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-depend ent Extracellular signal-regulated kinase/Mitogen-activated protein ki nase kinase (MEK) Stimulator), which is necessary for Ras-dependent ME K activation. In this study, we attempted to highly purify and charact erize REKS. We have highly purified REKS by successive column chromato graphies using a cell-free assay system in which REKS activates recomb inant extracellular signal-regulated kinase 2 through recombinant MER 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 co immunoprecipitated 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 ad sorbed to a GTP gamma S-glutathione S-transferase (GST)-Ha-Ras-coupled glutathione-agarose column but not to a GDP-GST-Ha-Ras-coupled glutat hione-agarose column and was coeluted with GTP gamma S-GST-Ha-Ras by r educed glutathione. The minimum molecular mass of REKS was estimated t o be about 98 kDa on SDS-polyacrylamide gel electrophoresis. REKS phos phorylated this 98-kDa protein as well as recombinant MEK. REKS was no t recognized by any of the anti-c-Raf-1, anti-Mos, and anti-mSte11 ant ibodies. These results indicate that REKS is a Ras-dependent MEK kinas e.