RESUMEN
NO, via its second messenger cGMP, activates protein kinase GI (PKGI) to induce vascular smooth muscle cell relaxation. The mechanisms by which PKGI kinase activity regulates cardiovascular function remain incompletely understood. Therefore, to identify novel protein kinase G substrates in vascular cells, a λ phage coronary artery smooth muscle cell library was constructed and screened for phosphorylation by PKGI. The screen identified steroid-sensitive gene 1 (SSG1), which harbors several predicted PKGI phosphorylation sites. We observed direct and cGMP-regulated interaction between PKGI and SSG1. In cultured vascular smooth muscle cells, both the NO donor S-nitrosocysteine and atrial natriuretic peptide induced SSG1 phosphorylation, and mutation of SSG1 at each of the two predicted PKGI phosphorylation sites completely abolished its basal phosphorylation by PKGI. We detected high SSG1 expression in cardiovascular tissues. Finally, we found that activation of PKGI with cGMP regulated SSG1 intracellular distribution.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Regulación de la Expresión Génica/fisiología , Glicoproteínas/biosíntesis , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Células Cultivadas , GMP Cíclico/genética , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Cisteína/análogos & derivados , Cisteína/farmacología , Proteínas de la Matriz Extracelular , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Donantes de Óxido Nítrico/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , S-Nitrosotioles/farmacología , Proteínas Supresoras de Tumor/genéticaRESUMEN
Nitric oxide (NO) inhibits vascular contraction by activating cGMP-dependent protein kinase I-alpha (PKGI-alpha), which causes dephosphorylation of myosin light chain (MLC) and vascular smooth muscle relaxation. Here we show that PKGI-alpha attenuates signaling by the thrombin receptor protease-activated receptor-1 (PAR-1) through direct activation of regulator of G-protein signaling-2 (RGS-2). NO donors and cGMP cause cGMP-mediated inhibition of PAR-1 and membrane localization of RGS-2. PKGI-alpha binds directly to and phosphorylates RGS-2, which significantly increases GTPase activity of G(q), terminating PAR-1 signaling. Disruption of the RGS-2-PKGI-alpha interaction reverses inhibition of PAR-1 signaling by nitrovasodilators and cGMP. Rgs2-/- mice develop marked hypertension, and their blood vessels show enhanced contraction and decreased cGMP-mediated relaxation. Thus, PKGI-alpha binds to, phosphorylates and activates RGS-2, attenuating receptor-mediated vascular contraction. Our study shows that RGS-2 is required for normal vascular function and blood pressure and is a new drug development target for hypertension.