Résumé
The cGMP-dependent protein kinase I (cGKI) is a key mediator of cGMP signaling, but the specific functions of its two isoforms, cGKI alpha and cGKI beta, are poorly understood. Recent studies indicated a novel cGMP-independent role for cGKI alpha in redox sensing. To dissect the effects of oxidative stress on the cGKI isoforms, we used mouse embryonic fibroblasts and vascular smooth muscle cells (VSMCs) expressing both, one, or none of them. In cGKI alpha-expressing cells, but not in cells expressing only cGKI beta, incubation with H2O2 induced the formation of a disulfide bond between the two identical subunits of the dimeric enzyme. Oxidation of cGKI alpha was associated with increased phosphorylation of its substrate, vasodilator-stimulated phosphoprotein. H2O2 did not stimulate cGMP production, indicating that it activates cGKI alpha directly via oxidation. Interestingly, there was a mutual influence of H2O2 and cGMP on cGKI activity and disulfide bond formation, respectively; preoxidation of the kinase with H2O2 slightly impaired its activation by cGMP, whereas preactivation of the enzyme with cGMP attenuated its oxidation by H2O2. To evaluate the functional relevance of the noncanonical H2O2-cGKI alpha pathway, we studied the regulation of the cytosolic Ca2+ concentration ([Ca2+](i)). H2O2 suppressed norepinephrine-induced Ca2+ transients in cGKI alpha-expressing VSMCs and, to a lower extent, in VSMCs expressing only cGKI beta or none of the isoforms. Thus, H2O2 lowers [Ca2+](i) mainly via a cGKI alpha-dependent pathway. These results indicate that oxidative stress selectively targets the cGKI alpha isoform, which then modulates cellular processes in a cGMP-independent manner. A decrease in [Ca2+-](i) in VSMCs via activation of cGKI alpha might be a major mechanism of H2O2-induced vasodilation. (C) 2012 Elsevier Inc. All rights reserved.