Temporal and spatial distribution of activated Pak1 in fibroblasts

p21-activated kinases (Paks) are effecters of the small GTPases Cdc42 and R ac, and are thought to mediate some of the cytoskeletal and transcriptional activities of these proteins. To localize activated Pak1 in cells, we deve loped an antibody directed against a phosphopeptide that is contained withi n the activation loop of Pak1. This antibody specifically recognizes the ac tivated form of Pak1. Immunofluorescence analysis of NIH-3T3 cells coexpres sing activated Cdc42 or Rad plus wild-type Pak1 shows that activated Pak1 a ccumulates at sites of focal adhesion, throughout filopodia and within the body and edges of lamellipodia. Platelet-derived growth factor stimulation of NIH-3T3 cells shows a pattern of Pak1 activation similar to that observe d with Rad. During closure of a fibroblast monolayer wound, Pak1 is rapidly activated and localizes to the leading edge of motile cells, then graduall y tapers off as the wound closes. The activation of Pak1 by wounding is blo cked by inhibitors of phosphatidylinositol 3-kinase, and Src family kinases , but not by an inhibitor of the epidermal growth factor receptor. These fi ndings indicate that activated Pak1, and by extension, probably activated C dc42 or Rac, accumulates at sites of cortical actin remodeling in motile fi broblasts.