Abstract-cAMP and cyclic GMP-dependent kinases (PKA and PKG) 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 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 because of 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 Ⅲ signal transduction Ⅲ phosphorylation Ⅲ cGMP Ⅲ ubiquitin T he small G proteins of the Rho family are identified as tightly regulated molecular switches that cycle between an active GTP-bound form, and an inactive GDP-bound form. Rho proteins are recognized as major regulators of the actin cytoskeleton. 1,2 A large body of evidence has now been obtained regarding the important functions of Rho proteins in the vasculature. [3][4][5] The activity of Rho is under the direct control of a large set of other regulatory proteins. 2,6 In the inactive GDP-bound form, Rho is locked in the cytosol by guanine dissociation inhibitors (RhoGDI). 7 The guanine nucleotide exchange factors (RhoGEFs) catalyze the exchange of GDP for GTP to activate RhoA. 8 In the active GTP-bound form, Rho translocates to plasma membrane where it interacts with effectors to transduce the signal downstream. GTPaseactivating proteins that hydrolyze GTP to GDP then turn off activation. In addition to this regulation, recent reports have proposed that the phosphorylation/dephosphorylation cycle also controls small G protein activity. cAMP-dependent protein kinase (PKA) has been shown to phosphorylate Ser180 of Rap1, Ser179 of Rap1B, and...

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