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.
PAK I is a member of the PAK (p21-activated protein kinase) family and is activated by Cdc42 (Jakobi, R., Chen, C.-J., Tuazon, P. T., and Traugh, J. A. (1996) J. Biol. Chem. 271, 6206-6211). To examine the effects of PAK I on cleavage arrest, subfemtomole amounts of endogenously active (58 kDa) and inactive (60 kDa) PAK I and a tryptic peptide (37 kDa) containing the active catalytic domain were injected into one blastomere of 2-cell frog embryos. Active PAK I resulted in cleavage arrest in the injected blastomere at mitotic metaphase, whereas the uninjected blastomere progressed through mid- to late cleavage. Injection of other protein kinases at similar concentrations had no effect on cleavage. Endogenous PAK I was highly active in frog oocytes, and antibody to PAK I reacted specifically with protein of 58-60 kDa. PAK I protein was decreased at 60 min post-fertilization, with little or no PAK I protein or activity detectable at 80 min post-fertilization or in 2-cell embryos. At the 4-cell stage PAK I protein increased, but the protein kinase was present primarily as an inactive form. Rac2 and Cdc42, but not Rac 1, were identified in oocytes and throughout early embryo development. Thus, PAK I appears to be a potent cytostatic protein kinase involved in maintaining cells in a non-dividing state. PAK I activity is high in oocytes and appears to be regulated by degradation/synthesis and through autophosphorylation via binding of Cdc42. PAK I may act through regulation of the stress-activated protein kinase signaling pathway and/or by direct regulation of multiple metabolic pathways.
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