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• W2113144462 abstract "A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105–33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Cα and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Cα and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factorin vitro. A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105–33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Cα and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Cα and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factorin vitro. The Rho family of small GTPases plays an important role in the regulation of several key cellular functions. Rho is involved in the formation of actin stress fibers and focal adhesions (1Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3832) Google Scholar, 2Miura Y. Kikuchi A. Musha T. Kurodi S. Yaku H. Sasaki T. Takai Y. J. Biol. Chem. 1993; 268: 510-515Abstract Full Text PDF PubMed Google Scholar, 3Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3735) Google Scholar), Rac is required in actin polymerization associated with membrane ruffling and lamellipodia formation in fibroblasts (3Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3735) Google Scholar, 4Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3076) Google Scholar), and Cdc42 is important in the formation of filopodia in fibroblasts (3Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3735) Google Scholar). Moreover, Rho family GTPases are involved in cell cycle progression (5Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1058) Google Scholar), stimulate gene transcription through activation of the serum response factor (6Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar), activate the Jun kinase and p38 mitogen-activated protein kinase signaling cascades (7Coso O.A. Chiariello M. Yu J-C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1567) Google Scholar, 8Minden A. Lin A. Claret F-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1446) Google Scholar, 9Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 10Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar), enhance Ras-triggered transformation of NIH3T3 fibroblasts (11Qiu R.-G. Chen J. McCormick F. Symons M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11781-11785Crossref PubMed Scopus (488) Google Scholar, 12Khosravi-Far R. Solski P.A. Clark G.J. Kinch M.S. Der C.J. Mol. Cell. Biol. 1995; 15: 6443-6454Crossref PubMed Scopus (640) Google Scholar), and are required in the NADPH oxidase-mediated phagocytic response in neutrophils (13Knaus U.G. Heyworth P.G. Evans T. Curnutte J.T. Bokoch G.M. Science. 1991; 254: 1512-1515Crossref PubMed Scopus (544) Google Scholar). During the past few years, a number of guanine nucleotide exchange factors for Rho family GTPases have been identified (14Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar). These exchange factors promote binding of GTP by facilitating the release of GDP from Rho proteins. Nucleotide exchange factors which act on Rho proteins contain two key conserved domains: a Dbl homology domain, which is believed to be responsible for catalyzing GDP/GTP exchange; and a pleckstrin homology domain, which seems to be important for cellular localization through interaction with lipids and/or proteins (14Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar). Relatively little is known concerning the specificity of these exchange factors in vivo, although it has been demonstrated that Tiam1 acts as a Rac1-specific exchange factor in NIH3T3 fibroblasts, stimulating membrane ruffling and Jun kinase (15Michiels F. Habets G.G.M. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (508) Google Scholar, 16Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar), Lbc acts as a Rho-specific exchange factor, inducing stress fiber formation in Swiss 3T3 cells and foci in NIH3T3 cells (17Zheng Y. Olson M.F. Hall A. Cerione R.A. Toksoz D. J. Biol. Chem. 1995; 270: 9031-9034Crossref PubMed Scopus (149) Google Scholar), and Dbl stimulates Jun kinase in HeLa cells (8Minden A. Lin A. Claret F-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1446) Google Scholar). The mechanism(s) of activation of Rho family nucleotide exchange factors is not yet evident. It has been demonstrated that membrane localization of Tiam1 is required for Rac-dependent membrane ruffling and Jun kinase activation in NIH3T3 cells (16Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar), and that the N-terminal pleckstrin homology domain and an adjacent protein interaction domain are required for membrane localization of the exchange factor (16Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar, 18Stam J.C. Sander E.E. Michiels F. van Leewen F.N. Kain H.E.T. van der Kammen R.A. Collard J.G. J. Biol. Chem. 1997; 272: 28447-28454Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Phospholipids may play an important role in determining the cellular localization of Tiam1, since both PIP2 1The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; CamKII, Ca2+/calmodulin-dependent protein kinase II, DMEM, Dulbecco's modified Eagle's medium; LPA, lysophosphatidic acid; PKC, protein kinase C; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; PP1, PP2A, and PP2B, protein phosphatases 1, 2A, and 2B, respectively; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid; PLC, phospholipase Cand PIP3 bind to its N-terminal pleckstrin homology domain (19Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.-S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Crossref PubMed Scopus (425) Google Scholar), and phosphoinositide 3-kinase activity is required for activation of Rac1 by Tiam1 (20Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der Kammen R.A. Michiels F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (587) Google Scholar). Reversible protein phosphorylation may also be involved in the regulation of Rho family exchange factors. It has been shown that Dbl (21Graziani G. Ron D. Eva A. Srivastava S.K. Oncogene. 1989; 4: 823-829PubMed Google Scholar) and Ost (22Horii Y. Beeler J.F. Sakaguchi K. Tachibana T. Miki T. EMBO J. 1994; 13: 4776-4786Crossref PubMed Scopus (185) Google Scholar) both exist as phosphoproteins in cells. Significantly, tyrosine phosphorylation of the oncogenes Vav (23Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (680) Google Scholar) and Vav2 (24Schuebel K.E. Movilla N. Rosa J.L. Bustelo X.R. EMBO. J. 1998; 17: 6608-6621Crossref PubMed Scopus (223) Google Scholar) by Lck results in increased GDP/GTP nucleotide exchange on Rac1 and RhoA-like GTPases, respectively, and PIP3 may enhance both phosphorylation and activation of Vav (25Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar). In addition, we have recently demonstrated that lysophosphatidic acid (LPA), platelet-derived growth factor, and several other agonists stimulate phosphorylation of Tiam1 in Swiss 3T3 fibroblasts, via activation of protein kinase C (PKC) (26Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 27Fleming I.N. Elliott C.M Exton J.H. FEBS Lett. 1998; 429: 229-233Crossref PubMed Scopus (38) Google Scholar), indicating that Rho exchange factors can also be phosphorylated on serine/threonine residues by a regulated mechanism. In this study we demonstrate that Tiam1 is phosphorylated by several PKC isozymes in vitro, but is selectively phosphorylated by a classical PKC isoform, PKCα, when Swiss 3T3 cells are treated with LPA. In addition, we present strong evidence that Ca2+/calmodulin-dependent protein kinase II (CamKII) also phosphorylates Tiam1 in Swiss 3T3 fibroblasts in response to LPA treatment and that this phosphorylation produces electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Finally, we show that phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not protein kinase Cα, enhances its nucleotide exchange rate toward Rac1, and that this can be abrogated by treatment with protein phosphatase 1. Swiss 3T3 fibroblasts were obtained from the American Type Culture Collection. Fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM), penicillin, and streptomycin were from Life Technologies, Inc. LPA (1-oleoyl) was from Avanti Polar lipids. Phorbol 12-myristate 13-acetate (PMA), sodium orthovanadate, leupeptin, antipain, phenylmethylsulfonyl fluoride, sodium fluoride, sodium pyrophosphate, Tween 20, Triton X-100, and fatty acid-free bovine serum albumin were obtained from Sigma. Ro-31-8220, KN93, bisindolylmaleimide I, ionomycin, A23187, BAPTA/AM, and purified protein phosphatase 1 (PP1) catalytic subunit were from Calbiochem. Tiam1 antibody was from Santa Cruz. The phosphothreonine-specific antibody was obtained fromZymed Laboratories Inc. GDP and GTP were from Roche Molecular Biochemicals. [γ-32P]ATP and [3H]GDP were from NEN Life Science Products, and protein kinase C isozymes were from Panvera. PIP3 was from Echelon Research Laboratories. Purified recombinant mouse brain Ca2+/calmodulin-dependent kinase IIα was a kind gift from Dr. R. Colbran (Vanderbilt University, Nashville, TN). Protein phosphatase-2A (PP2A) and -2B (PP2B) catalytic subunits were from Promega. Glutathione-Sepharose 4B beads were from Amersham Pharmacia Biotech. Nitrocellulose filters were from Whatman. GST-Rac1-expressing Escherichia coli were a kind gift from Prof. A. Hall (University College, London, United Kingdom). Swiss 3T3 fibroblasts were maintained in HEPES-buffered DMEM with 4 mm l-glutamine supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells were grown on 100-mm dishes for 1–2 days to subconfluence (60–70%). The medium was then replaced with a low serum medium (DMEM containing 1% fetal bovine serum, 0.5% (w/v) bovine serum albumin, 100 units/ml penicillin, and 100 μg/ml streptomycin) for 24 h to allow the cells to become quiescent. The cells were then treated with serum-free medium (DMEM containing 0.5% bovine serum albumin and antibiotics) for 1 h prior to agonist stimulation. Serum-starved cultures, on 100-mm dishes, were treated with various concentrations of LPA, PMA, or ionomycin at 37 °C for different times as noted in the experiments. The medium was removed, the cells washed three times with 5 ml of ice-cold PBS containing 500 μm sodium orthovanadate, and scraped in 400 μl/dish of lysis buffer (50 mm HEPES, pH 7.5, 50 mm NaCl, 1 mm MgCl2, 2 mm EDTA, 10 μg/ml antipain and leupeptin, 1 mm phenylmethylsulfonyl fluoride, 500 μm sodium orthovanadate, 10 mmpyrophosphate, 10 mm sodium fluoride, and 1 mmdithiothreitol). The cells were lysed by five passes through a 27-gauge needle (28Fleming I.N. Elliott C.M Exton J.H. J. Biol. Chem. 1996; 271: 33067-33073Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) at 4 °C. Lysates were centrifuged at 120,000 ×g for 45 min to prepare cytosolic and total particulate fractions. The membrane pellet was washed twice with lysis buffer to remove cytosolic proteins. Protein determination was done by the method of Bradford (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar). SDS-Polyacrylamide gel electrophoresis was performed on 6% or 4–12% gradient polyacrylamide gels (Novel Experimental Corp) and proteins transferred onto polyvinylidene difluoride membranes (Millipore) for 1.5 h at 20 V using a Novex wet transfer unit. The membranes were blocked overnight with 5% (w/v) nonfat dried milk. Blots were incubated for 1 h with Tiam1 antibody (diluted 1:2000) in 1% bovine serum albumin, then for 1 h with a horseradish peroxidase-conjugated secondary antibody (Vector Laboratories), prior to development using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). Phosphothreonine Western blots were carried out essentially as described above, using a 1:500 primary antibody dilution. An N-terminally truncated form of Tiam1, GST-C1199-Tiam1 (16Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar), was expressed in Cos-7 cells and purified using glutathione-Sepharose beads essentially as described (15Michiels F. Habets G.G.M. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (508) Google Scholar), in the presence of 0.1% (v/v) Triton X-100. Silver staining indicated that the purified GST-Tiam1 was almost homogeneous. Purified GST-Tiam1 (3 μl) was incubated for 20 min at 30 °C in the presence or absence of 0.06 units of various purified PKC isozymes. Assays were carried out in 20 mm MOPS buffer, pH 7.2, containing 25 mm glycerol 3-phosphate, 1 mmsodium orthovanadate, 1 mm dithiothreitol, 1 mmCaCl2, 0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 15 mm MgCl2, and 100 μm [γ-32P]ATP (specific activity 5 × 106 dpm/nmol) and phosphorylation analysis by autoradiography. Purified GST-Tiam1 (3 μl) was incubated for 20 min at 30 °C in the presence or absence of the indicated amounts of purified CamKIIα. Assays were carried out in 10 mm Tris buffer, pH 7.4, containing 0.1 mg/ml BSA, 1.25 mm CaCl2, 25 μg/ml calmodulin, 15 mm MgCl2 and 100 μm ATP. Assays were carried out either using non-radiolabeled ATP and phosphorylation analysis by Western blotting or with [γ-32P]ATP (specific activity 5 × 106 dpm/nmol) and phosphorylation analysis by autoradiography. Since the purified Tiam1 preparations contained detergent and some aggregated protein, the concentration of purified Tiam1 was estimated from silver-stained gels for the stoichiometry experiments, using BSA as a standard. 0.2 pmol of GST-Tiam1 was phosphorylated by PKCα (0.3 units) or CamKIIα (4 μg), for 1 h in the presence of [γ-32P]ATP (specific activity 2 × 107dpm/nmol), as described above. The samples were separated by SDS-PAGE on 6% gels, the Tiam1 band excised from the gel and 32P incorporation assessed by scintillation counting. Purified GST-Tiam1 (10 μl) was phosphorylated with PKCα (0.3 units) or CamKIIα (4 μg), for 1 h in the presence of [γ-32P]ATP (specific activity 2 × 107 dpm/nmol), as described above. Phosphorylated GST-Tiam1 was incubated with 30 μl of glutathione-Sepharose beads for 1 h at 30 °C, and the beads collected by centrifugation (3,000 × g for 5 min). The Tiam1-bound beads were washed three times with 200 μl of 50 mm Tris buffer, pH 7.0, containing 0.5 mg/ml BSA to remove the kinase, resuspended in 50 μl of the same buffer, and stored on ice until use. Tiam1-bound beads (5 μl) were incubated with 0.3 units of purified protein phosphatase 1, 2A or 2B at 30 °C for 0 and 5 min. Protein phosphatase 1-catalyzed dephosphorylation was carried out in 50 mm Tris buffer, pH 7.0, containing 0.5 mg/ml BSA and 0.2 mm MnCl2. Protein phosphatase 2A was incubated with Tiam1 in 50 mm Tris buffer, pH 7.0, containing 0.5 mg/ml BSA. Protein phosphatase 2B-catalyzed dephosphorylation was carried out in 50 mm Tris buffer, pH 7.0, containing 0.5 mg/ml BSA, 20 μg/ml calmodulin, and 1 mmCaCl2. The samples were separated by electrophoresis on 6% polyacrylamide gels and Tiam1 dephosphorylation analyzed by autoradiography. C1199-Tiam1 with an N-terminal hexahistidine tag was expressed in sf9 cells and purified using Talon metal affinity resin (CLONTECH) in a 25 mm Tris buffer, pH 8.0, containing 0.5 μmβ-mercaptoethanol and 100 mm NaCl. Tiam1 was eluted from the beads using 100 mm imidazole and dialyzed prior to freezing. GST-Rac1 was expressed in E. coli, and purified using glutathione-Sepharose beads in a 100 mm Tris buffer, pH 8.0, containing 250 mm NaCl and 0.1 mmdithiothreitol. GST-Rac1 was eluted from the beads with 10 mm glutathione, dialyzed, and frozen. The Tiam1 exchange assay was carried out essentially as described (15Michiels F. Habets G.G.M. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (508) Google Scholar). Purified GST-Rac1 (120 pmol) was preloaded with [3H]GDP (30 μm; 25 Ci/mmol) in 60 μl of binding buffer. Eight μl of the preloaded GTPase was added to 32 μl of exchange mixture, which contained 5 pmol of Tiam1 or BSA, 1 mm GTP, and 75 μm PIP3, in exchange buffer (15Michiels F. Habets G.G.M. