The c-Jun N-terminal kinases (JNKs) belonging to the mitogen-activated protein kinase (MAPK) superfamily play important roles in foam-cell formation, hypercholesterolemia-mediated endothelial dysfunction, and the development of obesity. Although decreased nitric oxide (NO) production via decreased phosphorylation of endothelial NO synthase at serine 1179 (eNOS-Ser(1179)) was reported to be partly involved in JNK2-derived endothelial dysfunction, JNK2 seems likely to be indirectly involved in this signaling pathway. Here, using bovine aortic endothelial cells, we examined whether JNK2 directly phosphorylated eN0S-Ser(116), a putative substrate site for the MAPK superfamily, and this phosphorylation resulted in decreased NO release. JNK inhibitor SP60012 increased NO release in a time- and dose-dependent manner, which was accompanied by increased eN0S-Ser(116) phosphorylation. Purified JNK2 directly phosphorylated eNOS-Ser(116) in vitro. Ectopic expression of dominant negative JNK2 repressed eNOS-Ser(116) phosphorylation and increased NO production. Coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and JNK2. However, all these observed effects were not manifested when JNK1 probes were used. Overall, this study indicates that JNK2 is a physiological kinase responsible for eNOS-Ser(116) phosphorylation and regulates NO production. (C) 2011 Elsevier Inc. All rights reserved.