HUMAN P21-ACTIVATED KINASE (PAK1) REGULATES ACTIN ORGANIZATION IN MAMMALIAN-CELLS

Background: The Rho family GTPases Cdc42, Rad and RhoA regulate the re organization of the actin cytoskeleton induced by extracellular signal s such as growth factors. In mammalian cells, Cdc42 regulates the form ation of filopodia, whereas Rac regulates lamellipodia formation and m embrane ruffling, and RhoA regulates the formation of stress fibers. R ecently, the serine/threonine protein kinase p65(pak) was isolated by virtue of its ability to bind to activated Cdc42 and Rad. Upon binding , p65(pak) autophosphorylates, thereby increasing its catalytic activi ty towards exogenous substrates. This kinase is therefore a candidate effector for the changes in cell shape induced by growth factors. Resu lts: Here, we report that the microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polariz ed filopodia and membrane ruffles. The prolonged overexpression of Pak 1 amino-terminal mutants that are unable to bind Cdc42 or Rad results in the accumulation of filamentous actin in large, polarized membrane ruffles and the formation of vinculin-containing focal complexes withi n these structures. This phenotype resembles that seen in motile fibro blasts. The amino-terminal Pak1 mutant displays enhanced binding to th e adaptor protein Nck, which contains three Src-homology 3 (SH3) domai ns, Mutation of a proline residue within a conserved SH3-binding regio n at the amino terminus of Pak1 interferes with SH3-protein binding an d alters the effects of Pak1 on the cytoskeleton. Conclusions: These r esults indicate that Pak1, acting through a protein that contains an S H3 domain, regulates the structure of the actin cytoskeleton in mammal ian cells, and may serve as an effector for Cdc42 and/or Rac1 in promo ting cell motility.