The Ras-related GTPase Rac1 binds tubulin.
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 [gamma-32P]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 alpha- and beta-tubulin. Indeed, beta-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.
Best
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Department of Neurochemistry, Institute of Neurology, 1, Wakefield St., London WC1N 1PJ, United Kingdom.
Ahmed
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Kozma
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Lim
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism; D010873:Pinocytosis; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013737:Testis / Q000378:metabolism; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein; D020703:ras GTPase-Activating Proteins; D020742:rhoA GTP-Binding Protein
D018797:Cell Cycle Proteins; D020690:GTPase-Activating Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein