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) (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.
Kuroda
S
S
Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Japan.
Shimizu
K
K
Yamamori
B
B
Matsuda
S
S
Imazumi
K
K
Kaibuchi
K
K
Takai
Y
Y
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D048049:Extracellular Signal-Regulated MAP Kinases; D005978:Glutathione; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020930:MAP Kinase Kinase Kinases; D009865:Oocytes; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins
D029867:Xenopus Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; C499530:REKS protein, Xenopus; C089319:Ras-dependent mitogen-activated protein kinase kinase kinase; D005978:Glutathione