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

To examine the requirements for activation of the p21-activated protein kin ase gamma-PAK (Pak2, PAK I) from rabbit reticulocytes by Cdc42(GTP gamma S) , autophosphorylation with ATP(Mg) or ATP(Mn) and its effects on protein ki nase activity were examined. Autophosphorylation with ATP(Mg) alone was min imal with negligible protein kinase activity; the rate of autophosphorylati on was increased 3-4-fold upon binding of Cdc42(GTP gamma S), resulting in a 3-fold stimulation of protein kinase activity with peptide and protein su bstrates. The rate of autophosphorylation with ATP(Mn) was 4.7-fold faster than with ATP(Mg) alone and was stimulated 2-fold by Cdc42(GTP gamma S). Ho wever, gamma-PAK autophosphorylated with ATP(Mn in the presence or absence of Cdc42(GTP gamma S) did not phosphorylate peptide or protein substrates i n the presence of ATP(Mn), indicating that gamma-PAK can utilize ATP(Mn) fo r autophosphorylation but not for phosphorylation of exogenous substrates. Tryptic phosphopeptide maps of gamma-PAK autophosphorylated with ATP(Mg) al one showed 3 phosphopeptides, while with Cdc42(GTP gamma S) a total of 9 ma jor phosphopeptides was observed. When gamma-PAK was autophosphorylated wit h ATP(Mn) in the presence or absence of Cdc42(GTP gamma S), 7 major phospho peptides were observed, which were identical to peptides obtained with Cdc4 2(GTP gamma S) and ATP(Mg). Utilizing a recombinant mutant of gamma-PAK wit h alanine replacing threonine 402 in the catalytic region (T402A), it was d etermined that the two additional phosphopeptides observed in active PAK (p eptides 7 and 8) were due to phosphorylation of threonine 402. These result s 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.