Published online before print May 12, 2005, doi: 10.1161/01.RES.0000170084.88780.ea
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Submitted on February 16, 2005
Revised on May 4, 2005
Accepted on May 4, 2005
Phosphorylation of Serine 188 Protects RhoA from Ubiquitin/Proteasome-Mediated Degradation in Vascular Smooth Muscle Cells
Malvyne Rolli-Derkinderen ;
From INSERM U-533 (M.R.-D., V.S., G.L., P.P.), Faculté des Sciences, Nantes, INSERM U-627 (L.B., E.L.), Faculté de Médecine, Nice and CNRS UMR 6188 (C.B., D.H.), Faculté de Médecine, Angers, France
* To whom correspondence should be addressed. E-mail: pierre.pacaud{at}univ-nantes.fr.
cAMP and cyclic GMP-dependent kinases phosphorylate the small G protein RhoA on Ser188. We have previously demonstrated that phosphorylation of Ser188 inhibits RhoA-dependent functions and positively regulates RhoA expression, and that the nitric oxide (NO)/cGMP-dependent protein kinase (PKG) pathway plays an essential role, both in vitro and in vivo, in the regulation of RhoA protein expression and functions in vascular smooth muscle cells. Here we analyze the consequences of Ser188 phosphorylation on RhoA protein degradation. By expressing Ser188 phosphomimetic wild-type (WT-RhoA-S188E) and active RhoA proteins (Q63L-RhoA-S188E), we show that phosphorylation of Ser188 of RhoA protects RhoA, particularly its active form, from ubiquitin-mediated proteasomal degradation. Coimmunoprecipitation experiments indicate that the resistance of the phosphorylated active form of RhoA to proteasome-mediated degradation is due to its cytoplasmic sequestration through enhanced RhoGDI interaction. In rat aortic smooth muscle cells, stimulation of PKG and inhibition of proteasome by lactacystin induce nonadditive increases in RhoA protein expression. In addition, stimulation of PKG leads to the accumulation of GTP-bound RhoA in the cytoplasm. In vivo stimulation of the NO/PKG signaling by treating rats with sildenafil increased RhoA level and RhoA phosphorylation, and enhanced its association to RhoGDI in the pulmonary artery, whereas opposite effects are induced by chronic inhibition of NO synthesis in N-
-nitro-L-arginine-treated rats. Our results thus suggest that Ser188 phosphorylation-mediated protection against degradation is a physiological process regulating the level of endogenous RhoA and define a novel function for RhoGDI, as an inhibitor of Rho protein degradation.
Key words: Rho-GTP-binding proteins • signal transduction • phosphorylation • cGMP • Ubiquitin
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