Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events1,2,3,4. In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury5,6 and in cytokine-stimulated hypertrophy7. Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS4,8; however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the βγ-subunit of G protein (Gβγ) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. The Gβγ-responsive ERK activation induced by H2O2 is independent of ligands binding to Gi-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H2O2 increases [35S]GTP-γS binding to cardiac membranes and directly activates purified heterotrimeric Gi and Go but not Gs. Analysis using heterotrimeric Go and its individual subunits indicates that H2O2 modifies Gαo but not Gβγ, which leads to subunit dissociation. We conclude that Gαi and Gαo are critical targets of oxidative stress for activation of ERK.
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We thank T. Katada for providing the purified heterotrimeric proteins (Gi, Go, Gs) Gαo and Gβγ. We thank RIKEN DNA Bank for recombinant adenovirus for LacZ. We also thank I. Saito for cosmid vector for the COS–TPC method, and T.-G. He for plasmids of pAdEasy system.
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