TI - Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells . AB - The pathways by which activation of the small GTP -binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin -dependent ATPase activity of myosin II . Here we show that expression of active RacQ61 in growing HeLa cells , in addition to inducing ruffling , substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain ( MLC ) , which would increase actomyosin II ATPase and motor activities . Phosphorylated myosin was localized to RacQ61 -induced ruffles and stress fibers . RacQ61 -induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase ( PAK ) , about 35% by an inhibitor (Y-27632) of Rho kinase , 51% by Tat-PAK plus Y-27632 , and 10% by an inhibitor (ML7) of myosin light chain kinase . Staurosporine , a non-specific inhibitor of serine/threonine kinases , reduced RacQ61 -induced phosphorylation of MLC by about 58% , at the maximum concentration that did not kill cells . Since Rac activates PAK and PAK can phosphorylate MLC , these data strongly suggest that PAK is responsible for a significant fraction of RacQ61 -induced MLC phosphorylation . To our knowledge , this is the first evidence that active Rac causes phosphorylation of MLC in cells , thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes .