Characterization of Ral GDP Dissociation Stimulator-like (RGL) Activities to Regulate c-fos Promoter and the GDP/GTP Exchange of Ral

doi:10.1074/jbc.272.16.10483

Characterization of Ral GDP Dissociation Stimulator-like (RGL) Activities to Regulate c-fos Promoter and the GDP/GTP Exchange of Ral*

From the Department of Biochemistry, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan and the^§Department of Virology II, National Institute of Health, 1-23-1 Toyama, Shinjyuku-ku, Tokyo 162, Japan

Abstract

Ral GDP dissociation stimulator-like (RGL) has been identified to be a possible effector protein of Ras. RGL shares 50% amino acid identity with Ral GDP dissociation stimulator and contains the CDC25-like domain in the central region and the Ras-interacting domain in the C-terminal region. Since the modes of activation and action of RGL have not yet been clarified, in this paper we have analyzed the functions of RGL. In COS cells, RGL interacted with Ras^G12V/E37G (a Ras mutant in which Gly-12 and Glu-37 were changed to Val and Gly, respectively) which failed to bind to Raf, but not with Ras^G12V/T35S which bound to Raf. Raf did not inhibit the binding of RGL to Ras^G12V/E37G under the condition that Raf inhibited that of RGL to Ras^G12V. Expression of either RGL or Raf into NIH3T3 cells slightly activated c-fos promoter, while coexpression of both proteins greatly stimulated the c-fos promoter activity. RGL stimulated the GDP/GTP exchange of Ral and this action was enhanced by the post-translational modification of Ral. However, RGL was not active on Ras, Rac, CDC42, Rap, or Rho. Furthermore, this action of RGL to stimulate the GDP/GTP exchange of Ral was dependent on Ras in COS cells. These results suggest that RGL constitutes another Ras-signaling pathway which is distinct from the Raf pathway and indicate that the RGL pathway regulates the c-fos promoter activity and the GDP/GTP exchange of Ral.

Footnotes

↵* This work was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture, Japan (1995, 1996), by grants from the Yamanouchi Foundation for Research on Metabolic Disorders (1996), Kanae Foundation of Research for New Medicine (1995), Japan Research Foundation for Clinical Pharmacology (1995), Uehara Memorial Foundation (1995), and ONO Medical Research Foundation (1995).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Contributed equally to the results of this report.
↵¶ To whom correspondence should be addressed: Dept. of Biochemistry, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan. Tel.: 81-82-257-5130; Fax: 81-82-257-5134; E-mail: akikuchi{at}mcai.med.hiroshima-u.ac.jp.
↵1 The abbreviations used are: G protein, GTP-binding protein; RalGDS, Ral GDP dissociation stimulator; RGL, RalGDS-like; MAP kinase, mitogen-activated protein kinase; RID, Ras-interacting domain; HA, hemagglutinin; GTPγS, guanosine 5′-O-(thiotriphosphate); PCR, polymerase chain reaction; GST, glutathione S-transferase; MBP, maltose-binding protein; DTT, dithiothreitol; CHAPS, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate; GDI, GDP dissociation inhibitor; Rlf, RalGDS-like factor.
- Received August 23, 1996.
- Revision received December 24, 1996.