Molecular cloning of a new member of the p21-Cdc42/Rac-activated kinase (PAK) family

Abstract

A number of 'target' proteins for the Rho family of small GTP-binding proteins have now been identified, including the protein kinases ACK and p65(PAK) (Manser, E., Leung, T., Salihuddin, H, Zhao, Z.-S., and Lim, L. (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 β-PAK, which shares 79% amino acid identity with the previously described α-isoform. Their mRNAs are differentially expressed in the brain, with α-PAK mRNA being particularly abundant in motor-associated regions. In vitro translation products of the α- and β-PAK cDNAs exhibited relative molecular masses of 68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A specific β-PAK peptide sequence was obtained from rat brain-purified p65(PAK). Recombinant α- and β-PAKs exhibited an increase in kinase activity mediated by GTP-p21 induced autophosphorylation. Cdc42 was a more potent activator in vitro of α-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 β-PAK and brain p65(PAK) which is observed upon kinase activation does not occur with recombinant α-PAK.