Purification and characterization of REKS from Xenopus eggs. Identification of REKS as a Ras-dependent mitogen-activated protein kinase kinase kinase

• Published in: The Journal of biological chemistry Vol. 270; no. 6; pp. 2460 - 2465
• Main Authors: Kuroda, S, Shimizu, K, Yamamori, B, Matsuda, S, Imazumi, K, Kaibuchi, K, Takai, Y
• Format: Journal Article
• Language: English
• Published: United States 10.02.1995
• Subjects: Animals; Calcium-Calmodulin-Dependent Protein Kinases - isolation & purification; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Chromatography, Affinity; Chromatography, Ion Exchange; Extracellular Signal-Regulated MAP Kinases; Glutathione; Guanosine 5'-O-(3-Thiotriphosphate); MAP Kinase Kinase Kinases; Oocytes; Phosphorylation; Precipitin Tests; Protein-Serine-Threonine Kinases - metabolism; Xenopus; Xenopus Proteins
• ISSN: 0021-9258
• DOI: 10.1074/jbc.270.6.2460

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) (GTP gamma S)-Ki-Ras-dependent manner. REKS formed a stable complex with GTP gamma S-Ras; REKS was coimmunoprecipitated with GTP gamma S-Ki-Ras or GTP gamma S-Ha-Ras, but not with GDP-Ki-Ras or GDP-Ha-Ras by an anti-Ras antibody. REKS was absorbed to a GTP gamma S-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 GTP gamma S-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.