Wang J, Frost J A, Cobb M H, Ross E M
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
J Biol Chem. 1999 Oct 29;274(44):31641-7. doi: 10.1074/jbc.274.44.31641.
p21-activated protein kinase (PAK)-1 phosphorylated Galpha(z), a member of the Galpha(i) family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of Galpha(z) in vitro. In transfected cells, Galpha(z) was phosphorylated both by wild-type PAK1 when stimulated by the GTP-binding protein Rac1 and by constitutively active PAK1 mutants. In vitro, phosphorylation occurred only at Ser(16), one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other Galpha subunits (i1, i2, i3, o, s, or q). PAK1-phosphorylated Galpha(z) was resistant both to RGSZ1, a G(z)-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the G(i) and G(q) families of G proteins. Phosphorylation of Ser(27) by PKC did not alter sensitivity to either GAP. The previously described inhibition of G(z) GAPs by PKC is therefore mediated by phosphorylation of Ser(16). Phosphorylation of either Ser(16) by PAK1 or Ser(27) by PKC decreased the affinity of Galpha(z) for Gbetagamma; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates Galpha(z) function by attenuating the inhibitory effects of both GAPs and Gbetagamma. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gbetagamma, suggesting that PAK1 acts as a Gbetagamma-regulated effector protein. This inhibition of mammalian PAK1 by Gbetagamma contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gbetagamma homolog Ste4p/Ste18p.
p21激活蛋白激酶（PAK）-1使Gαz发生磷酸化，Gαz是Gαi家族的成员之一，存在于大脑、血小板和肾上腺髓质中。在体外，磷酸化程度接近1摩尔磷酸盐/摩尔Gαz。在转染细胞中，当受到GTP结合蛋白Rac1刺激时，野生型PAK1以及组成型活性PAK1突变体均可使Gαz发生磷酸化。在体外，磷酸化仅发生在丝氨酸（Ser）16位点，这是蛋白激酶C（PKC）的两个主要底物位点之一。PAK1不会使其他Gα亚基（i1、i2、i3、o、s或q）发生磷酸化。PAK1磷酸化的Gαz对RGSZ1（一种G(z)选择性GTP酶激活蛋白（GAP））和RGS4（一种对G蛋白的G(i)和G(q)家族相对非选择性的GAP）均具有抗性。PKC对丝氨酸（Ser）27的磷酸化不会改变对任何一种GAP的敏感性。因此，先前描述的PKC对G(z) GAP的抑制作用是由丝氨酸（Ser）16的磷酸化介导的。PAK1对丝氨酸（Ser）16的磷酸化或PKC对丝氨酸（Ser）27的磷酸化均会降低Gαz对Gβγ的亲和力；PKC对这两个位点的磷酸化不会产生进一步影响。因此，PAK1通过减弱GAP和Gβγ的抑制作用来调节Gαz的功能。在这种情况下，Gβγ直接抑制了PAK1对几种蛋白质底物的激酶活性，这表明PAK1作为一种受Gβγ调节的效应蛋白发挥作用。Gβγ对哺乳动物PAK1的这种抑制作用与酿酒酵母中Gβγ同源物Ste4p/Ste18p对PAK同源物Ste20p的刺激作用形成对比。
