Analysis of RhoA-binding Proteins Reveals an Interaction Domain Conserved in Heterotrimeric G Protein β Subunits and the Yeast Response Regulator Protein Skn7*
- From the ‡Transcription Laboratory, Imperial Cancer Research Fund Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3PX and ‖Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
Abstract
To identify potential RhoA effector proteins, we conducted a two-hybrid screen for cDNAs encoding proteins that interact with a Gal4-RhoA.V14 fusion protein. In addition to the RhoA effector ROCK-I we identified cDNAs encoding Kinectin, mDia2 (a p140 mDia-related protein), and the guanine nucleotide exchange factor, mNET1. ROCK-I, Kinectin, and mDia2 can bind the wild type forms of both RhoA and Cdc42 in a GTP-dependent manner in vitro. Comparison of the ROCK-I and Kinectin sequences revealed a short region of sequence homology that is both required for interaction in the two-hybrid assay and sufficient for weak interaction in vitro. Sequences related to the ROCK-I/Kinectin sequence homology are present in heterotrimeric G protein β subunits and in theSaccharomyces cerevisiae Skn7 protein. We show that β2 and Skn7 can interact with mammalian RhoA and Cdc42 and yeast Rho1, both in vivo and in vitro. Functional assays in yeast suggest that the Skn7 ROCK-I/Kinectin homology region is required for its function in vivo.
Footnotes
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↵* This work was funded in part by the Imperial Cancer Research Fund and the Medical Research Council of Great Britain.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.
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↵§ Supported in part by a postdoctoral fellowship from the Howard Hughes Medical Institute. To whom correspondence should be addressed. Present address: UCSF Cancer Center, Box 0128, San Francisco, CA 94143-0128. Tel.: 415-502-1713; Fax: 415-502-3179; E-mail:alberts{at}cc.ucsf.edu.
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↵‖ Supported in part by an International Travelling Research Fellowship from the Wellcome Trust.
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↵** Howard Hughes Medical Insitute International Research Scholar.
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↵1 The abbreviations used are: MBS, myosin-binding subunit; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GEF, guanine nucleotide exchange factor.
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↵2 Flynn, P., Mellor, H., Palmer, R., Panayotou, G., and Parker, P. J. (1998) J. Biol. Chem. 273,2698–2705.
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↵3 A. S. Alberts and R. Treisman, unpublished data.
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↵4 A. S. Alberts and R. Treisman, manuscript in preparation.
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↵5 A. S. Alberts, unpublished data.
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↵6 N. Bouquin and L. H. Johnston, unpublished data.
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↵7 N. Bouquin and L. H. Johnston, manuscript in preparation.
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↵8 N. Bouquin, unpublished data.
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↵9 J. Sgouros, unpublished observations.
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- Received November 20, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
