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• W2077671521 abstract "Eleven isoforms of G protein γ subunit have been found thus far, but the precise roles of individual γ subunits are not known. The γ12 subunit has two unique properties: phosphorylation by protein kinase C and association with F-actin. To elucidate the role of γ12, we overexpressed γ12 and other γ subunits in NIH 3T3 cells together with the β1 subunit. The overexpressed γ12 as well as endogenous γ12, but not γ2, γ5, and γ7 subunits, associated with cytoskeletal components. Expression of γ12 induced remarkable changes including cell rounding, disruption of stress fibers, and enhancement of cell migration, but expression of other γ subunits did not induce significant changes. Deletion of the N-terminal region of γ12 decreased the abilities of γ12 to associate with cytoskeletal fractions, to induce cell rounding, and to increase cell motility. Replacement by alanine of Ser2 of γ12 (Ser1 of a mature γ12 protein), a phosphorylation site for protein kinase C, eliminated these effects of γ12, whereas a mutant in which Ser2 was replaced with glutamic acid showed effects equivalent to wild-type γ12. These results indicate that phosphorylation of γ12 at Ser2 enhances the motility of cells. Eleven isoforms of G protein γ subunit have been found thus far, but the precise roles of individual γ subunits are not known. The γ12 subunit has two unique properties: phosphorylation by protein kinase C and association with F-actin. To elucidate the role of γ12, we overexpressed γ12 and other γ subunits in NIH 3T3 cells together with the β1 subunit. The overexpressed γ12 as well as endogenous γ12, but not γ2, γ5, and γ7 subunits, associated with cytoskeletal components. Expression of γ12 induced remarkable changes including cell rounding, disruption of stress fibers, and enhancement of cell migration, but expression of other γ subunits did not induce significant changes. Deletion of the N-terminal region of γ12 decreased the abilities of γ12 to associate with cytoskeletal fractions, to induce cell rounding, and to increase cell motility. Replacement by alanine of Ser2 of γ12 (Ser1 of a mature γ12 protein), a phosphorylation site for protein kinase C, eliminated these effects of γ12, whereas a mutant in which Ser2 was replaced with glutamic acid showed effects equivalent to wild-type γ12. These results indicate that phosphorylation of γ12 at Ser2 enhances the motility of cells. guanine nucleotide-binding protein protein kinase C Dulbecco's modified essential medium C-terminal fragment of the β-adrenergic receptor kinase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine Heterotrimeric G proteins1 play a major role in signal transduction from cell surface receptors to intracellular effectors. The βγ complexes, as well as α subunits, directly regulate various effectors, including adenylyl cyclase, phospholipase C-β, phosphatidylinositol 3-kinase, K+ channels, and Ca2+ channels (1Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1290) Google Scholar, 2Iñiguez-Lluhi J. Kleuss C. Gilman A.G. Trends Cell Biol. 1993; 3: 230-236Abstract Full Text PDF PubMed Scopus (111) Google Scholar). At present, 11 isoforms of the γ subunit have been found (2Iñiguez-Lluhi J. Kleuss C. Gilman A.G. Trends Cell Biol. 1993; 3: 230-236Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 3Gautam N. Baetscher M. Aebersold R. Simon M.I. Science. 1989; 244: 971-974Crossref PubMed Scopus (93) Google Scholar, 4Fisher K.J. Aronson Jr., N.N. Mol. Cell. Biol. 1992; 12: 1585-1591Crossref PubMed Scopus (51) Google Scholar, 5Cali J.J. Balcueva E.A. Rybalkin I. Robishaw J.D. J. Biol. Chem. 1992; 267: 24023-24027Abstract Full Text PDF PubMed Google Scholar, 6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Although the biological properties of the βγ complex containing γ1 are noticeably different from those of βγ complexes containing the other γ subunits (7Asano T. Morishita R. Matsuda T. Fukada Y. Yoshizawa T. Kato K. J. Biol. Chem. 1993; 268: 20512-20519Abstract Full Text PDF PubMed Google Scholar,8Ueda N. Iñiguez-Lluhi J.A. Lee E. Smrcka A.V. Robishaw J.D. Gilman A.G. J. Biol. Chem. 1994; 269: 4388-4395Abstract Full Text PDF PubMed Google Scholar), the precise roles of individual γ subunits are not known. The γ12 subunit, which is widely distributed and especially rich in fibroblasts and smooth muscle cells (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), has unique properties. First, γ12 is a selective substrate for protein kinase C (PKC) (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 9Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar). In Swiss 3T3 cells, γ12 is phosphorylated upon exposure of cells to various reagents such as phorbol 12-myristate 13-acetate, serum, lysophosphatidic acid, endothelines, and growth factors (9Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar). Phosphorylated γ12enhanced the association of βγ12 with Goα (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and weakened the ability of βγ12 to stimulate type II adenylyl cyclase (10Yasuda H. Lindorfer M.A. Myung C.S. Garrison J.C. J. Biol. Chem. 1998; 273: 21958-21965Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), but the magnitudes of the changes induced by phosphorylation were relatively small. The second unique property is that γ12 associates with F-actin in cells and in a cell-free system (11Ueda H. Saga S. Shinohara H. Morishita R. Kato K. Asano T. J. Cell Sci. 1997; 110: 1503-1511Crossref PubMed Google Scholar). In addition to γ12, various α subunits, such as Gi2α, Gsα (12Sarndahl E. Bokoch G.M. Stendahl O. Andersson T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6552-6556Crossref PubMed Scopus (36) Google Scholar), and Gq/11α (13Ibarrondo J. Joubert D. Dufour M.N. Cohen-Solal A. Homburger V. Jard S. Guillon G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8413-8417Crossref PubMed Scopus (64) Google Scholar), and the β subunit (14Chiba K. Longo F.J. Kontani K. Katada T. Hoshi M. Dev. Biol. 1995; 169: 415-420Crossref PubMed Scopus (19) Google Scholar) as well as enzymes involved in signal transduction, such as phospholipase C, phosphoinositide 3-kinase, and PKC (15Grondin P. Plantavid M. Sultan C. Breton M. Mauco G. Chap H. J. Biol. Chem. 1991; 266: 15705-15709Abstract Full Text PDF PubMed Google Scholar, 16Payrastre B. van Bergen en Henegouwen P.M. Breton M. den Hartigh J.C. Plantavid M. Verkleij A.J. Boonstra J. J. Cell Biol. 1991; 115: 121-128Crossref PubMed Scopus (174) Google Scholar, 17Blobe G.C. Stribling D.S. Fabbro D. Stabel S. Hannun Y.A. J. Biol. Chem. 1996; 271: 15823-15830Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) are found associated with the cytoskeleton in a variety of cells, but the physiological significance of these associations is unclear. To examine the role of γ12, we overexpressed γ12, other γ subunits and their mutants together with β1 in NIH 3T3 cells. The results indicate that only γ12 subunit induces cell rounding and enhances cell migration and that phosphorylation of γ12 is involved in these processes. cDNAs of several γ12 and γ2 mutants were prepared using synthetic polymerase chain reaction primers (3Gautam N. Baetscher M. Aebersold R. Simon M.I. Science. 1989; 244: 971-974Crossref PubMed Scopus (93) Google Scholar, 6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). cDNAs of bovine β1 (18Fong H.K. Hurley J.B. Hopkins R.S. Miake-Lye R. Johnson M.S. Doolittle R.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2162-2166Crossref PubMed Scopus (324) Google Scholar) and γ2 (3Gautam N. Baetscher M. Aebersold R. Simon M.I. Science. 1989; 244: 971-974Crossref PubMed Scopus (93) Google Scholar) were generously provided by Dr. M. I. Simon (California Institute of Technology) and Dr. T. Nukada (Tokyo Institute of Psychiatry), respectively. All cDNAs of G protein subunits (3Gautam N. Baetscher M. Aebersold R. Simon M.I. Science. 1989; 244: 971-974Crossref PubMed Scopus (93) Google Scholar, 4Fisher K.J. Aronson Jr., N.N. Mol. Cell. Biol. 1992; 12: 1585-1591Crossref PubMed Scopus (51) Google Scholar, 5Cali J.J. Balcueva E.A. Rybalkin I. Robishaw J.D. J. Biol. Chem. 1992; 267: 24023-24027Abstract Full Text PDF PubMed Google Scholar, 6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and a C-terminal fragment (amino acids 495–689) of the β-adrenergic receptor kinase (βARKct) (19Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar) were subcloned into pCMV5 vector as described previously (20Yamauchi J. Kaziro Y. Itoh H. Biochem. Biophys. Res. Commun. 1995; 214: 694-700Crossref PubMed Scopus (14) Google Scholar). Antibodies against the β and γ subunits of G protein have been described previously (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 9Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar, 21Morishita R. Kato K. Asano T. Eur. J. Biochem. 1988; 174: 87-94Crossref PubMed Scopus (46) Google Scholar, 22Asano T. Morishita R. Ohashi K. Nagahama M. Miyake T. Kato K. J. Neurochem. 1995; 64: 1267-1273Crossref PubMed Scopus (37) Google Scholar). NIH 3T3 cells were grown in Dulbecco's modified essential medium (DMEM) supplemented with 10% calf serum. Transfection was performed using LipofectAMINE Plus reagent (Life Technologies, Inc.) as described. After 48 h, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline and immunostained using antibodies against γ or β, followed by fluorescein isothiocyanate-labeled secondary antibody (MBL) as described (11Ueda H. Saga S. Shinohara H. Morishita R. Kato K. Asano T. J. Cell Sci. 1997; 110: 1503-1511Crossref PubMed Google Scholar). The cells were also stained for F-actin with tetramethylrhodamine isothiocyanate-phalloidin. After transfection, the cells (5 × 106 cells) washed with phosphate-buffered saline were incubated for 5 min at 0 °C with 0.5% Triton X-100 in 20 mm Hepes, pH 7.5, 50 mm NaCl, 1 mm EDTA, 0.2 mmphenylmethylsulfonyl fluoride, and 2 μg/ml trypsin inhibitor and centrifuged at 12,000 × g for 3 min at 4 °C (11Ueda H. Saga S. Shinohara H. Morishita R. Kato K. Asano T. J. Cell Sci. 1997; 110: 1503-1511Crossref PubMed Google Scholar). The pellet was washed once with the same buffer. The supernatant and pellet were used as the Triton X-100-soluble and Triton X-100-insoluble fractions, respectively (11Ueda H. Saga S. Shinohara H. Morishita R. Kato K. Asano T. J. Cell Sci. 1997; 110: 1503-1511Crossref PubMed Google Scholar). 24 h after transfection, the culture medium was replaced with DMEM for 24 h prior to the 32P incorporation experiment. The cells were washed twice with phosphate-free medium (Eagle's minimum essential medium without sodium phosphate), preincubated for 1 h at 37 °C in the same medium containing [32P]orthophosphate (0.2 mCi), and then incubated for 30 min with 10% calf serum in DMEM. After labeling, the cells were washed with the ice-cold phosphate-buffered saline and then suspended in 0.2 ml of a solution containing 20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 100 mm NaCl, 0.2 mmphenylmethylsulfonyl fluoride, 1 μg/ml trypsin inhibitor, 10 nm calyculin A, and 1% CHAPS. The suspension was mixed with a vortex mixer and centrifuged at 100,000 × g for 15 min at 4 °C. The supernatant was incubated at 4 °C for 2 h with 5 μg of affinity-purified antibody against γ7, which had been preincubated with protein A-Sepharose beads. The Sepharose beads were washed with the solution containing 20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 100 mm NaCl, 0.2 mm phenylmethylsulfonyl fluoride, 1 μg/ml trypsin inhibitor, 10 nm calyculin A, and 0.1% CHAPS. The beads were suspended in 30 μl of sample buffer for electrophoresis, and each resultant supernatant was subjected to Tricine/SDS-polyacrylamide gel electrophoresis (23Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10527) Google Scholar) with subsequent autoradiography (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). For immunoblotting, cells were trypsinized 48 h after transfection, seeded into culture dishes, and incubated for 2 h at 37 °C in DMEM with 10% calf serum. Then cell lysates were subjected to Tricine/SDS-polyacrylamide gel electrophoresis for immunoblotting with the antibody against phosphorylated γ12 (p-γ12) (9Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar). Migration through a membrane in response to serum was assayed with the use of a Chemotaxicell chamber (Kurabo) with an 8-μm polycarbonate filter (24Cunningham C.C. Stossel T.P. Kwiatkowski D.J. Science. 1991; 251: 1233-1236Crossref PubMed Scopus (259) Google Scholar). Transfected cells were trypsinized and counted, and 5 × 104 cells/well were loaded into the top wells in DMEM. The bottom wells were similarly filled with DMEM with 10% calf serum so that the cells were exposed to a gradient of serum factors. The chambers were incubated for 2 h at 37 °C, and the membranes were removed and stained. Under these conditions, a negligible number of cells fell off the bottom of the filter. The number of cells that had migrated through the membrane was counted for each well. To determine cell migration by chemokinesis, top and bottom wells were filled with media with 10% calf serum so that the cells were exposed to no