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VASCULAR BIOLOGY

Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA

¹Department of Pharmacology, Rush University Medical Center and ²Research Resources Center, Protein Core Facility, University of Illinois, Chicago, Illinois

Submitted 6 March 2008 ; accepted in final form 30 August 2008

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 GDI 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 GDI-GST fusion protein and PKA motif-containing GDI peptide at Ser¹⁷⁴, but not Thr¹⁸². Transfection of COS-7 cells with mutated full-length GDI at Ser¹⁷⁴ to Ala¹⁷⁴ (GDI-Ser^174A) abrogated the ability of cAMP to phosphorylate GDI. However, mutation of Thr¹⁸² to Ala¹⁸² (GDI-Thr^182A) did not abrogate, and cAMP increased phosphorylation of GDI to a similar extent as wild-type GDI transfectants. The mutant GDI-Ser^174A, but not GDI-Thr^182A, was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDI-Ser^174A was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDI binding with RhoA, suggesting that phosphorylation of Ser¹⁷⁴ regulated preformed GDI-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.

adenosine 3',5'-cyclic monophosphate-dependent protein kinase; protein kinase A consensus phosphorylation sites; single-site mutated GDI