RhoE binds to ROCK I and inhibits downstream signaling.
RhoE belongs to the Rho GTPase family, the members of which control actin cytoskeletal dynamics. RhoE induces stress fiber disassembly in a variety of cell types, whereas RhoA stimulates stress fiber assembly. The similarity of RhoE and RhoA sequences suggested that RhoE might compete with RhoA for interaction with its targets. Here, we show that RhoE binds ROCK I but none of the other RhoA targets tested. The interaction of RhoE with ROCK I was confirmed by coimmunoprecipitation of the endogenous proteins, and the two proteins colocalized on the trans-Golgi network in COS-7 cells. Although RhoE and RhoA were not able to bind ROCK I simultaneously, RhoE bound to the amino-terminal region of ROCK I encompassing the kinase domain, at a site distant from the carboxy-terminal RhoA-binding site. Overexpression of RhoE inhibited ROCK I-induced stress fiber formation and phosphorylation of the ROCK I target myosin light chain phosphatase. These data suggest that RhoE induces stress fiber disassembly by directly binding ROCK I and inhibiting it from phosphorylating downstream targets.
Riento
Kirsi
K
Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, London, United Kingdom.
Guasch
Rosa M
RM
Garg
Ritu
R
Jin
Boquan
B
Ridley
Anne J
AJ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D020741:rho GTP-Binding Proteins; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; C085379:rho GTPase-activating protein; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; C513341:Rnd3 protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein