Post-transcriptional Down-regulation of ROCKI/Rho-kinase through an MEK-dependent Pathway Leads to Cytoskeleton Disruption in Ras-transformed Fibroblasts

Abstract

Transformation by oncogenic Ras profoundly alters actin cytoskeleton organization. We investigated Ras-dependent signaling pathways involved in cytoskeleton disruption by transfecting normal rat kidney (NRK) cells with different Ras mutants. RasV12S35, a mutant known to activate specifically the Raf/MAPK pathway, led to stress fiber and focal contact disruption, whereas the adherens junctions remained intact. Next, we found that pharmacological inhibition of MEK was sufficient to restore the cytoskeletal defects of ras-transformed NRK cells, including assembly of stress fibers and focal contacts, but it did not induce reorganization of the cell-cell junctions. Investigating the mechanism underlying this phenotypic reversion, we found that the sustained MAPK signaling resulting from Ras-transformation down-regulated the expression of ROCKI and Rho-kinase, two-Rho effectors required for stress fiber formation, at the post-transcriptional level. On MEK inhibition, ROCKI/Rho-kinase expression and cofilin phosphorylation were increased, demonstrating that the Rho-kinase/LIM-kinase/cofilin pathway was functionally restored. Finally, using dominant negative or constitutively active mutants, we demonstrated that expression of ROCKI/Rho-kinase was both necessary and sufficient to promote cytoskeleton reorganization in NRK/ras cells. These findings further establish the Ras/MAPK pathway as the critical pathway involved in cytoskeleton disruption during Ras-transformation, and they suggest a new mechanism, involving alteration in ROCKI/Rho-kinase expression, by which oncogenic Ras can specifically target the actin-based cytoskeleton and achieve morphological transformation of the cells.

5‐Hydroxytryptamine as a potent migration enhancer of human aortic endothelial cells

“…Association of the Rho and ERK pathways for regulation of actin filaments has also been shown in a variety of cells, including vascular cells [19][20][21][22][23]. In the present study, expression of phosphorylated ERK (p44/p42) was increased from 2.5 min to 15 min after stimulation with 5-HT in a time-dependent manner (Fig.…”

ALK5 promotes tumor angiogenesis by upregulating matrix metalloproteinase-9 in tumor cells

“…The p38 MAPK pathway may enhance cell motility by regulating actin remodeling factor HSP27 (Hedges et al, 1999) and by affecting actin polymerization and cell contractility (Huot et al, 1998;Hedges et al, 1999;Bakin et al, 2002). MEK-ERK may regulate cell motility by preventing formation of extensive actin stress fibers via suppression of tropomyosin induction by TGF-b1 (Bakin et al, 2004) or inhibition of RhoA-Rho kinase pathway (Sahai et al, 2001;Pawlak and Helfman, 2002). JNK may regulate motility by modulating the dynamics of microtubules (Huang et al, 2004) or by affecting the assembly/ stability of focal adhesion complexes via phosphorylation of paxillin and Spir, a WASP family protein (reviewed by Huang et al (2004)).…”

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

“…In HT1080 human fibrosarcoma cells containing oncogenic N-Ras, both Rac and RhoA activity were increased compared with cells lacking the mutant N-Ras; Rac and RhoA activities were also increased when cells lacking the mutant N-Ras were stably transfected with constitutively active Raf or MEK (18). In K-Ras(V12)-transformed normal rat kidney cells, no significant change of RhoA activity was observed compared with untransformed cells (23). Several older studies reported loss of stress fibers in Ras-transformed Rat1 cells, with restoration of stress fibers upon transfection of constitutively active RhoA, suggesting loss of RhoA activity in the Ras-transformed cells (7,24); others reported increased stress fibers in Rastransformed breast cancer cells and NIH 3T3 cells without direct measurement of Rho activity (14,15).…”