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (508) Google Scholar). At the indicated times, 8-μl aliquots were pipetted into 1 ml stopping buffer (50 mm Tris, pH 7.4, 5 mmMgCl2, 50 mm NaCl), and [3H]GDP bound to Rac1 analyzed by filtering through nitrocellulose. For some exchange experiments, Tiam1 (5 pmol) was prephosphorylated with 0.1 units of protein kinase C or 2 μg of CamKII, as described above. In some experiments Tiam1 was assayed after prephosphorylation with 2 μg of CamKII in the presence or absence of 0.2 units of PP1. In Swiss 3T3 fibroblasts, LPA stimulates threonine phosphorylation of Tiam1 through activation of PKC, and causes its electrophoretic retardation on SDS-PAGE (26Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). To understand further the mechanism of Tiam1 phosphorylation, we incubated purified GST-C1199-Tiam1 with several PKC isozymes to determine which isoform(s) phosphorylates the exchange factor. As shown in Fig.1, all of the kinases tested phosphorylate Tiam1, indicating that PKC isozymes of the classical, novel, and atypical families can phosphorylate the protein in vitro. However, the different PKC isoforms phosphorylated Tiam1 to different extents. The exchange factor was preferentially phosphorylated by PKCα, -γ, and -ζ, moderately phosphorylated by PKCε, and only weakly phosphorylated by PKCβ1, -β2, and -δ. Significantly, none of the PKC isozymes tested decreased the electrophoretic mobility of Tiam1, suggesting that this was probably caused by a kinase from a different family in vivo. Down-regulation of non-atypical PKC isozymes by long term PMA pretreatment, or preincubation with the protein kinase C inhibitor Ro-31-8220, reduces LPA- or platelet-derived growth factor-stimulated Tiam1 phosphorylation by approximately 75% in Swiss 3T3 cells (26Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 27Fleming I.N. Elliott C.M Exton J.H. FEBS Lett. 1998; 429: 229-233Crossref PubMed Scopus (38) Google Scholar) suggesting that another protein kinase is also involved. Therefore, purified GST-C1199-Tiam1 was incubated with Ca2+/calmodulin-dependent protein kinase II (CamKII), a kinase with a very broad substrate specificity and widespread expression (30Fujisawa H. BioEssays. 1990; 12: 27-29Crossref PubMed Scopus (35) Google Scholar), to determine whether this kinase phosphorylated the exchange factor. Although some phosphorylation of Tiam1 was observed in the absence of CamKII (Fig.2 A), perhaps due to a protein kinase which co-purifies with the GST-Tiam1 (26Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), addition of the kinase significantly enhanced 32P phosphorylation of the exchange factor (Fig. 2 A), demonstrating that this kinase can phosphorylate Tiam1. Indeed, Western blotting with antibodies confirmed that CamKII stimulated phosphorylation of Tiam1 on threonine (Fig. 2 B). Significantly, in addition to phosphorylating Tiam1, CamKII induced electrophoretic retardation of the exchange factor (Fig. 2), such as is observed upon stimulation of Swiss 3T3 cells with LPA (26Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Ca2+/calmodulin-dependent protein kinase II induced the Tiam1 bandshift in a concentration-dependent (Fig. 2 B) and time-dependent (Fig. 2 C) manner, but only in the presence of Ca2+ and calmodulin (data not shown). Intriguingly, the Tiam1 bandshift occurred in a gradual manner with time, and not as one step, suggesting that the exchange factor probably exists in several different phosphorylation states and has multiple phosphorylation sites which serve as substrates for CamKII. Indeed, when the Tiam1 protein concentration was estimated by silver staining, using BSA as a standard, stoichiometry experiments indicated that under maximal phosphorylating conditions, Tiam1 contains 10.1 ± 2.7 PKCα and 3.7 ± 0.6 CamKII phosphorylation sites. Swiss 3T3 cells were stimulated with LPA, in the presence and absence of the intracellular Ca2+ chelator BAPTA/AM, to investigate the importance of this metal ion in Tiam1 phosphorylation. The results (Fig. 3 A) show that Tiam1 phosphorylation is totally abolished in the presence of the chelator, indicating that Ca2+ plays an essential role in this pathway. Together with the results obtained using protein kinase inhibitors, and PKC down-regulation (26Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), this suggests that LPA stimulates Tiam1 phosphorylation through activation of a classical PKC isoform and another Ca2+-dependent enzyme. Therefore, since Swiss 3T3 cells only contain PKCα, -δ, -ε, and -ζ (31Olivier A.R. Parker P.J. J. Cell. Physiol. 