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• W2031556543 abstract "We have previously identified a protein factor, named REKS (Ras-dependent Extracellular signal-regulated kinase/Mitogen-activated protein kinase kinase (MEK) Stimulator), which is necessary for Ras-dependent MEK activation. In this study, we attempted to highly purify and characterize REKS. We have highly purified REKS by successive column chromatographies using a cell-free assay system in which REKS activates recombinant extracellular signal-regulated kinase 2 through recombinant MEK in a guanosine 5′-O-(thiotriphosphate) (GTPγS)-Ki-Ras-dependent manner. REKS formed a stable complex with GTPγS-Ras; REKS was coimmunoprecipitated with GTPγS-Ki-Ras or GTPγS-Ha-Ras, but not with GDP-Ki-Ras or GDP-Ha-Ras by an anti-Ras antibody. REKS was adsorbed to a GTPγS-glutathione S-transferase (GST)-Ha-Ras-coupled glutathione-agarose column but not to a GDP-GST-Ha-Ras-coupled glutathione-agarose column and was coeluted with GTPγS-GST-Ha-Ras by reduced glutathione. The minimum molecular mass of REKS was estimated to be about 98 kDa on SDS-polyacrylamide gel electrophoresis. REKS phosphorylated this 98-kDa protein as well as recombinant MEK. REKS was not recognized by any of the anti-c-Raf-1, anti-Mos, and anti-mSte11 antibodies. These results indicate that REKS is a Ras-dependent MEK kinase. We have previously identified a protein factor, named REKS (Ras-dependent Extracellular signal-regulated kinase/Mitogen-activated protein kinase kinase (MEK) Stimulator), which is necessary for Ras-dependent MEK activation. In this study, we attempted to highly purify and characterize REKS. We have highly purified REKS by successive column chromatographies using a cell-free assay system in which REKS activates recombinant extracellular signal-regulated kinase 2 through recombinant MEK in a guanosine 5′-O-(thiotriphosphate) (GTPγS)-Ki-Ras-dependent manner. REKS formed a stable complex with GTPγS-Ras; REKS was coimmunoprecipitated with GTPγS-Ki-Ras or GTPγS-Ha-Ras, but not with GDP-Ki-Ras or GDP-Ha-Ras by an anti-Ras antibody. REKS was adsorbed to a GTPγS-glutathione S-transferase (GST)-Ha-Ras-coupled glutathione-agarose column but not to a GDP-GST-Ha-Ras-coupled glutathione-agarose column and was coeluted with GTPγS-GST-Ha-Ras by reduced glutathione. The minimum molecular mass of REKS was estimated to be about 98 kDa on SDS-polyacrylamide gel electrophoresis. REKS phosphorylated this 98-kDa protein as well as recombinant MEK. REKS was not recognized by any of the anti-c-Raf-1, anti-Mos, and anti-mSte11 antibodies. These results indicate that REKS is a Ras-dependent MEK kinase. INTRODUCTIONThree ras genes encode proteins with Mr values of about 21,000, named Ha-Ras, Ki-Ras, and N-Ras. Ras exhibits GDP/GTP binding and GTPase activities. They have two interconvertible forms: GDP-bound inactive and GTP-bound active forms. The GDP-bound form is converted to the GTP-bound form by the GDP/GTP exchange reaction, which is regulated by GDP/GTP exchange protein, whereas the GTP-bound form is converted to the GDP-bound form by the GTPase reaction, which is regulated by GTPase-activating protein (for review, see (1Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2660) Google Scholar). Four GDP/GTP exchange proteins, including mCdc25(2Martegani E. Vanoni M. Zippel R. Coccetti P. Brambilla R. Ferrari C. Sturani E. Alberghina L. EMBO J. 1992; 11: 2151-2157Crossref PubMed Scopus (188) Google Scholar, 3Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (289) Google Scholar), mSos(4Bowtell D. Fu P. Simon M. Senior P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6511-6515Crossref PubMed Scopus (237) Google Scholar), C3G(5Tanaka S. Morishita T. Hashimoto Y. Hattori S. Nakamura S. Shibuya M. Matuoka K. Takenawa T. Kurata T. Nagashima K. Matsuda M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3443-3447Crossref PubMed Scopus (358) Google Scholar), and Smg GDP dissociation stimulator(6Mizuno T. Kaibuchi K. Yamamoto T. Kawamura M. Sakoda T. Fujioka H. Matsuura Y. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6442-6446Crossref PubMed Scopus (167) Google Scholar), and two GTPase-activating proteins, Ras GTPase-activating protein (7Trahey M. Wong G. Halenbeck R. Rubinfeld B. Martin G.A. Ladner M. Long C.M. Crosier W.J. Watt K. Koths K. McCormick F. Science. 1988; 242: 1697-1700Crossref PubMed Scopus (302) Google Scholar, 8Vogel U.S. Dixon R.A.F. Schaber M.D. Diehl R.E. Marshall M.S. Scolnick E.M. Sigal I.S. Gibbs J.B. Nature. 1988; 335: 90-93Crossref PubMed Scopus (395) Google Scholar) and neurofibromin(9Xu G. O'Connell P. Viskochil D. Cawthon R. Robertson M. Culver M. Dunn D. Stevens J. Gesteland R. White R. Weiss R. Cell. 1990; 62: 599-608Abstract Full Text PDF PubMed Scopus (895) Google Scholar), have thus far been identified. GTP-Ras interacts with its specific target protein. A Ras target molecule has first been identified to be adenylate cyclase in Saccharomyces cerevisiae(10Toda T. Uno I. Ishikawa T. Powers S. Kataoka T. Broek D. Cameron S. Broach J. Matsumoto K. Wigler M. Cell. 1985; 40: 27-36Abstract Full Text PDF PubMed Scopus (706) Google Scholar). However, the target molecule of Ras in higher eukaryotes still remains to be identified. Recent studies indicate that Ras positions upstream of the MEK( 1The abbreviations used are: MEKmitogen-activated protein kinase kinase/ERK kinaseERKextracellular signal-regulated kinaseGTPγSguanosine 5′-O-(thiotriphosphate)GSTglutathione S-transferasePAGEpolyacrylamide gel electrophoresis. )/ERK cascade in Xenopus oocytes (11Leevers S.J. Marshall C.J. EMBO J. 1992; 11: 569-574Crossref PubMed Scopus (382) Google Scholar, 12Shibuya E.K. Polverino A.J. Chang E. Wigler M. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9831-9835Crossref PubMed Scopus (104) Google Scholar, 13Hattori S. Fukuda M. Yamashita T. Nakamura S. Gotoh Y. Nishida E. J. Biol. Chem. 1992; 267: 20346-20351Abstract Full Text PDF PubMed Google Scholar) and mammalian cells(14Thomas S.M. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (502) Google Scholar, 15Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (656) Google Scholar). ERK is phosphorylated and activated by MEK in response to many extracellular signals (for a review, see (16Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1152) Google Scholar). In this signal cascade, MEK is phosphorylated and activated by its kinases (MEK kinases)(17Dent P. Haser W. Haystead T.A.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (496) Google Scholar, 18Howe L.R. Leevers S.J. Gómez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar, 19Kyriakis J.M. App H. Zhang X. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (966) Google Scholar, 20Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (869) Google Scholar, 21Matsuda S. Gotoh Y. Nishida E. J. Biol. Chem. 1993; 268: 3277-3281Abstract Full Text PDF PubMed Google Scholar, 22Nebreda A.R. Hunt T. EMBO J. 1993; 12: 1979-1986Crossref PubMed Scopus (251) Google Scholar, 23Posada J. Yew N. Ahn N.G. Vande Woude G.F. Cooper J.A. Mol. Cell. Biol. 1993; 13: 2546-2553Crossref PubMed Scopus (335) Google Scholar, 24Lange-Carter C.A. Johnson G.L. Science. 1994; 265: 1458-1461Crossref PubMed Scopus (294) Google Scholar). Raf is one of the MEK kinases(17Dent P. Haser W. Haystead T.A.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (496) Google Scholar, 18Howe L.R. Leevers S.J. Gómez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar, 19Kyriakis J.M. App H. Zhang X. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (966) Google Scholar). Raf has been positioned downstream of Ras in many signal transduction pathways. Genetic analyses of eye development and embryonic structure formation in Drosophila and of vulval induction in Caenorhabditis elegans have clarified that Raf functions downstream of Ras(25Dickson B. Sprenger F. Morrison D. Hafen E. Nature. 1992; 360: 600-603Crossref PubMed Scopus (242) Google Scholar, 26Han M. Golden A. Han Y. Sternberg P.W. Nature. 1993; 363: 133-140Crossref PubMed Scopus (191) Google Scholar). Moreover, several groups have reported that c-Raf-1 directly binds to GTP-Ras in a cell-free system (27Moodie S.A. Willumsen B.M. Weber M.J. Wolfman A. Science. 1993; 260: 1658-1661Crossref PubMed Scopus (775) Google Scholar, 28Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (581) Google Scholar, 29Zhang X. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-313Crossref PubMed Scopus (684) Google Scholar, 30Koide H. Satoh T. Nakafuku M. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8683-8686Crossref PubMed Scopus (157) Google Scholar) and to wild-type Ras and dominant active Ras in a yeast two-hybrid system(31Aelst L.V. Barr M. Marcus S. Polverino A. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6213-6217Crossref PubMed Scopus (503) Google Scholar, 32Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1655) Google Scholar). Experiments using antisense c-Raf-1 expression constructs have positioned c-Raf-1 downstream of Ras in proliferation and transformation of NIH/3T3 cells(33Kolch W. Heidecker G. Lioyd P. Rapp U.R. Nature. 1991; 349: 426-428Crossref PubMed Scopus (353) Google Scholar). Recently, it has been shown that c-Raf-1 is activated as a result of its recruitment to the plasma membrane(34Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (877) Google Scholar, 35Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (836) Google Scholar). Although these results strongly suggest that Ras, c-Raf-1, MEK, and ERK function in the same signaling pathway, no evidence has so far been obtained that GTP-Ras directly activates c-Raf-1 in a cell-free system. Mos is a germ cell-specific kinase that is synthesized to initiate maturation of Xenopus oocytes(36Sagata N. Daar I. Oskarsson M. Showalter S.D. Vande Woude G.F. Science. 1989; 245: 643-646Crossref PubMed Scopus (249) Google Scholar). Mos has also been shown to be a MEK kinase(22Nebreda A.R. Hunt T. EMBO J. 1993; 12: 1979-1986Crossref PubMed Scopus (251) Google Scholar, 23Posada J. Yew N. Ahn N.G. Vande Woude G.F. Cooper J.A. Mol. Cell. Biol. 1993; 13: 2546-2553Crossref PubMed Scopus (335) Google Scholar). The relationship between Mos and Ras has not yet been clarified. On the other hand, the cDNA of the mammalian Ste11 homologue, termed mSte11, has been isolated from NIH/3T3 cells by use of the reverse-transcriptase polymerase chain reaction, and mSte11 has been shown to phosphorylate and activate MEK (20Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (869) Google Scholar). Recently, it has been shown that another MEK kinase, immunoprecipitated by an anti-mSte11 antibody, and B-Raf phosphorylate MEK and that the expression of oncogenic Ras in PC12 cells results in the activation of this MEK kinase and B-Raf(24Lange-Carter C.A. Johnson G.L. Science. 1994; 265: 1458-1461Crossref PubMed Scopus (294) Google Scholar). Moreover, it has been shown that phosphatidylinositol 3-kinase directly interacts with GTP-Ras but not with GDP-Ras (37Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1717) Google Scholar) and that GTP-Ras slightly activates phosphatidylinositol 3-kinase in a cell-free system (38Kodaki T. Woscholski R. Hallberg B. Rodriguez-Viciana P. Downward J. Parker P.J. Curr. Biol. 1994; 4: 798-806Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Thus, the direct target molecule of Ras in higher eukaryotes still remains to be fully understood.To identify a direct target molecule of Ras, we have established a cell-free assay system using Xenopus oocyte extract in which Ras activates ERK through MEK(39Itoh T. Kaibuchi K. Masuda T. Yamamoto T. Matsuura Y. Maeda A. Shimizu K. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 975-979Crossref PubMed Scopus (50) Google Scholar). By use of this assay system, we have identified a protein factor, tentatively named REKS (Ras-dependent ERK Kinase Stimulator), for the Ras-dependent MEK activation(39Itoh T. Kaibuchi K. Masuda T. Yamamoto T. Matsuura Y. Maeda A. Shimizu K. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 975-979Crossref PubMed Scopus (50) Google Scholar). Recently, we have modified this cell-free assay system by use of recombinant MEK and recombinant ERK(40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar). We have, moreover, shown that posttranslationally lipid-modified Ras is far more effective on the activation of REKS (41Itoh T. Kaibuchi K. Masuda T. Yamamoto T. Matsuura Y. Maeda A. Shimizu K. Takai Y. J. Biol. Chem. 1993; 268: 3025-3028Abstract Full Text PDF PubMed Google Scholar) and yeast adenylate cyclase (42Horiuchi H. Kaibuchi K. Kawamura M. Matsuura Y. Suzuki N. Kuroda Y. Kataoka T. Takai Y. Mol. Cell. Biol. 1992; 12: 4515-4520Crossref PubMed Scopus (25) Google Scholar) than lipid-unmodified Ras in cell-free assay systems. Kataoka's group (43Kuroda Y. Suzuki N. Kataoka T. Science. 1993; 259: 683-686Crossref PubMed Scopus (119) Google Scholar) has also reported the similar results for the Ras-dependent activation of yeast adenylate cyclase. It has also been reported that lipid modification of Ras is necessary for the activation of c-Raf-1 in insect cells overexpressing Ras and c-Raf-1(44Kikuchi A. Williams L.T. J. Biol. Chem. 1994; 269: 20054-20059Abstract Full Text PDF PubMed Google Scholar).In these earlier reports, it has not been examined, however, whether GTP-Ki-Ras or GTP-Ha-Ras directly interacts with REKS, whether REKS is a protein kinase, or whether REKS is the same as or different from other MEK kinases including c-Raf-1, Mos, and mSte11. In the present study, we have first attempted to highly purify REKS and have addressed these important issues by use of the purified sample.EXPERIMENTAL PROCEDURESMaterials and ChemicalsPost-translationally lipid-modified Ki-Ras and Ha-Ras were purified from the membrane fraction of insect cells, which were infected with baculovirus carrying the cDNAs of Ki-Ras and Ha-Ras, respectively(6Mizuno T. Kaibuchi K. Yamamoto T. Kawamura M. Sakoda T. Fujioka H. Matsuura Y. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6442-6446Crossref PubMed Scopus (167) Google Scholar). GTPγS-Ki-Ras, GTPγS-Ha-Ras, GDP-Ki-Ras, and GDP-Ha-Ras were prepared as described previously(6Mizuno T. Kaibuchi K. Yamamoto T. Kawamura M. Sakoda T. Fujioka H. Matsuura Y. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6442-6446Crossref PubMed Scopus (167) Google Scholar). The cDNA of mouse MEK was cloned as described(40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar). The kinase-negative MEK was generated by the site-directed mutagenesis of Lys97 to Trp(45Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1574-1579Google Scholar). Recombinant wild-type MEK, kinase-negative MEK, ERK2, and Ha-Ras were purified from overexpressing Escherichia coli as GST fusion proteins using a glutathione-Sepharose 4B column as described(46Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5035) Google Scholar). An anti-Ras monoclonal antibody (RASK-4) was kindly provided by H. Shiku (Nagasaki University, Nagasaki, Japan). An anti-Xenopus c-Raf-1 monoclonal antibody was kindly provided by L.T. Williams (University of California, San Francisco, CA). An anti-Xenopus Mos polyclonal antibody was kindly provided by N. Sagata (Kurume University, Fukuoka, Japan). An anti-Xenopus ERK polyclonal antibody was kindly provided by E. Nishida (University of Kyoto, Kyoto, Japan). An anti-MEK polyclonal antibody was generated as described(40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar). An anti-mSte11 polyclonal antibody was purchased from