Rho GTPases including Rho, Rac, and Cdc42 are key modulators of the actin cytoskeleton (1-3). They are critical for the cell shape changes and adhesion dynamics that drive cell migration (4 -7). Among the Rho GTPase family, Rho induces the formation of focal adhesions and stress fibers (7,8). Interestingly, although a basal level of Rho activity is needed for fibroblast migration, too much Rho activity impedes migration (4, 9, 10). It has been shown that the concerted action of two of the immediate Rho targets, Rho-associated kinase (Rho-kinase) 1 /ROCK and the formin homology protein mDia1, mediate the effect of Rho on matrix adhesion and the actin cytoskeleton (11). In particular, Rho-kinase was shown to stimulate myosindriven contractility in smooth muscle and nonmuscle cells by phosphorylating, thereby inactivating myosin light chain (MLC) phosphatase (12, 13), and possibly by direct phosphorylation of MLC (14 -16). In addition to Rho-kinase, MLC kinase (MLCK) is another kinase that phosphorylates the MLC in both smooth muscle and nonmuscle cells (17, 18). The phosphorylation of MLC on Ser-19 and to a lesser extent on Thr-18 by MLCK promotes the assembly of myosin II into filaments and activates its ATPase activity, which stabilizes the actin-myosin interaction and promotes cell contractility (19 -22). Recently, Rho-kinase and MLCK were suggested to play distinct roles in spatial regulation of MLC phosphorylation. Rho-kinase appears to be important for MLC phosphorylation in the center of cells, and MLCK is responsible for phosphorylating MLC at the cell periphery (16). Focal adhesion kinase (FAK), a 125-kDa cytoplasmic tyrosine kinase localized in focal contacts, has been known to play an important role in integrin-mediated cell migration (23). Fibroblasts derived from FAK-null mouse embryos are more rounded and poorly spread than their wild-type counterparts (23). They show an overabundance of focal adhesions, enriched cortical actin filaments at the cell periphery, and a decreased migration rate (23,24). It has been suggested that the increase in peripheral adhesions results from an inhibition of turnover in FAK Ϫ/Ϫ cells (23), which may result from constitutive activation of Rho (24). Because of the known involvement of Rhokinase and MLCK in cell contractility, a major factor controlling cell migration, we hypothesize that abnormal regulation of Rho-kinase and MLCK may underlie the migratory defect of FAK Ϫ/Ϫ cells. EXPERIMENTAL PROCEDURESMaterials-Fetal bovine serum, non-essential amino acids, sodium pyruvate, and 2-mercaptoethanol were purchased from Invitrogen. Y27632, a specific inhibitor of Rho-kinase, was purchased from Calbiochem. The monoclonal anti-MLCK, monoclonal anti-MLC, monoclonal anti-␤-tubulin, bovine MLC, myelin basic protein (MBP), cytochalasin D, and 2,3-butanedione monoxime (BDM) were purchased from SigmaAldrich. The polyclonal anti-Rho-kinase and monoclonal anti-paxillin were purchased from Transduction Laboratories (Lexington, KY). The plasmid pEGFP-N1-MLCK and polyclonal anti-MLCK ...