LOCALIZATION OF P21-ACTIVATED KINASE-1 (PAK1) TO PINOCYTIC VESICLES AND CORTICAL ACTIN STRUCTURES IN STIMULATED CELLS

The mechanisms through which the small GTPases Rad and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are cur rently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytos keletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskel etal dynamics. We have examined the subcellular localization of endoge nous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK 1 is detected in submembranous vesicles in both unstimulated and stimu lated fibroblasts that colocalize with a marker for fluid-phase uptake . In cells stimulated with PDGF, in v-Src-transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymer ized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appea rs to precede F-actin in translocating to cytoskeletal structures form ed at the cell periphery. The association of PAK1 with the actin cytos keleton is prevented by the actin filament-disrupting agent cytochalas in D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co -immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection o f a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpre ssing the insulin receptor (HIRcB cells) induced the formation of F-ac tin-and PAK1-containing structures reminiscent of dorsal ruffles. Thes e data indicate a close correlation between the subcellular distributi on of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoske letal structures and support an active role for PAK1 in regulating cor tical actin rearrangements.