Transduction Laboratories (Lexington, KY). Anti-A-Raf and anti-B-Raf polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Myelin basic protein was purchased from Sigma.REKS AssayREKS activity was assayed by measuring the phosphorylation of myelin basic protein by recombinant GST-ERK2 in the presence of recombinant GST-MEK as described(40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar). GTPγS-Ki-Ras, GTPγS-Ha-Ras, GDP-Ki-Ras, or GDP-Ha-Ras was added as indicated. Namely, a REKS sample to be assayed was incubated for 10 min at 30°C in a final volume of 50 μl containing 20 mM Tris/HCl, pH 8.0, 120 μM ATP, 10 mM MgCl2, 6 mM EGTA, 80 nM recombinant GST-MEK, and 100 nM GTPγS-Ki-Ras, GTPγS-Ha-Ras, GDP-Ki-Ras, or GDP-Ha-Ras. After the 10-min incubation, 10 μl of 3 μM recombinant GST-ERK2 was added. The reaction mixture was incubated for additional 20 min at 30°C. Then, 20 μl of a reaction mixture containing 20 mM Tris/HCl, pH 8.0, 100 μM [γ-32P]ATP (600 cpm/pmol), 220 μM myelin basic protein, 10 mM MgCl2, and 6 mM EGTA was added. Incubation was continued for another 10 min at 30°C, after which 30 μl of the reaction mixture was spotted onto a phosphocellulose paper sheet. The sheet was washed with 75 mM phosphoric acid, and the radioactivity was measured by liquid scintillation spectrometry.Preparation of the Cytosol of Xenopus EggsEggs were obtained from fully mature Xenopus laevis females as described(39Itoh T. Kaibuchi K. Masuda T. Yamamoto T. Matsuura Y. Maeda A. Shimizu K. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 975-979Crossref PubMed Scopus (50) Google Scholar, 47Kishi K. Sasaki T. Kuroda S. Itoh T. Takai Y. J. Cell Biol. 1993; 120: 1187-1195Crossref PubMed Scopus (308) Google Scholar). Eggs, dejellied with cysteine and washed with modified modified Ringer's solution containing 5.0 mM HEPES/NaOH, pH 7.8, 0.1 M NaCl, 2.0 mM KCl, 1.0 mM MgSO4, 2.0 mM CaCl2, and 0.1 mM EDTA were activated by electric shock as described (48Gerhart J. Wu M. Kirschner M. J. Cell Biol. 1984; 98: 1247-1255Crossref PubMed Scopus (385) Google Scholar) to make them enter into interphase and inactivate endogenous ERK and MEK activities(49Ferrell Jr., J.E. Wu M. Gerhart J.C. Martin G.S. Mol. Cell. Biol. 1991; 11: 1965-1971Crossref PubMed Scopus (273) Google Scholar, 50Shibuya E.K. Boulton T.G. Cobb M.H. Ruderman J.V. EMBO J. 1992; 11: 3963-3975Crossref PubMed Scopus (141) Google Scholar). The cytosol of activated eggs was obtained by centrifugation as described previously(40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar).Partial Purification of REKS from the Cytosol by Mono Q Column ChromatographyThe cytosol of activated eggs (24 mg of protein, 6 ml) was applied to a Mono Q column (0.5 × 5 cm) equilibrated with Buffer A containing 20 mM Tris/HCl, pH 8.0, 1 mM dithiothreitol, 5 mM MgCl2, 10 mM EGTA, and 10 μM (p-amidinophenyl)methanesulfonyl fluoride. After the column was washed with 50 ml of Buffer A, elution was performed with a 15-ml linear gradient of NaCl (0-0.5 M) in Buffer A, and fractions of 1 ml each were collected. An aliquot of each fraction (15 μl) was assayed for the REKS activity.Coimmunoprecipitation of REKS with GTPγS-Ras by an Anti-Ras AntibodyThe peak 2 (67.5 μl) of the Mono Q column chromatography was incubated with an anti-Ras antibody coupled to Protein A-Sepharose beads (Pharmacia Biotech Inc.) in the presence of [35S]GTPγS-Ki-Ras, [35S]GTPγS-Ha-Ras, [3H]GDP-Ki-Ras, or [3H]GDP-Ha-Ras (22.5 μl) at the final concentration of 500 nM. After the incubation for 30 min at 4°C, the immunocomplex was precipitated and washed 3 times with Buffer A containing 0.5% Nonidet P-40. The precipitate and the supernatant were assayed for the REKS activity. The amounts of Ki-Ras or Ha-Ras in the precipitate and the supernatant were determined by measuring the radioactivity of [35S]GTPγS or [3H]GDP bound to Ki-Ras or Ha-Ras.GTPγS-GST-Ha-Ras-coupled Glutathione-Agarose Column Affinity Chromatography of REKSTo make GTPγS-GST-Ha-Ras-coupled or GDP-GST-Ha-Ras-coupled glutathione-agarose column, GTPγS-GST-Ha-Ras or GDP-GST-Ha-Ras was prepared as described(6Mizuno T. Kaibuchi K. Yamamoto T. Kawamura M. Sakoda T. Fujioka H. Matsuura Y. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6442-6446Crossref PubMed Scopus (167) Google Scholar). GTPγS-GST-Ha-Ras or GDP-GST-Ha-Ras (5 nmol each) was separately applied to a glutathione-agarose column (200 μl) preequilibrated with 3.6 ml of Buffer B containing 20 mM Tris/HCl, pH 8.0, 1 mM dithiothreitol, 5 mM MgCl2, and 1 mM EGTA(46Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5035) Google Scholar), and the column was washed with 5.0 ml of Buffer B. About 95% of GTPγS-GST-Ha-Ras or GDP-GST-Ha-Ras was adsorbed to the column. The REKS sample applied to this column was purified by the Mono S column chromatography as described (40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar) except that the cytosol of activated eggs (160 mg of protein, 40 ml) was applied and that the elution was performed with a 15-ml linear gradient of NaCl (0-1.0 M). After MgCl2 and GTPγS or GDP were added to this REKS sample (3 ml) to give the final concentrations of 15 mM and 10 μM, respectively, in order to prevent the dissociation of guanine nucleotides from GST-Ha-Ras during the application of the REKS sample, the REKS sample was applied to the GTPγS-GST-Ha-Ras-coupled glutathione-agarose column or the GDP-GST-Ha-Ras-coupled glutathione-agarose column. After the column was washed with 4 ml of Buffer B, elution was performed with 600 μl of Buffer B containing 20 mM reduced glutathione. The eluate fraction of the GTPγS-GST-Ha-Ras-coupled glutathione-agarose column chromatography was used for the experiments as affinity-purified REKS.Phosphorylation of MEK by REKSAffinity-purified REKS (5 μl) was supplemented with 200 ng of each of GST alone, wild-type GST-MEK, or kinase-negative GST-MEK (40 μl each). The reaction was started by adding the mixture (5 μl) containing 20 mM Tris/HCl, pH 8.0, 200 μM [γ-32P]ATP (5,000 cpm/pmol), and 15 mM MgCl2 and continued for 10 min at 30°C. The reaction was terminated by the addition of Laemmli's sample buffer (25 μl) and subjected to SDS-PAGE. The radioactivity of 32P incorporated into MEK was detected by bioimaging analyzer BAS2000 (Fujix, Tokyo) as described previously (39Itoh T. Kaibuchi K. Masuda T. Yamamoto T. Matsuura Y. Maeda A. Shimizu K. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 975-979Crossref PubMed Scopus (50) Google Scholar).Phosphorylation of Affinity-purified REKSAffinity-purified REKS (50 μl) was incubated for 2 min at 30°C with the reaction mixture (5 μl) containing 20 mM Tris/HCl, pH 8.0, 110 μM [γ-32P]ATP (50,000 cpm/pmol), 1 mM dithiothreitol, 5 mM MgCl2, and 1 mM EGTA. The reaction was stopped by the addition of Laemmli's sample buffer (27.5 μl) and was subjected to SDS-PAGE followed by autoradiography.Other ProceduresSDS-PAGE was performed by the method of Laemmli(51Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). Protein concentrations were determined with bovine serum albumin as a standard protein by the method of Bradford(52Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar). Immunoblot was carried out as described(40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar).RESULTSPartial Purification of REKS by Mono Q Column ChromatographyThe cytosol of activated eggs was subjected to a Mono Q column chromatography. When each fraction was assayed for the REKS activity, three peaks (peaks 1-3) were detected in the presence of both recombinant MEK and recombinant ERK2 (Fig. 1A). The activity of only peak 2 was enhanced by GTPγS-Ki-Ras but not by GDP-Ki-Ras. In the presence of recombinant ERK2 alone, two peaks were detected in the same fractions as those of peaks 1 and 3 of Fig. 1A (Fig. 1B). The