gradient of serum factors. Cell migration in the absence of a gradient was about 40% of that in a gradient, indicating that the observed migration consisted of both chemotaxis and chemokinesis. Immunocytochemical double staining of normal NIH 3T3 cells with phalloidin showed the complete overlap of γ12 staining with staining of actin stress fibers, which were found crossing the entire cell (Fig. 1 A) as previously reported for Swiss 3T3 cells (11Ueda H. Saga S. Shinohara H. Morishita R. Kato K. Asano T. J. Cell Sci. 1997; 110: 1503-1511Crossref PubMed Google Scholar). To investigate the role of γ12, we co-transfected γ12 or various γ subunits with β1 in NIH 3T3 cells. Most cells overexpressing γ12 were rounded, with disruption of F-actin architecture (Fig. 1 B), whereas some cells overexpressing γ12 had decreased stress fibers with a flattened shape (Fig. 1 C). Because the fluorescence of γ subunits in transfected cells was much stronger than that in normal cells, photographs with short exposure were shown in Fig. 1(B–G). Therefore, the staining of endogenous γ12 in normal cells was faint or not observed in Fig. 1(B and C). In contrast with γ12, overexpression of γ2, γ5, and γ7 did not induce such dramatic changes in cell shape, but some of cells overexpressing these γ subunits showed decreased stress fibers in the center and adopted a flattened, rounded shape (Fig. 1, D–F). Immunoblotting analyses showed that similar amounts of β1 and transfected γ subunits were expressed in these experiments, whereas basal levels of γ5 and γ12, major endogenous γ subunits in NIH 3T3 cells, were detected in all cells (Fig.2 A). To examine whether the expressed γ subunits associated with cytoskeletal components, transfected cells were fractionated using Triton X-100. A large portion of the expressed γ12 was present in the Triton X-100-insoluble fraction in transfected cells, and a similar distribution of endogenous γ12 was observed in control cells, whereas most γ2, γ5, and γ7 were present in Triton X-100-soluble fractions (Fig.2 B). These results indicate that expressed γ12, but not other isoforms, associates with cytoskeletal components, which is consistent with the localization of endogenous γ subunits in Swiss 3T3 cells (11Ueda H. Saga S. Shinohara H. Morishita R. Kato K. Asano T. J. Cell Sci. 1997; 110: 1503-1511Crossref PubMed Google Scholar).Figure 2Expression of β and γ subunits in transfected cells and association of expressed γ subunits with cytoskeletal components. A, expression of β and γ subunits in transfected cells. NIH 3T3 cells were co-transfected with β1 and the various γ subunits as indicated. Equal amounts of cell lysates were subjected to Tricine/SDS-polyacrylamide gel electrophoresis for γ subunits or SDS-polyacrylamide gel electrophoresis for β subunit and immunoblotted with antibodies against β subunit and various γ subunits. The standards (Std, from top to bottom) were purified bovine βγ2 (10 ng), βγ5 (5 ng), βγ7 (5 ng), βγ12 (5 ng), and βγ2 (5 ng). Because the antibody used to detect γ7 cross-reacted with γ2, γ3and γ12 (22Asano T. Morishita R. Ohashi K. Nagahama M. Miyake T. Kato K. J. Neurochem. 1995; 64: 1267-1273Crossref PubMed Scopus (37) Google Scholar), staining for γ7 artifactually stained γ2 and γ12 as well as γ7. The other antibodies used to stain the γ subunits were specific for their respective isoforms (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 22Asano T. Morishita R. Ohashi K. Nagahama M. Miyake T. Kato K. J. Neurochem. 1995; 64: 1267-1273Crossref PubMed Scopus (37) Google Scholar). B, association of transfected γ subunits with cytoskeletal components. Transfected cells were fractionated into Triton X-100-soluble (Sol) and -insoluble (Insol) fractions. Fractions were then subjected to immunoblotting with respective antibodies.View Large Image Figure ViewerDownload (PPT) In contrast with the cells co-transfected with β1 and γ12, the cells transfected with γ12 alone were unchanged, suggesting that β1 and γ12were co-expressed in cells and the β1γ12complex induced cell rounding (data not shown). To test further whether the β1γ12 complex was indeed involved in induction of cell rounding, we expressed βARKct (19Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar) and Gi2α, both of which are expected to bind and sequester free βγ. Co-expression of βARKct (Fig. 1 G) or Gi2α (data not shown) prevented β1γ12-induced cell rounding, supporting the involvement of the βγ complex in these changes. The round shape of the cells suggested a decrease of cell adhesion, which might influence cell migration (24Cunningham C.C. Stossel T.P. Kwiatkowski D.J. Science. 