TI - Autophosphorylation and protein kinase activity of p21-activated protein kinase gamma-PAK are differentially affected by magnesium and manganese . AB - 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 .