Oncogenic Ras Leads to Rho Activation by Activating the Mitogen-activated Protein Kinase Pathway and Decreasing Rho-GTPase-activating Protein Activity

Abstract

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 ϳ2-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.

Inhibition of Geranylgeranylation Mediates the Effects of 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase Inhibitors on Microglia

“…Activation of ERK1/2 during periods of Rho family inhibition, although not widely reported, has been described. Decreases in the GTP-bound forms of Rac1 and Cdc42 are associated with ERK activation in fibroblasts (48), RhoB inhibits constitutive activation of ERK in tumor cells (49), and inhibition of RhoA stimulates ERK in MCF-7 breast cancer cells (50). Particularly relevant to the present results, a recent study found that inhibition of Rho up-regulated TNF-␣ in macrophages and that this effect was mediated by ERK1/2 activation (43).…”

Oncogenic H-Ras V12 promotes anchorage-independent cytokinesis in human fibroblasts

“…Thus, active H-Ras might abrogate checkpoints sensing anchorage and/or cytoskeletal organization in addition to promoting cell proliferation. One mediator of H-Ras-induced cytoskeletal rearrangements is RhoA, which is activated upon overexpression of activated H-Ras (27,28). RhoA is also the only member of the Rhoprotein family of small GTPases that is required for cytokinesis in mammalian cells (29,30).…”

Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibitors

“…Rho standards were purified from bacteria to >90% homogeneity as judged by PAGE/Coomassie staining, and were quantified by comparison to bovine serum albumin. Glutathione-S-transferase(GST)-tagged rhotekin Rho binding domain (RBD) was purified from bacteria as described previously [3,17]. Vectors encoding Rho A and B with the N-terminal epitope tag EEEEYMPME (EE-tag) were constructed as described [18]; vectors encoding hemagglutinin (HA)-tagged RhoC (wild type and V14) were purchased from the University of Misouri-Rolla cDNA Resource Center.…”

“…To determine the activation state of each Rho isoform separately, we performed the assay as described by Ren et al [17], assessing the amount of GTP-bound RhoA, B, or C by Western blotting. To quantify absolute amounts of GTP and GTP+GDP bound to Rho, we employed a coupled enzymatic assay using nucleoside diphosphate kinase and luciferase as described previously [3,18].…”

“…In fibroblasts and cancer cells, RhoA and C are predominantly proproliferative and anti-apoptotic, while RhoB is anti-proliferative and pro-apoptotic [1]. RhoA is required for Ras-induced cellular transformation, and shows increased activation in Rastransformed cells [1,3]. Activating Rho mutations have not been reported in human malignancies, but several Rho-regulatory proteins were found in screens for transforming genes, and in leukemia-associated chromosomal translocations [1].…”