ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

Abstract

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632–induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632–induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.

R-Ras Controls Membrane Protrusion and Cell Migration through the Spatial Regulation of Rac and Rho

“…In addition, although activated Rac can lead to increased Rho activity in some cells, this newly active Rho then inhibits Rac activation, suggesting a negative feedback loop by which Rho and Rac activity are precisely regulated (Li et al, 2002). In addition to this work, Tsuji et al (2002) have revealed that downstream of activated Rho, its effectors ROCK and mDia antagonize each other so that ROCK inhibits, and mDia promotes, Rac activation. They also propose that Rho-dependent Rac activation is controlled spatially so that mDia activation of Rac may occur preferentially at the leading edge, whereas ROCK's inhibition of Rac may occur at the trailing edge (Tsuji et al, 2002).…”

“…Recent work has identified a mechanism by which active Rho activates its effector, ROCK, which then inhibits Rac (Katsumi et al, 2002;Tsuji et al, 2002). To determine if a similar mechanism controls Rac activity downstream of R- Cell lysates from pseudopodia and cell bodies were prepared as previously described (Cho and Klemke, 2002).…”

The effects of modifying RhoA and Rac1 activities on heterotypic contact inhibition of locomotion

“…RhoA was thought to be predominantly involved in tail retraction of migrating cells [24], however, recent studies suggest RhoA may participate in both membrane protrusion and retraction at the leading edge [20,25]. Cells expressing dominant active mDia became more invasive, possibly because mDia activates Rac1 [26]. N17Rac1 cells had decreased invasion indices and reduced levels of active RhoA, probably because mDia is activating endogenous Rac1.…”

Cell surface dynamics – how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton

“…The different Rho GTPases might operate in a coordinated fashion to affect the actin cytoskeleton. In particular, RhoA often acts as an antagonist of Rac1 and Cdc42 (Kozma et al, 1997;Tsuji et al, 2002;Ohta et al, 2006; reviewed by Brown et al, 2006;Guilluy et al, 2011). Moreover, the small GTPases regulate the activity of effector enzymes, such as the protein kinases Rho-associated coiled-coil forming kinase (ROCK) (activated by RhoA) and p21-activated kinase (PAK) (activated by Rac and Cdc42).…”

“…These effectors, in turn, can then contribute to the coordinated control of the GTPases. For example, in various cell types, Rac1 operates under the stable inhibitory control of ROCK (Yamaguchi et al, 2001;Tsuji et al, 2002;Takefuji et al, 2007). Therefore, inhibition of either RhoA or its effector ROCK can result in Rac1 activation (Jeon et al, 2010;Jeon et al, 2012).…”