EXPRESSION OF CONSTITUTIVELY ACTIVE ALPHA-PAK REVEALS EFFECTS OF THE KINASE ON ACTIN AND FOCAL COMPLEXES

The family of p21-activated protein kinases (PAKs) appear to be presen t in all organisms that have Cdc42-like GTPases, In mammalian cells, P AKs have been implicated in the activation of mitogen-activated protei n kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is e nriched in the leading edge of embryonic epithelial cells undergoing d orsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leu ng, E. Manser, and L. Lim, Mel. Cell, Biol, 16:1896-1908, 1996), where it colocalizes,vith structures resembling focal complexes, We show he re by transfection that in epithelial HeLa cells alpha-PAK is recruite d from the cytoplasm to distinct focal complexes by both Cdc42(G12V) a nd Rac1(G12V), which themselves colocalize to these sites, By deletion analysis, the N terminus of PAK is shown to contain targeting sequenc es for focal adhesions which indicate that these complexes are the sit e of kinase function in vivo. Cdc42 and Rad cause alpha-PAK autophosph orylation and kinase activation, Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant , By fusing regions of Cdc42 to the C terminus of PAK, activated chime ras were also obtained, Plasmids encoding these different constitutive ly active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of i ntroducing CdC42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed, These combined effects resulted i n retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rad and indicate that PAK functions include the dissol ution of stress fibers and reorganization of focal complexes.