MEK Mediates v-Src-induced Disruption of the Actin Cytoskeleton via Inactivation of the Rho-ROCK-LIM Kinase Pathway

Abstract

Organization of actin filaments is controlled by the Rho family of small GTPases, Rho, Rac, and Cdc42 (1). Among them, Rho regulates the assembly of actin stress fibers and focal contacts, through activation of the downstream effectors mDia and the closely related kinases ROCKI and ROCKII (2, 3). Activation of ROCK by Rho is implicated in stress fiber and focal contact formation (2, 4). ROCK increases the phosphorylation of myosin light chains by directly phosphorylating myosin light chains and negatively regulating myosin phosphatase and thus increases actomyosin-based contractility (5, 6). The resulting contractile forces are thought to contribute to the formation of stress fibers and focal contacts (7,8). ROCK also activates LIMK, 1 which subsequently phosphorylates cofilin and thereby inhibits its actin-depolymerizing activity, thus contributing to actin stress fiber stabilization (9 -11).In cells transformed by the product of the Rous sarcoma virus, the v-Src tyrosine kinase, the shape and cytoskeletal architecture are dramatically altered, with a disruption of actin stress fibers and focal contacts and the formation of dot-like actin-associated adhesions called podosomes. This disruption of the cytoskeleton is believed to contribute to several aspects of the transformed phenotype, including adhesion-independent cell growth and increased migration abilities (12). However, the mechanism by which v-Src disrupts actin stress fibers and focal contacts is not clear. Stimulation of a Rho-GAP (GTPase-activating protein) activity was reported upon v-Src activation in chicken embryo fibroblasts (13), suggesting that v-Src-induced cytoskeleton disruption is linked to inhibition of Rho activity. In addition, an active mutant form of Rho was shown to restore stress fibers and adhesion plaques in v-Src-transformed fibroblasts (14). Despite these experiments, the relationship between Rho-mediated stress fiber assembly and v-Src-induced morphological transformation, as well as the signaling pathways mediating v-Src effects on the cytoskeleton, have not been clearly identified. Expression of v-Src results in tyrosine phosphorylation of numerous substrates and simultaneous activation of several signaling pathways, including the Ras/MEK and PI3K pathways (15). Both pathways have been implicated in cytoskeleton regulation in normal cells. Activation of PI3K is necessary for Rac-and Rho-mediated rearrangements of the actin cytoskeleton induced by platelet-derived growth factor and other growth factors (16,17). ERK activity has been implicated in cell migration and was shown to directly phosphorylate myosin light chain kinase, suggesting a role for ERK in the regulation of cytoskeleton and focal contact dynamics (18,19). In v-Srctransformed chicken embryo fibroblasts, both ERK and PI3K are translocated to focal adhesions upon v-Src activation (20,21), raising the possibility that both pathways participate in cellular adhesion dynamic during transformation.We report here that activation of MEK, but not PI3K, is required for the ...

Disruption of c-Jun Reduces Cellular Migration and Invasion through Inhibition of c-Src and Hyperactivation of ROCK II Kinase

“…ROCK activates LIMK which phosphorylates cofilin, inhibiting its actin-depolymerizing activity, thereby stabilizing actin stress fibers. Consistent with our findings, v-Src inhibited cofilin phosphorylation and the mechanism involved a MEK-dependent and PI3 kinase-independent pathway (Pawlak and Helfman, 2002). Rho mutants restore stress fiber formation in v-Src-transformed cells (Mayer et al, 1999), consistent with a model in which Rho proteins serve as downstream targets of v-Src.…”

“…c-Src inhibits ROCK II activity (Pawlak and Helfman, 2002). We therefore examined the relative abundance of c-Src, in c-jun Ϫ/Ϫ and wt 3T3 cells.…”

Thrombospondin Induces RhoA Inactivation through FAK-dependent Signaling to Stimulate Focal Adhesion Disassembly

“…However, calpain has numerous other targets, including both signaling and structural proteins within the focal adhesion plaque, suggesting that calpain could mediate focal adhesion turnover through a variety of mechanisms (68 -70). In addition to epidermal growth factor signaling, sustained ERK activation during cellular transformation by oncogenic Ras and v-Src stimulates focal adhesion disassembly by uncoupling the RhoA-ROCK pathway (71,72). TSP1 stimulates a biphasic activation of ERK, with both an early peak in ERK activity at 10 min and a late peak in ERK activity at 2 h following treatment (42).…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…Integrin activation of MAPK requires c-Src and FAK, and ERK activity is required for FAK stimulated focal adhesion disassembly to promote cell migration [79,89]. Several studies have shown that FAK or v-Src can suppress the activities of Rho [48,87] and its effector, ROCK [88], and this pathway requires ERK signaling [79,104] to modulate actin and focal adhesion dynamics. Consistent with these observations, activated FAK and c-Src and activated ERK localize to focal adhesions [89].…”