1991; 251: 1233-1236Crossref PubMed Scopus (259) Google Scholar, 25Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1311) Google Scholar, 26Anand-Apte B. Zetter B.R. Viswanathan A. Qiu R.G. Chen J. Ruggieri R. Symons M. J. Biol. Chem. 1997; 272: 30688-30692Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). To examine this possibility, Boyden chamber cell migration assays were performed for cells transfected with various γ subunits. Cell migration markedly increased in cells transfected with γ12 but did not significantly change in cells transfected with γ2, γ5, or γ7 (Fig.3). Co-transfection of βARKct again suppressed the increase of cell motility induced by γ12. These results suggested that γ12, but not other γ subunits, was involved in enhancement of cell motility. Expression of βARKct alone in normal cells decreased cell motility, suggesting that βγ12-induced cell migration occurred in normal cells (data not shown). Comparison of amino acid sequences of various isoforms of γ revealed diverged residues concentrated at the N-terminal region (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). To test whether the ability of γ12 to associate with F-actin is determined by its N-terminal sequence, the N-terminal truncated γ12 (γ12ΔN5, Fig.4 A) was transfected into NIH 3T3 cells. The deletion of the N terminus decreased the ability of γ12 to associate with cytoskeletal fractions (Fig.4 B), indicating that the N-terminal region of γ12 is important for the association with F-actin. The weak association of γ12ΔN5 with cytoskeletal fractions was not due to an inability to form the βγ complex, because γ12ΔN5 was coimmunoprecipitated with an antibody against Gi2α when co-transfected with β and Gi2α but not when co-transfected with only Gi2α (data not shown). The γ12ΔN5 mutant neither caused cell rounding nor enhanced cell migration (Fig. 4,C and F), suggesting that the association of γ12 with F-actin is important to induce these changes. On the other hand, the N-terminal region contains a site phosphorylated by PKC (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 9Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar), and therefore it is possible that phosphorylation of γ12 is involved in induction of these changes. When phosphorylation of γ12 was analyzed by immunoblotting with the antibody against p-γ12, the expressed γ12 was strongly phosphorylated, whereas weak phosphorylation of endogenous γ12 was observed (Fig.4 D). The phosphorylation of the expressed γ12determined with 32P incorporation was also greater than that of the endogenous γ12 observed in control cells, but the increase appeared to be smaller than that observed in immunoblots (Fig. 4 E). The conditions of these experiments were not identical, but the main reason for the small increase of phosphorylation was that some γ12 subunits in transfected cells had already been phosphorylated before the 32P incorporation experiment (data not shown). We then transfected NIH 3T3 cells with the mutant γ12S2A, in which Ser 2A phosphorylation site of γ12 for PKC is the first serine residue from the N terminus of γ12, which was previously designated Ser1from the sequence of a mature γ12 protein (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 9Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar, 10Yasuda H. Lindorfer M.A. Myung C.S. Garrison J.C. J. Biol. Chem. 1998; 273: 21958-21965Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) but is designated Ser2 in this paper. , a phosphorylation site,2 is replaced with alanine (Fig. 4 A). The γ12S2A, which was not phosphorylated, could associate with the cytoskeleton but had no effect on morphology and motility of cells (Fig. 4). Because glutamic acid can substitute for phosphoserine in some proteins activated by phosphorylation, we next transfected cells with mutant γ12S2E, in which Ser2 is replaced with glutamate (Fig. 4 A). This mutant caused similar effects in transfected cells to wild-type γ12, except of the increase of phosphorylation (Fig. 4). These results supported the idea that phosphorylation of γ12 induced cell rounding and enhanced cell motility. In Fig. 4 (D and E), smaller increases