Signal Crosstalk between Rho and Tyrosine phosphorylation

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The small GTPase Rho acts on two effectors, ROCK and mDial, and induces stress fibers and focal adhesions. However, how ROCK and mDial 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 FAK 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 mDial mutant inhibited also the membrane ruffle formation by Y-27632. These results have thus revealed the Rho-dependent Rac activation signaling that is mediated by mDial through Cas phosphorylation, and is antagonized by the action of ROCK.