RhoA effector mutants reveal distinct effector pathways for cytoskeletal reorganization, SRF activation and transformation.
The RhoA GTPase regulates diverse cellular processes including cytoskeletal reorganization, transcription and transformation. Although many different potential RhoA effectors have been identified, including two families of protein kinases, their roles in RhoA-regulated events remain unclear. We used a genetic screen to identify mutations at positions 37-42 in the RhoA effector loop that selectively disrupt effector binding, and used these to investigate the role of RhoA effectors in the formation of actin stress fibres, activation of transcription by serum response factor (SRF) and transformation. Interaction with the ROCK kinase and at least one other unidentified effector is required for stress fibre formation. Signalling to SRF by RhoA can occur in the absence of RhoA-induced cytoskeletal changes, and did not correlate with binding to any of the effectors tested, indicating that it may be mediated by an unknown effector. Binding to ROCK-I, but not activation of SRF, correlated with the activity of RhoA in transformation. The effector mutants should provide novel approaches for the functional study of RhoA and isolation of effector molecules involved in specific signalling processes.
Sahai
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Transcription Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
Alberts
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AS
Treisman
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D016475:3T3 Cells; D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D002471:Cell Transformation, Neoplastic; D004268:DNA-Binding Proteins / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D009154:Mutation / Q000502:physiology*; D009687:Nuclear Proteins / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D015398:Signal Transduction / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D054460:rho-Associated Kinases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein