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

Abstract

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 phosphorylation of a series of synthetic peptides patterned after the sequence KKRKSGL, which is the site phosphorylated by gamma-PAK in the Rous sarcoma virus nucleocapsid protein NC in vivo and in vitro. With these peptides, the recognition sequence for gamma-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 Vmax and decreasing the Km. 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 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 kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) have been compared with gamma-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 gamma-PAK.

Phosphorylation of Mnk1 by Caspase-activated Pak2/γ-PAK Inhibits Phosphorylation and Interaction of eIF4G with Mnk

“…A schematic representation of Mnk1 is shown in Fig. 9, in which (51). This region is a nuclear localization sequence, and Mnk1 has been identified in the nucleus (52).…”

Phosphorylation of Estrogen Receptor α Blocks Its Acetylation and Regulates Estrogen Sensitivity

“…These serines are all within the cAMP-dependent protein kinase phosphorylation sequences XRRXSX or SKKXSX. Interestingly, it has been shown that mutation of the SKKXSX recognition sequence to SKRXSX increases the kinetics of PKA phosphorylation (37) and predicts that the K303R mutation in ER␣ may generate a more efficient substrate for PKA phosphorylation at S305, a hypothesis that was addressed in the study herein in the context of its effects on hormone sensitivity and acetylation status.…”

The Mechanism of PAK Activation

“…Equivalent threonine residues are present in a motif that is conserved in all the PAK family kinases, and corresponding peptides function as efficient substrates for PAK (30). Recent experiments with ␥PAK have demonstrated a peculiar difference between autophosphorylation of the N-terminal serine residues and the single-threonine residue (18).…”