of phosphorylation were observed in the cells transfected with γ12ΔN5, γ12S2A, and γ12S2E, in comparison with control cells. The reason for these increases will be discussed later (see “Discussion”). To further evaluate the effect of phosphorylation on the activity of γ subunits, we mutated the N-terminal region of a different γ, γ2, to introduce SSK, the phosphorylation motif for PKC (γ2SSK, Fig. 4 A) (9Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar). The γ2SSK was phosphorylated as well as γ12 in cells when judged by32P incorporation, whereas the antibody against p-γ12 weakly recognized phosphorylated γ2SSK, probably due to a low reactivity with the mutant. However, the γ2SSK, which hardly associated with cytoskeletal fractions, neither induced cell rounding nor increased cell migration (Fig. 4), suggesting that phosphorylation is not sufficient for the changes induced by phosphorylated γ12. The weak association of this mutant with cytoskeletal fractions (Fig.4 B) suggested that the association with the cytoskeleton might be essential for enhancing cell migration. The present study showed that transfection of βγ12markedly induced cell rounding and increased cell migration, but transfection of βγ2, βγ5, and βγ7 induced only minor changes, clearly indicating a functional difference among γ subunits. This specific function of γ12 is derived from its unique property of selective phosphorylation by PKC. However, the phosphorylation is not sufficient to account for the activity of γ12, because the γ2SSK mutant, which could be phosphorylated, did not induce these changes in cells. This is in contrast to the result that the activity of the βγ complex containing phosphorylated γ10G4K (γ10SSK) mutant was similar to that of the phosphorylated βγ12 in stimulating type II adenylyl cyclase (10Yasuda H. Lindorfer M.A. Myung C.S. Garrison J.C. J. Biol. Chem. 1998; 273: 21958-21965Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Some part(s) of the structure of γ12 other than the N terminus might also be important for enhancing cell migration or/and some other characteristic of γ12 important for associating with F-actin might be essential for inducing these changes. The present results demonstrated that γ subunits unable to associate with cytoskeletal factions did not increase cell migration, but further experiments are necessary to show whether association with F-actin is essential. Although the effects of γ2, γ5, and γ7 were not very remarkable, decreases of stress fibers (Fig. 1, E–G) and small increases of cell motility (Fig. 3) were observed in cells transfected with these γ subunits. One previous report showed that transfection of β1γ2 did not induce morphological changes in Swiss 3T3 cells (27Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar), but another report indicated that microinjection of β1γ2 reduced stress fibers in CV-1 cells (28Lin H.C. Duncan J.A. Kozasa T. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5057-5060Crossref PubMed Scopus (47) Google Scholar), which is basically consistent with our present observations. These isoforms may have weak activities, but it is also possible that the apparent effects of γ2, γ5, and γ7 are due to βγ12released from endogenous G protein due to displacement by the overexpressed βγ. This speculation might be supported by the evidence that phosphorylation of γ12 slightly increased in the cells transfected with γ subunits other than wild-type γ12 in comparison with control cells (Fig. 4,D and E). Because similar amounts of endogenous γ12 exist in all these cells, the increase of phosphorylated γ12 suggests the replacement of the βγ12 in endogenous G protein and an increase of the free βγ12. We have shown that phosphorylation of γ12 enhances fibroblast migration in response to serum. A variety of growth factors, including platelet-derived growth factor (26Anand-Apte B. Zetter B.R. Viswanathan A. Qiu R.G. Chen J. Ruggieri R. Symons M. J. Biol. Chem. 1997; 272: 30688-30692Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 29Derman M.P. Toker A. Hartwig J.H. Spokes K. Falck J.R. Chen C.S. Cantley L.C. Cantley L.G. J. Biol. Chem. 1997; 272: 6465-6470Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), basic fibroblast growth factor (30Daviet I. Herbert J.M. Maffrand J.P. FEBS Lett. 1990; 259: 315-317Crossref PubMed Scopus (35) Google Scholar), lysophosphatidic acid (26Anand-Apte B. Zetter B.R. Viswanathan A. Qiu R.G. Chen J. Ruggieri R. Symons M. J. Biol. Chem. 1997; 272: 30688-30692Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), and serum (24Cunningham C.C. Stossel T.P. Kwiatkowski D.J. Science. 