid micelles were allowed to bind to peptides corresponding either to the full-length cytoplasmic domain of syndecan-4 or syndecan-2, and the unbound peptide recovered and quantified after centrifugation through a molecular mass cut-off filter. Affinities of the lipids for the peptides are shown in Table I. There was a significant preference of syndecan-4 cytoplasmic domain for PtdIns(4,5)P2 and PtdIns(4)P over inositol phospholipids containing phosphate at the 3-position on the inositol ring. Phosphatidylserine had weak affinity for syndecan-4, while no binding to phosphatidylethanolamine or two forms of diacylglycerol was noted. Since diacylglycerol is a cleavage product of PtdIns(4,5)P2generated by phospholipase C, the data suggest that interactions of the inositol phosphate moiety of PtdIns(4,5)P2 with syndecan-4 are key. Very little binding of any lipid to syndecan-2 cytoplasmic domain was seen, confirming that only the central V region of syndecan-4 contains the lipid binding site(s), since the C1 and C2 regions of these two core proteins are highly homologous. This was confirmed with a synthetic peptide corresponding to the V region of syndecan-4 cytoplasmic domains, which had similar affinity to 4L for PtdIns(4,5)P2 (not shown).Table ILipid affinities for syndecan-4Lipid4Lp-4L4ΔE2LPtdIns4,5P255704.56100PtdIns4P4ND1065PtdIns3,4P2480NDNDPtdIns3,4,5P3NDNDNDDAG1-aDAG, 1-stearoyl-2-linoleoyl-sn-glycerol.NDNDDAG1-bDAG, 1-stearoyl-2-arachidonyl-sn-glycerol.NDNDPtd ethanolamineNDNDPtd serine3653700Lipid affinities (μm) for cytoplasmic domains of wild-type syndecan-4 (4L), phosphorylated syndecan-4 (p-4L), syndecan-4 with a truncation of the C-terminal last three amino acids (4ΔE), or wild-type syndecan-2 (2L). ND-not detectable.1-a DAG, 1-stearoyl-2-linoleoyl-sn-glycerol.1-b DAG, 1-stearoyl-2-arachidonyl-sn-glycerol. Open table in a new tab Lipid affinities (μm) for cytoplasmic domains of wild-type syndecan-4 (4L), phosphorylated syndecan-4 (p-4L), syndecan-4 with a truncation of the C-terminal last three amino acids (4ΔE), or wild-type syndecan-2 (2L). ND-not detectable. The almost identical affinities of PtdIns(4,5)P2 and PtdIns(4)P for the cytoplasmic domain of syndecan-4 (4L) raised the question of whether both could stabilize the peptide in dimeric conformation. Fig. 2 shows that both phospholipids markedly promoted 4L dimer formation, which is only limited in their absence. Syndecan-2 cytoplasmic domain (2L) showed no tendency to form dimers (Fig. 2), even in the presence of PtdIns (4Cockcroft S. De Matteis M.A. J. Membrane Biol. 2001; 180: 187-194Crossref PubMed Scopus (69) Google Scholar)P or PtdIns(4,5)P2 (not shown). Phosphorylation of the single Ser183 residue in syndecan-4 cytoplasmic domain has been reported to decrease phospholipid binding and subsequent PKCα activity (24Horowitz A. Murakami M. Gao Y. Simons M. Biochemistry. 1999; 38: 15871-15877Crossref PubMed Scopus (78) Google Scholar). A synthetic peptide corresponding to the entire cytoplasmic domain of syndecan-4, but incorporating phosphorylated Ser183 had markedly lower affinity for PtdIns(4,5)P2 than the unphosphorylated peptide (570 μm compared with 5 μm; Table I). Moreover, this decrease was not accompanied by any change in preference for phospholipid interactions. There was still no detectable binding to D3 inositol phospholipids, and the affinity of the 4L peptides for PtdIns (4Cockcroft S. De Matteis M.A. J. Membrane Biol. 2001; 180: 187-194Crossref PubMed Scopus (69) Google Scholar)P was also reduced to undetectable levels (Table I). One-dimensional NMR spectroscopy was used as a further sensitive indicator of syndecan cytoplasmic domain interactions with PtdIns(4,5)P2 (Fig. 3). The spectra demonstrated that each of the three peptides, 4L, p-4L and 2L, have different characteristics with respect to PtdIns(4,5)P2 binding. Most resonances for 4L and p-4L were changed and broadened upon inositide titration, indicative of oligomerization in the presence of the phospholipid. Therefore, even though the membrane filter assay showed a much decreased affinity of p-4L for PtdIns(4,5)P2, an interaction was still clearly detectable by NMR spectroscopy. However, the spectra of 4L and p-4L were distinct, which indicate differences in oligomer organization. Consistent with results of gel chromatography and lipid binding assays, the spectra of 2L peptide were unchanged with increasing inositide, indicative of no detectable interaction (Fig. 3 C).Figure 3Syndecan cytoplasmic domain interactions with PtdIns(4,5)P2. Proton one-dimensional NMR spectra of 4L peptide (A), p-4L peptide (B), and 2L peptide (C) with titration of PtdIns(4,5)P2. Molar ratios of peptide to inositide are shown in the upper leftof each panel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Published data suggest that the C2 region of syndecan-4 cytoplasmic domain is flexible and may not participate in interactions with inositides (22Shin J. Lee W. Lee D. Koo B.-K. Han I. Lim Y. Woods A. Couchman J.R. Oh E.-S. Biochemistry. 2001; 40: 8471-8478Crossref PubMed Scopus (43) Google Scholar). This region, and particularly its terminal FYA motif, does interact with PDZ domain proteins (28Zimmerman P. David G. FASEB J. 1999; 13: S91-S100Crossref PubMed Google Scholar). These may stabilize the C2 structure in situ. Deletion of the FYA sequence did not alter in vitro binding affinity for PtdIns(4,5)P2 (Table I). Peptides corresponding to the V region and entire cytoplasmic domains of syndecan-2 and −4 were exposed to [3H]BZDC-InsP4 or [3H]BZDC-InsP6. Fig.4 shows the structure of these compounds. Covalent cross-linking was achieved by UV irradiation, with subsequent SDS-PAGE, fluorography, and quantitation. Control experiments contained an ∼400-fold excess of unlabeled InsP4 or InsP6. The results in Fig.5 A show that both the full-length cytoplasmic domain of syndecan-4 and its central V region specifically bound the [3H]BZDC-InsP6 probe. Quantification of the InsP6 probe bound to 4V and 4L peptides, by scanning densitometry, showed reductions of 92–94% in the presence of excess unlabeled InsP6. In contrast, there was limited binding of the [3H]BZDC-InsP4, and this was reduced by 58–61% by excess unlabeled compound. Consiste" @default.
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• W1974604649 title "Regulation of Inositol Phospholipid Binding and Signaling through Syndecan-4" @default.
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