Protein kinase A-mediated phosphorylation of the Galpha13 switch I region alters the Galphabetagamma13-G protein-coupled receptor complex and inhibits Rho activation.
The present studies mapped the protein kinase A (PKA) phosphorylation site of Galpha(13) and studied the consequences of its phosphorylation. Initial experiments using purified human Galpha(13) and the PKA catalytic subunit established that PKA directly phosphorylates Galpha(13). The location of this phosphorylation site was next investigated with a new synthetic peptide (G(13)SRI(pep)) containing the PKA consensus sequence (Arg-Arg-Pro-Thr(203)) within the switch I region of Galpha(13). G(13)SRI(pep) produced a dose-dependent inhibition of PKA-mediated Galpha(13) phosphorylation. On the other hand, the Thr-phosphorylated derivative of G(13)SRI(pep) possessed no inhibitory activity, suggesting that Galpha(13) Thr(203) may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the Galpha(13)-T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that Galpha(13) phosphorylation stabilized coupling of Galpha(13) with platelet thromboxane A(2) receptors but destabilized coupling of Galpha(13) to its betagamma subunits. In order to determine the functional consequences of this phosphorylation on Galpha(13) signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells overexpressing human Galpha(13) wild type (Galpha(13)-WT) or Galpha(13)-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 Galpha(13) 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 Galpha(13) Thr(203).
Manganello
Jeanne M
JM
Department of Pharmacology, University of Illinois, Chicago, Illinois 60612, USA.
Huang
Jin-Sheng
JS
Kozasa
Tohru
T
Voyno-Yasenetskaya
Tatyana A
TA
Le Breton
Guy C
GC
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D017482:Receptors, Thromboxane / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; D017482:Receptors, Thromboxane; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins; D020741:rho GTP-Binding Proteins