Reciprocal signaling between heterotrimeric G proteins and the p21- stimulated protein kinase

p21-activated protein kinase (PAK)-1 phosphorylated Gα(z), a member of the Gα(i) family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of Gα(z) in vitro. In transfected cells, Gα(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 Ser¹⁶, one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other Gα subunits (i1, i2, i3, o, s, or q). PAK1- phosphorylated Gα(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 Set²⁷ 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 Ser¹⁶. Phosphorylation of either Ser¹⁶ by PAK1 or Ser²⁷ by PKC decreased the affinity of Gα(z) for Gβγ; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates Gα(z) function by attenuating the inhibitory effects of both GAPs and Gβγ. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gβγ, suggesting that PAK1 acts as a Gβγ-regulated effector protein. This inhibition of mammalian PAK1 by Gβγ contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gβγ homolog Ste4p/Ste18p.