Abstract
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.
Highlights
Stimulated by G␥ subunits (Ste4p/Ste18p) in response to mating pheromones [1, 2], and in Schizosaccharomyces pombe, the G␣ subunit (Gpa1p) is the signal transducer to the Ste20p homolog Shk1p [3]
PAK1-catalyzed phosphorylation of G␣z was efficient relative to that catalyzed by protein kinase C (PKC)␣, and the truncated protein PAK1-(232–544)
In contrast to the site specificity displayed by PAK1, PKC phosphorylated at least one additional site C-terminal to Gln30, usually accounting for ϳ10% of the total incorporation of phosphate (Fig. 2A, lane 12)
Summary
Stimulated by G␥ subunits (Ste4p/Ste18p) in response to mating pheromones [1, 2], and in Schizosaccharomyces pombe, the G␣ subunit (Gpa1p) is the signal transducer to the Ste20p homolog Shk1p [3]. The PAKs are mammalian homologs of Ste20p and Shk1p that were initially recognized as kinases that are activated by Rac and Cdc, members of the Rho family of monomeric GTP-binding proteins [4]. We report here that G␣z is phosphorylated at Ser by PAK1, inhibiting its interaction with both G␥ and RGS proteins. We have used this specificity to distinguish and delineate the functional consequences of phosphorylation at Ser and Ser, which we show to be the two principal substrate sites for PKC. An unexpected outcome is the finding that G␥, which stimulates the PAK homolog Ste20p in Saccharomyces, inhibits the activity of mammalian PAK1 toward both G␣z and other substrates
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