activities of both peaks were independent of GTPγS-Ki-Ras. In the absence of recombinant MEK and recombinant ERK2, only one peak was detected at the same position as that of peak 3 of Fig. 1A (Fig. 1C). This peak was also independent of GTPγS-Ki-Ras. The similar results were obtained when Ha-Ras was used instead of Ki-Ras (data not shown). Immunoblot analysis of MEK and ERK revealed that the immunoreactivities of MEK and ERK were detected in fractions 4-6 and 10-12, respectively. These results indicate that peaks 1 and 2 are MEK and REKS, respectively, and that peak 3 contains ERK and an unknown myelin basic protein kinase. These results are essentially consistent with our earlier observations that REKS is required for the Ras-dependent activation of ERK2 through MEK(39Itoh T. Kaibuchi K. Masuda T. Yamamoto T. Matsuura Y. Maeda A. Shimizu K. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 975-979Crossref PubMed Scopus (50) Google Scholar, 40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar).Coimmunoprecipitation of REKS with GTPγS-Ras by an Anti-Ras AntibodyTo show interaction of REKS with GTPγS-Ras, we examined whether REKS is coimmunoprecipitated with GTPγS-Ki-Ras or GTPγS-Ha-Ras by an anti-Ras antibody. GTPγS-Ki-Ras was incubated with the peak 2 of the Mono Q column chromatography and anti-Ras antibody-coupled Protein A-agarose beads. After the incubation for 30 min at 4°C, the mixture was centrifuged. The REKS activity was recovered in the precipitate but not in the supernatant (Table 1). GTPγS-Ki-Ras was also mostly recovered in the precipitate. In contrast, when the similar experiment was done with GDP-Ki-Ras, GDP-Ki-Ras was mostly recovered in the precipitate, whereas the REKS activity was recovered in the supernatant but not in the precipitate. The similar results were obtained when Ha-Ras was used instead of Ki-Ras (data not shown). We have purified REKS by the Mono S column chromatography as described previously(40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar). The similar results were obtained when REKS purified by the Mono S column chromatography was used instead of that purified by the Mono Q column chromatography (data not shown).Tabled 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab GTPγS-GST-Ha-Ras-coupled Glutathione-Agarose Column Affinity Chromatography of REKSTo obtain another line of evidence for interaction of REKS with GTPγS-Ras, we first prepared a large amount of REKS by a large scale of a Mono S column chromatography. By use of this sample, we examined whether REKS interacts with GTPγS-GST-Ha-Ras coupled to a glutathione-agarose column. In this large scale experiment, 40 ml of the cytosol of activated eggs was subjected to a Mono S column chromatography under the same conditions as described (40Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar), except that the elution was performed with a 15-ml linear gradient of NaCl (0-1.0 M). In the large scale of the Mono S column chromatography, the REKS activity became mostly Ras-independent for an unknown reason. However, the activity still absolutely required both recombinant MEK and recombinant ERK2. This REKS sample was collected and subjected to the GTPγS-GST-Ha-Ras-coupled glutathione-agarose column. The column was washed with Buffer B and was eluted by reduced glutathione. About 60% of the REKS activity was adsorbed to the column and was eluted by reduced glutathione (Fig. 2). This eluate fraction was used for the experiments as affinity-purified REKS. About 80% of GTPγS-GST-Ha-Ras was also eluted by reduced glutathione (data not shown). About 40% of the REKS activity was detected in the pass fraction. This reason was not known, but REK" @default.
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• W2031556543 title "Purification and Characterization of REKS from Xenopus Eggs" @default.
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