Purification and Characterization of REKS from Xenopus Eggs
IDENTIFICATION OF REKS AS A Ras-DEPENDENT MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE (*)
- Shinya Kuroda(1)(2)(§),
- Kazuya Shimizu(1)(2)(§),
- Bunpei Yamamori(1)(2)(§),
- Shuji Matsuda(1)(2)(§),
- Katsunori Imazumi(1)(2)(§),
- Kozo Kaibuchi(2)(3)(¶) and
- Yoshimi Takai(1)(2)(§)(3)(**)
- From the (1)Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565,
- the (2)Department of Biochemistry, Kobe University School of Medicine, Kobe 650, and
- the (3)Department of Cell Physiology, National Institute of Physiological Sciences, Okazaki 444, Japan
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↵¶ Present address: Div. of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-01, Japan.
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↵§ Present address: Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan.
- ** To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan. Tel.: 81-6-879-3410; Fax: 81-6-879-3419; ytakai{at}molbio.med.osaka-u.ac.jp
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
S)-Ki-Ras-dependent manner. REKS formed a stable complex with GTP
S-Ras; REKS was coimmunoprecipitated with GTP
S-Ki-Ras or GTP
S-Ha-Ras, but not with GDP-Ki-Ras or GDP-Ha-Ras by an anti-Ras antibody. REKS was adsorbed to a GTP
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
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.
Footnotes
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↵* This investigation was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture, Japan(1993, 1994), by grants-in-aid for abnormalities in hormone receptor mechanisms and for aging and health from the Ministry of Health and Welfare, Japan(1993, 1994), and by grants from the Yamanouchi Foundation for Research on Metabolic Disease(1993, 1994), the Research Program on Cell Calcium Signal in the Cardiovascular System(1993), Setsuro Fujii Memorial, the Osaka Foundation for Promotion of Fundamental Medical Research(1993), Uehara Memorial Foundation(1994), and Nissan Science Foundation(1994). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- MEK
-
mitogen-activated protein kinase kinase/ERK kinase
- ERK
-
extracellular signal-regulated kinase
- GTP
S -
guanosine 5′-O-(thiotriphosphate)
- GST
-
glutathione S-transferase
- PAGE
-
polyacrylamide gel electrophoresis.
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- Received October 4, 1994.
- Revision received November 8, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