1992; 152: 240-244Crossref PubMed Scopus (77) Google Scholar), LPA must stimulate Tiam1 phosphorylation through activation of PKCα, which is the only classical Ca2+-dependent enzyme present. Significantly, BAPTA treatment also inhibited the LPA-stimulated Tiam1 bandshift (Fig.3 B), indicating that Ca2+ is required for this effect. However, the selective PKC inhibitors bisindolylmaleimide I (Fig. 3 C) and Ro-31-8220 (data not shown) had no effect on the LPA-induced Tiam1 bandshift, providing further evidence that PKC does not cause this. To confirm that CamKII is involved in LPA-induced Tiam1 phosphorylation, Swiss 3T3 cells were preincubated with the CamKII inhibitor KN93 (20 μm) for 24 h, in the presence and absence of the PKC inhibitor Ro-31-8220 (5 μm) for 1 h. As expected (26Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), Ro-31-8220 greatly reduced LPA-stimulated Tiam1 phosphorylation (Fig. 3 D). KN93 also significantly reduced LPA-induced Tiam1 phosphorylation, and the two inhibitors together almost completely eliminated the phosphorylation (Fig. 3 D). Therefore, these data strongly suggest that CamKII and PKC both contribute to the phosphorylation studied here. To provide additional evidence that PKCα and CamKII phosphorylate Tiam1 in vivo, Swiss 3T3 cells were treated with the Ca2+ ionophore ionomycin, in the presence and absence of PMA. PMA (1 μm) alone induced limited threonine phosphorylation of Tiam1 (Fig. 3 E; Ref. 26Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Ionomycin (1 μm) alone stimulated Tiam1 phosphorylation to a greater extent (Fig. 3 E), and enhanced the PMA-stimulated Tiam1 phosphorylation. Similar results were obtained with the ionophore A23187 (data not shown). Therefore, the observation that PMA and a Ca2+ ionophore are sufficient to stimulate Tiam1 phosphorylation is consistent with a classical PKC isozyme and CamKII phosphorylating the exchange factor in vivo. We have previously established that LPA-stimulated Tiam1 phosphorylation is maximal at 2.5 min, begins to decrease after 10 min LPA treatment, but is still readily detectable after 60 min of LPA treatment (26Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Further experiments showed that Tiam1 phosphorylation was still detectable after 3 h of LPA treatment, but that the stimulation was lost after 4 h (data not shown), presumably because of dephosphorylation. To elucidate further the mechanisms involved in controlling the level of Tiam1 phosphorylation, we investigated which phosphatases are involved in the dephosphorylation process. The results show that Tiam1 is preferentially dephosphorylated by the catalytic subunit of PP1in vitro, when the exchange factor is phosphorylated by PKCα or CamKII (Fig. 4). Tiam1 was also dephosphorylated by protein phosphatase 2B in vitro, but at a much slower rate (Fig. 4). Interestingly, protein phosphatase 2A slowly dephosphorylated Tiam1 when it was phosphorylated by CamKII, but not when it was phosphorylated by PKCα. Since Tiam1 acts as a Rac1-specific exchange factor in NIH3T3 fibroblasts, stimulating membrane ruffling and Jun kinase activity (15Michiels F. Habets G.G.M. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (508) Google Scholar, 16Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar), we investigated whether protein phosphorylation could affect Tiam1 GDP/GTP exchange activity toward Rac1. Purified hexahistidine-tagged Tiam1 protein was incubated with ATP in the presence or absence of purified CamKII, and the GDP/GTP exchange rate of Tiam1 assessed by following the dissociation of [3H]GDP from Rac1. As expected (15Michiels F. Habets G.G.M. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (508) Google Scholar), Tiam1" @default.
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• W2113144462 title "Ca2+/Calmodulin-dependent Protein Kinase II Regulates Tiam1 by Reversible Protein Phosphorylation" @default.
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