A NOVEL SERINE KINASE ACTIVATED BY RAC1 CDC42HS-DEPENDENT AUTOPHOSPHORYLATION IS RELATED TO PAK65 AND STE20/

We identified three proteins in neutrophil cytosol of molecular size 6 5, 62 and 68 kDa which interact in a GTP-dependent manner with rad and CDC42Hs, but not with rho. Purification of p65 and subsequent peptide sequencing revealed identity to rat brain PAK65 and to yeast STE20 ki nase domains, Based on these sequences we screened a human placenta li brary and cloned the full-length cDNA. The complete amino acid sequenc e of the human cDNA shares similar to 73 % identity with rat brain PAK 65; within the kinase domain the human protein shares >95% and similar to 63% identity with rat PAK65 and yeast STE20 respectively. The new human (h)PAK65 mRNA is ubiquitously expressed and hPAK65 protein is di stinct from either human or rat brain PAK65. Recombinant hPAK65 exhibi ts identical specificity to the endogenous p65; both can bind rad and CDC42Hs in a GTP-dependent manner. The GTP-bound forms of rad and CDC4 2Hs induce autophosphorylation of hPAK65 on serine residues only, hPAK 65 activated by either rad or CDC42Hs is phosphorylated on the same si tes. Induction of hPAK65 autophosphorylation by rad or CDC42Hs stimula tes hPAK65 kinase activity towards myelin basic protein and once hPAK6 5 is activated, rad or CDC42Hs are no longer required to keep it activ e. The affinities of rac/CDC42Hs for the non-phosphorylated and phosph orylated hPAK65 were similar, hPAK65 had only a marginal effect on the intrinsic GTPase activity of CDC42Hs, but significantly affected the binding and GAP activity of p190. These data are consistent with a mod el in which hPAK65 functions as an effector molecule for rad and CDC42 Hs.