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

S. Kuroda, K. Shimizu, B. Yamamori, S. Matsuda, K. Imazumi, K. Kaibuchi, Y. Takai

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2460-2465
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number6
DOIs
Publication statusPublished - 1995
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Purification and characterization of REKS from Xenopus eggs. Identification of REKS as a Ras-dependent mitogen-activated protein kinase kinase kinase'. Together they form a unique fingerprint.

Cite this