p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts

The p21 (Cdc42/Rac) activated kinase Pak1 regulates cell morphology and pol arity in most, if not all, eukaryotic cells. We and others have established that Pak's effects on these parameters are mediated by changes in the orga nization of cortical actin. Because cell motility requires polarized rearra ngements of the actin/myosin cytoskeleton, we examined the role of Pak 1 in regulating cell movement. We established clonal tetracycline-regulated NIH -3T3 cell lines that inducibly express either wild-type Pak 1, a kinase-dea d, or constitutively-active forms of this enzyme, and examined the morpholo gy, F-actin organization, and motility of these cells. Expression of any of these forms of Pak1 induced dramatic changes in actin organization which w ere not inhibited by coexpression of a dominant-negative form of Rad. Cells inducibly expressing wild-type or constitutively-active Pak1 had large, po larized lamellipodia at the leading edge, were more motile than their norma l counterparts when plated on a fibronectin-coated surface, and displayed e nhanced directional movement in response to an immobilized collagen gradien t. In contrast, cells expressing a kinase-dead form of Pak1 projected multi ple lamellipodia emerging from different Darts of the cell simultaneously. These cells, though highly motile, displayed reduced persistence of movemen t when plated on a fibronectin-coated surface and had defects in directed m otility toward immobilized collagen. Expression of constitutively activated Pak1 was accompanied by increased myosin light chain (MLC) phosphorylation , whereas expression of kinase-dead Pak1 had no effect on MLC. These result s suggest that Pak1 affects the phosphorylation state of MLC, thus linking this kinase to a molecule that directly affects cell movement.