Abstract 703: Angiotensin II Type 1 Receptor Induces PP2B-mediated Serine 188 Dephosphorylation and Accelerated Activation of RhoA in Vascular Smooth Muscle Cells
Although over-activation of RhoA is recognized as a common component for the pathogenesis of hypertension and vascular proliferative disorders, the molecular mechanisms regulating RhoA activity in vascular smooth muscle cells (VSMC) are still unknown. In VSMC, RhoA is basally phosphorylated on Ser188 by NO-stimulated cGMP-dependent kinases (PKG). Ser188 phosphorylated-RhoA is sequestrated in the cytosol by its interaction with guanine dissociation inhibitor (GDI), which leads to inhibition of RhoA functions. As membrane translocation of RhoA is required for its activation, our hypothesis is that, by releasing RhoA from GDI, dephosphor-ylation of RhoA would participate to its activation. To address this hypothesis, we analyzed the effect of angiotensin II (Ang II) type 1 receptor (AT1R) activation on RhoA phosphorylation in rat aortic VSMC. In the presence of the AT2R antagonist PD 123319 (10 μM), Ang II (0.1 μM) induces a transient dephosphorylation of PKG-phosphorylated RhoA (84 ± 3%, n = 4), without modification of RhoA/PKG interaction. Phosphoresistant (S188A)- or phosphomimetic (S188E)-RhoA mutants expressed in VSMC were not dephosphorylated by AT1R. AT1R-mediated dephosphorylation of PKG-phosphorylated RhoA was prevented by the type 2B serine/threonine phosphatase (PP2B) inhibitor cypermethrin (20 nM) but unaffected by inhibition of PP2A and PP1 by 10 and 1000 nM okadaic acid, respectively. Co-immunoprecipitation and dephosphorylation assays indicated that, under basal conditions, PKG-phosphorylated RhoA was associated with, and slowly dephosphorylated by PP2A. AT1R stimulation induced the release of PKG-phosphorylated RhoA from PP2A, its association with PP2B and its fast dephosphorylation. Time-course analysis of AT1R-mediated RhoA activation showed that AT1R stimulation induced faster activation of RhoA pathway when RhoA is phosphorylated by PKG. This observation correlated with AT1R-mediated decrease in RhoA/GDI interaction. Our work thus identifies RhoA dephosphorylation as a new process involved in RhoA activation. AT1R activation by Ang II induces PP2B-mediated dephosphorylation of Ser 188 of RhoA. This leads to the release of RhoA from its cytosolic inhibitor GDI and accelerated activation of RhoA pathway.