No abstract

(1994) Nature 367, 40 -46). The purified serine/threonine kinase p65 PAK has been shown to be directly activated by GTP-Rac1 or GTP-Cdc42. Here we report the cDNA sequence encoding a new brain-enriched PAK isoform ␤-PAK, which shares 79% amino acid identity with the previously described ␣-isoform. Their mRNAs are differentially expressed in the brain, with ␣-PAK mRNA being particularly abundant in motor-associated regions. In vitro translation products of the ␣-and ␤-PAK cDNAs exhibited relative molecular masses of 68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A specific ␤-PAK peptide sequence was obtained from rat brainpurified p65 PAK . Recombinant ␣-and ␤-PAKs exhibited an increase in kinase activity mediated by GTP-p21 induced autophosphorylation. Cdc42 was a more potent activator in vitro of ␣-PAK kinase, and the fully activated enzyme is 300 times more active than the unphosphorylated form. Interestingly the down-regulation in the binding of p21s to recombinant ␤-PAK and brain p65 PAK

The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc42-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck. Here we investigate in detail how autophosphorylation events affect the catalytic activity of PAK by altering the autophosphorylation sites in both ␣-and ␤PAK. Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAK Nterminal regulatory domain play no direct role in activation, a phosphorylation of ␣PAK serine 144 or ␤PAK serine 139, which lie in the kinase inhibitory domain, significantly contribute to activation. By contrast, sphingosine-mediated activation is independent of this residue, indicating a different mode of activation. Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck.