Determinants for Substrate Phosphorylation by p21-Activated Protein Kinase (γ-PAK)

Abstract

γ-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 γ-PAK have been identified by examining the kinetics of phosphorylation of a series of synthetic peptides patterned after the sequence KKRKSGL, which is the site phosphorylated by γ-PAK in the Rous sarcoma virus nucleocapsid protein NC in vivo and in vitro. With these peptides, the recognition sequence for γ-PAK has been shown to contain two basic amino acids in the −2 and −3 positions, as represented by (K/R)RXS, in which the −2 position is an arginine, the −3 position is an arginine or a lysine, and X can be an acidic, basic, or neutral amino 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 +1 position has a deleterious effect and inhibits phosphorylation by γ-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 kinase (PKA) and Ca²⁺/phospholipid-dependent protein kinase (PKC) have been compared with γ-PAK using the same peptides. An acidic residue in the −1 position negatively affects PKA and PKC; thus, peptides containing the sequence KRES can be used to identify γ-PAK.