ACTIVATION OF AN S6 H4 KINASE (PAK-65) FROM HUMAN PLACENTA BY INTRAMOLECULAR AND INTERMOLECULAR AUTOPHOSPHORYLATION/

The S6/H4 kinase purified from human placenta catalyzes phosphorylatio n of the S6 ribosomal protein, histone H4, and myelin basic protein. I n vitro activation of the p60 S6/H4 kinase requires removal of an auto inhibitory domain by mild trypsin digestion and autophosphorylation of the catalytic domain (p40 S6/H4 kinase). The two autophosphorylation/ autoactivation sites contain the sequences SSMVGTPY (site 1) and SVIDP VPAPVGD-SHVDGAAK (site 2). These sequences identify S6/H4 kinase as th e rac-activated PAK65 (Martin, G. A., Bollag, G., McCormick, F. and Ab o, A. (1995) EMBO J. 14, 1971-1978). Site 1 phosphorylation is most ra pid, but activation does not occur until site 2 is autophosphorylated. The site 1 phosphorylation occurs by an intramolecular mechanism wher eas site 2 autophosphorylation occurs by an intermolecular mechanism. A model is proposed in which phosphorylation of sites 1 and 2 occurs s equentially. The model proposes that trypsin treatment of the inactive holoenzyme removes an inhibitory rac-binding domain which blocks MgAT P access to the catalytic site. The pseudosubstrate domain at site 1 i s autophosphorylated and subsequent bimolecular autophosphorylation at site 2 fully opens the catalytic site. Phosphorylation by a regulator y protein kinase may occur at site 2 in vivo.