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