DETERMINANTS FOR SUBSTRATE PHOSPHORYLATION BY P21-ACTIVATED PROTEIN-KINASE (GAMMA-PAK)

gamma-PAK, originally designated PAK I and subsequently identified as a member of the p21-activated protein kinase family, has been shown to have cytostatic properties and to be involved in maintaining cells in a nondividing state [Rooney, R. D., et al., (1996) J, Biol. Chem. 271 , 21498-21504]. The determinants for phosphorylation of substrates by gamma-PAK have been identified by examining the kinetics of phosphoryl ation of a series of synthetic peptides patterned after the sequence K KRKSGL, which is the site phosphorylated by gamma-PAK in the Rous sarc oma virus nucleocapsid protein NC in vivo and in vitro. With these pep tides, the recognition sequence for gamma-PAK has been shown to contai n two basic amino acids in the -2 and -3 positions, as represented by (WR)RXS, in which the -2 position is an arginine, the -3 position is a n arginine or a lysine, and X can be an acidic, basic, or neutral amin o acid. A basic amino acid in the -1 or -4 position improves the rate of phosphorylation by increasing the V-max and decreasing the K-m. An acidic amino acid in the -1 position increases the rate (2.5-fold), as does an acidic residue in the -4 position, although to a lower extent (1.6-fold). Proline in the -1 or fl position has a deleterious effect and inhibits phosphorylation by gamma-PAK. The substrate requirements of protein kinases that recognize basic amino acids on the N-terminal side of the phosphorylatable residue such as cAMP-dependent protein ki nase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) have b een compared with gamma-PAK using the same peptides. An acidic residue in the -1 position negatively affects PKA and PKC; thus, peptides con taining the sequence KRES can be used to identify gamma-PAK.