Substrates enhance autophosphorylation and activation of p21-activated protein kinase gamma-PAK in the absence of activation loop phosphorylation

The p21-activated protein kinase gamma-PAK from rabbit, expressed in insect cells, is activated following binding of Cdc42(GTP gamma S). The rate of a utophosphorylation is increased fivefold and the protein kinase activity 13 -fold, as measured with the synthetic heptapeptide (AKRESAA). The mutant K2 78R, where the invariant lysine in the catalytic site is replaced by argini ne, shows neither autophosphorylation nor activity. Replacement of the cons erved threonine in the catalytic domain with alanine (T402A) reduces autoph osphorylation and protein kinase activity to 1% that of the wild-type gamma -PAK, indicating autophosphorylation of Thr402 in the activation loop is es sential for protein kinase activity. In contrast, certain protein substrate s such as histone 2B, histone 4 and myelin basic protein, stimulate both au tophosphorylation and protein kinase activity to levels similar to those ob served with Cdc42(GTP gamma S). This substrate-level activation does not re quire autophosphorylation of Thr402 in the activation loop. As shown with T 402A, the protein kinase activity with histone 4 is similar to that observe d with recombinant wild-type gamma-PAK. Basic proteins or peptides which ar e not substrates of gamma-PAK, such as histone 1 and polylysine, do not sti mulate autophosphorylation or activity. Other substrates such as the Rous s arcoma virus protein NC are phosphorylated by gamma-PAK following activatio n by Cdc42(GTP gamma S), but are not phosphorylated by T402A. The data sugg est that some substrates can override the requirement for Cdc42(GTP gamma S ), by activating gamma-PAK directly.