TY - JOUR
T1 - Purification and characterization of REKS from Xenopus eggs. Identification of REKS as a Ras-dependent mitogen-activated protein kinase kinase kinase
AU - Kuroda, S.
AU - Shimizu, K.
AU - Yamamori, B.
AU - Matsuda, S.
AU - Imazumi, K.
AU - Kaibuchi, K.
AU - Takai, Y.
PY - 1995
Y1 - 1995
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.270.6.2460
DO - 10.1074/jbc.270.6.2460
M3 - Article
C2 - 7852306
AN - SCOPUS:0028847672
VL - 270
SP - 2460
EP - 2465
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 6
ER -