The Ras-related GTPase Rac1 Binds Tubulin (*)
- From the (1)Department of Neurochemistry, Institute of Neurology, 1, Wakefield St., London WC1N 1PJ, United Kingdom and
- (2)Glaxo-IMCB Group, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0411
Abstract
The Ras-related Rho family are involved in controlling actin-based changes in cell morphology. Microinjection of Rac1, RhoA,
and Cdc42Hs into Swiss 3T3 cells induces pinocytosis and membrane ruffling, stress fiber formation, and filopodia formation,
respectively. To identify target proteins involved in these signaling pathways cell extracts immobilized on nitrocellulose
have been probed with [
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P]GTP-labeled Rac1, RhoA, and Cdc42Hs. We have identified two 55-kDa brain proteins which bind Rac1 but not RhoA or Cdc42Hs.
These 55-kDa proteins were abundant, had pI values of around 5.5, and could be purified by Q-Sepharose chromatography. The
characteristics on two-dimensional gel analysis suggested the proteins comprised α- and β-tubulin. Indeed, β-tubulin specific
antibodies detected one of the purified 55-kDa proteins. Rac1 bound pure tubulin (purified by cycles of polymerization and
depolymerization) only in the GTP-bound state. The GTPase negative Rac1 point mutants, G12V and Q61L, did not significantly
affect the ability of Rac1 to interact with tubulin while the “effector-site” mutant D38A prevented interaction. These results
suggest that the Rac1-tubulin interaction may play a role in Rac1 function.
Footnotes
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↵* This work was supported in part by the Glaxo-Singapore Research Fund. 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:
- GAP
-
GTPase activating protein
- MES
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4-morpholineethanesulfonic acid
- Pipes
-
1,4-piperazinediethanesulfonic acid
- PAGE
-
polyacrylamide gel electrophoresis
- PAK
-
serine/threonine protein kinases.
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↵2R. Kozma, A. Best, and S. Ahmed, unpublished data.
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↵3A. Best, S. Ahmed, and R. Kozma, unpublished data.
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↵4S. Ahmed, A. Best, E. Prigmore, and R. Kozma, unpublished data.
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- Received May 23, 1995.
- Revision received November 7, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