1991; 251: 1233-1236Crossref PubMed Scopus (259) Google Scholar), were shown to stimulate migration of fibroblasts. Down-regulation or inhibition of PKC abolished the effect of platelet-derived growth factor and basic fibroblast growth factor on cell migration, suggesting the role of PKC in these processes (29Derman M.P. Toker A. Hartwig J.H. Spokes K. Falck J.R. Chen C.S. Cantley L.C. Cantley L.G. J. Biol. Chem. 1997; 272: 6465-6470Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 30Daviet I. Herbert J.M. Maffrand J.P. FEBS Lett. 1990; 259: 315-317Crossref PubMed Scopus (35) Google Scholar). Our previous observation that platelet-derived growth factor, basic fibroblast growth factor, lysophosphatidic acid, and serum stimulated phosphorylation of γ12 in Swiss 3T3 cells (9Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar) strongly suggests the involvement of phosphorylation of γ12 in cell migration stimulated by these growth factors. For phosphorylation of γ12, activation of G proteins is important as well as PKC activation, because free βγ12 was a better substrate for PKC than the trimer form (6Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Lysophosphatidic acid could stimulate Gi and Gq, so that both G protein and PKC could be activated by this agonist. By contrast, receptor tyrosine kinases are well known to stimulate PKC via activation of phospholipase Cγ, and the evidence that basic fibroblast growth factor-stimulated migration of endothelial cells was reduced by pertussis toxin suggests the involvement of G protein activation in this process (31Sa G. Fox P.L. J. Biol. Chem. 1994; 269: 3219-3225Abstract Full Text PDF PubMed Google Scholar). Neptune and Bourne (32Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (246) Google Scholar) and Arai et al. (33Arai H. Tsou C.L. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14495-14499Crossref PubMed Scopus (151) Google Scholar) reported that expressed Gi-coupled receptors, such as D2 dopamine and opioid receptors, induced cell migration, which was prevented by βARKct and Gtα, suggesting involvement of βγ subunits. However, they also suggest that Gi activation is necessary but probably not sufficient for chemotaxis. Activation of PKC to phosphorylate γ12 might be necessary for maximal stimulation of chemotaxis. Cell migration requires dynamic and coordinated disassembly and reassembly of stress fibers and focal adhesions, but the precise mechanisms regulating these processes are not clear (25Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1311) Google Scholar). The present observations that overexpression of γ12 decreases stress fibers and induces cell rounding suggest that γ12 may be involved in disassembly of stress fibers. Because PKC and phosphoinositide 3-kinase, of which activation increases cell migration, have also been found to associate with the cytoskeleton (16Payrastre B. van Bergen en Henegouwen P.M. Breton M. den Hartigh J.C. Plantavid M. Verkleij A.J. Boonstra J. J. Cell Biol. 1991; 115: 121-128Crossref PubMed Scopus (174) Google Scholar,17Blobe G.C. Stribling D.S. Fabbro D. Stabel S. Hannun Y.A. J. Biol. Chem. 1996; 271: 15823-15830Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), the γ12-associated cytoskeleton may be easily accessed by these enzymes, facilitating their interaction. Recent studies showed the role of Rho family GTPases such as Rho, Rac, and Cdc42 in regulation of assembly and organization of the actin cytoskeleton (34Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5242) Google Scholar). In the budding yeast Saccharomyces cerevisiae, βγ complex has been shown to associate with Cdc24, a guanine nucleotide exchange factor for Cdc42, suggesting a cascade from βγ to actin organization via Cdc42 (35Nern A. Arkowitz R.A. Nature. 1998; 391: 195-198Crossref PubMed Scopus (113) Google Scholar). At present, this cascade has not been shown in mammalian cells, but future experiments will elucidate downstream signaling molecules linking βγ12 to cell migration, possibly including this cascade. We thank Drs. M. I. Simon and T. Nukada for supplying the plasmids." @default.
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