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Purification and characterization of REKS from Xenopus eggs. Identification of REKS as a Ras-dependent
S Kuroda1, K Shimizu, B Yamamori
1Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Japan.
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) (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.
Insights
Researchers purified REKS, a protein essential for Ras-dependent signaling. REKS directly activates MEK, acting as a Ras-dependent MEK kinase.
Area of Science:
- Molecular Biology
- Cell Signaling
- Protein Biochemistry
Background:
- Ras signaling pathways are crucial for cell growth and differentiation.
- Mitogen-activated protein kinase kinase (MEK) is a key component in Ras-mediated signaling.
- REKS (Ras-dependent Extracellular signal-regulated kinase/Mitogen-activated protein kinase kinase (MEK) Stimulator) was previously identified as necessary for Ras-dependent MEK activation.
Purpose of the Study:
- To highly purify and characterize the protein factor REKS.
- To elucidate the mechanism by which REKS mediates Ras-dependent MEK activation.
Main Methods:
- Successive column chromatography for protein purification.
- Cell-free assay system utilizing recombinant proteins.
- Coimmunoprecipitation assays.
- Glutathione S-transferase (GST) pull-down assays.
- SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Main Results:
- REKS was highly purified and found to be approximately 98 kDa.
- REKS activated recombinant extracellular signal-regulated kinase 2 (ERK2) through recombinant MEK in a GTP gamma S-Ki-Ras-dependent manner.
- REKS formed a stable complex with GTP gamma S-Ras and was coimmunoprecipitated with GTP gamma S-Ki-Ras and GTP gamma S-Ha-Ras.
- REKS bound to GTP gamma S-Ras but not GDP-Ras, indicating Ras-GTP binding specificity.
- REKS phosphorylated both itself (98-kDa protein) and recombinant MEK.
- REKS did not react with antibodies against c-Raf-1, Mos, or mSte11.
Conclusions:
- REKS is a novel Ras-dependent MEK kinase.
- REKS plays a direct role in the Ras/MEK signaling cascade.
- The characterization of REKS provides new insights into the regulation of MAPK pathways.