Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA

Abstract

The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDIα showed two potential protein kinase A (PKA) phosphorylation motifs, Ser174 and Thr182. Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDIα-GST fusion protein and PKA motif-containing GDIα peptide at Ser174, but not Thr182. Transfection of COS-7 cells with mutated full-length GDIα at Ser174 to Ala174 (GDIα-Ser174A) abrogated the ability of cAMP to phosphorylate GDIα. However, mutation of Thr182 to Ala182 (GDIα-Thr182A) did not abrogate, and cAMP increased phosphorylation of GDIα to a similar extent as wild-type GDIα transfectants. The mutant GDIα-Ser174A, but not GDIα-Thr182A, was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDIα-Ser174A was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIα binding with RhoA, suggesting that phosphorylation of Ser174 regulated preformed GDIα-RhoA complexes. The findings provide strong support that the selective phosphorylation at Ser174 by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury.