Identification and molecular cloning of a p21cdc42/rac1-activated serine/threonine kinase that is rapidly activated by thrombin in platelets

Abstract

The brain-enriched p21cdc42/rac1-activated serine/threonine kinase, p65PAK, was identified and purified on the basis of overlays with [γ-32P]GTP-Cdc42 onto SDS-fractionated proteins (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). In this study, the ubiquitously expressed P21cdc42/rac1 binding protein with relative molecular weight of 62,000 was purified from rat testes and shown to contain peptides related to PAK. It has thus been designated as the γ-PAK isoform (α- and β-isoforms being brain enriched). Isolation of γ-PAK cDNAs show that the kinase is highly conserved with α-PAK in both the p21 binding and kinase domains. The purified protein exhibited kinase activity that was activated by GTP-Cdc42 or GTP-Rac1 in vitro. In platelets, a p62 in situ renaturable kinase was recognized by antibodies raised against γ-PAK. This thrombin-activated protein kinase appears to coprecipitate with another kinase of Mr 86,000, suggesting that PAK may be part of a thrombin-responsive signaling complex.