Protein kinase A-mediated phosphorylation of the Gα13 switch I region alters the Gαβγ13-G protein-coupled receptor complex and inhibits Rho activation

Abstract

The present studies mapped the protein kinase A (PKA) phosphorylation site of Gα13 and studied the consequences of its phosphorylation. Initial experiments using purified human Gα13 and the PKA catalytic subunit established that PKA directly phosphorylates Gα13. The location of this phosphorylation site was next investigated with a new synthetic peptide (G13SRIpep) containing the PKA consensus sequence (Arg-Arg-Pro-Thr203) within the switch I region of Gα13. G13SRIpep produced a dose-dependent inhibition of PKA-mediated Gα13 phosphorylation. On the other hand, the Thr-phosphorylated derivative of G13SRIpep possessed no inhibitory activity, suggesting that Gα13 Thr203 may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the Gα13-T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that Gα13 phosphorylation stabilized coupling of Gα13 with platelet thromboxane A2 receptors but destabilized coupling of Gα13 to its βγ subunits. In order to determine the functional consequences of this phosphorylation on Gα13 signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells over-expressing human Gα13 wild type (Gα13-WT) or Gα13-T203A mutant were generated and assayed for Rho activation. It was found that 8-bromo-cyclic AMP caused a significant decrease (50%; p < 0.002) of Rho activation in Gα13 wild type cells but produced no change of basal Rho activation levels in the mutant (p > 0.4). These results therefore suggest that PKA blocks Rho activation by phosphorylation of Gα13 Thr203.