Oncogenic Ras leads to Rho activation by activating the mitogen-activated protein kinase pathway and decreasing Rho-GTPase-activating protein activity.
Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity approximately 2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21(WAF1/CIP1) promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP.
Chen
Jeffrey C
JC
Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California 92093-0652, USA.
Zhuang
Shunhui
S
Nguyen
Tony H
TH
Boss
Gerry R
GR
Pilz
Renate B
RB
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021381:Protein Transport; D020703:ras GTPase-Activating Proteins / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D020690:GTPase-Activating Proteins; D020703:ras GTPase-Activating Proteins; C085379:rho GTPase-activating protein; D006153:Guanosine Diphosphate; D000255:Adenosine Triphosphate; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins; D020742:rhoA GTP-Binding Protein