Identification and Characterization of a Novel Protein Interacting with Ral-binding Protein 1, a Putative Effector Protein of Ral*
- From the Department of Biochemistry, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan
Abstract
Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral.
Footnotes
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↵* This work was supported by grants-in-aid for scientific research (B) and for scientific research on priority areas from the Ministry of Education, Science, and Culture, Japan (1996, 1997) and by grants from Yamanouchi Foundation for Research on Metabolic Disorders (1996), Fukuyama Transporting Shibuya Longevity and Health Foundation (1996), and Kato Memorial Bioscience Foundation (1997).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF010233.
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↵‡ To whom all 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.
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↵1 The abbreviations used are: G protein, GTP-binding protein; RalGDS, Ral GDP dissociation stimulator; GAP, GTPase-activating protein; RalBP1, Ral-binding protein 1; EGF, epidermal growth factor; SH3, Src homology 3; GST, glutathioneS-transferase; HA, hemagglutinin 1; PCR, polymerase chain reaction; GTPγS, guanosine-5′-0-(3-thiotriphosphate); MBP, maltose-binding protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; kb, kilobase(s).
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- Received August 22, 1997.
- Revision received October 16, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
