p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous ␥-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant ␥-PAK, while native ␥-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of ␥-PAK followed by immunoblot analysis revealed a similar profile for native and

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... binant ␥-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant ␥-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, ␥-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated ␥-PAK, six of which are in common with those previously reported in ␣-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the ␥-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with ␥-PAK activation.