To examine the requirements for activation of the p21-activated protein kinase gamma-PAK (Pak2, PAK I) from rabbit reticulocytes by Cdc42(GTPgammaS), autophosphorylation with ATP(Mg) or ATP(Mn) and its effects on protein kinase activity were examined. Autophosphorylation with ATP(Mg) alone was minimal with negligible protein kinase activity; the rate of autophosphorylation was increased 3-4-fold upon binding of Cdc42(GTPgammaS), resulting in a 3-fold stimulation of protein kinase activity with peptide and protein substrates. The rate of autophosphorylation with ATP(Mn) was 4.7-fold faster than with ATP(Mg) alone and was stimulated 2-fold by Cdc42(GTPgammaS). However, gamma-PAK autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS) did not phosphorylate peptide or protein substrates in the presence of ATP(Mn), indicating that gamma-PAK can utilize ATP(Mn) for autophosphorylation but not for phosphorylation of exogenous substrates. Tryptic phosphopeptide maps of gamma-PAK autophosphorylated with ATP(Mg) alone showed 3 phosphopeptides, while with Cdc42(GTPgammaS) a total of 9 major phosphopeptides was observed. When gamma-PAK was autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS), 7 major phosphopeptides were observed, which were identical to peptides obtained with Cdc42(GTPgammaS) and ATP(Mg). Utilizing a recombinant mutant of gamma-PAK with alanine replacing threonine 402 in the catalytic region (T402A), it was determined that the two additional phosphopeptides observed in active PAK (peptides 7 and 8) were due to phosphorylation of threonine 402. These results show that Mn sustains autophosphorylation on serine but does not support autophosphorylation of threonine 402, which is required for activity toward exogenous substrates, or phosphorylation of these substrates.