A conserved negative regulatory region in αPAK:: Inhibition of PAK kinases reveals their morphological roles downstream of Cdc42 and Rac1

alpha PAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y, Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim, Mol. Cell, Biol. 11:1129-1143, 1947) resembling those of Cdc42(G12V). PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rad through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in alpha PAK). alpha PAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42(G12V)-driven focal complexes, Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region, Indeed, a 67-residue polypeptide encoding alpha PAK(83-149) potently inhibited GTP gamma S-bound Cdc42-mediated kinase activation of both alpha PAK and beta PAK. Coexpression of this PAK inhibitor with Cdc42(G12V) prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1(G12V) also prevented loss of stress fibers but not ruffling induced by the p21, Coexpression of alpha PAK(83-149) completely blocked the phenotypic effects of hyperactive alpha PAK(L107F) in promoting dissolution of focal adhesions and actin stress fibers, These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rad in cytoskeletal reorganization.