Ca2+-dependent activation of c-jun NH2-terminal kinase in primary rabbit proximal tubule epithelial cells

Previous work from this laboratory demonstrated that arachidonic acid activates c-jun NH2-terminal kinase (JNK) through oxidative intermediates in a Ca2+-independent manner (Cui X and Douglas JG. Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells. Proc Natl Acad Sci USA 94: 3771-3776, 1997.). We now report that JNK can also be activated via a Ca2+-dependent mechanism by agents that increase the cytosolic Ca2+ concentration (Ca2+ ionophore A(23187),Ca2+-ATPase inhibitor thapsigargin) or deplete intracellular Ca2+ stores [intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N, N,N',N'-tetraacetic acid (BAPTA)-AM]. The activation of JNK by BAPTA-AM occurs despite a decrease in cytosolic Ca2+ concentration as detected by the indicator dye fura 2, but appears to be related to Ca2+ metabolism, because modification of BAPTA with two methyl groups increases not only the chelation affinity for Ca2+, but also the potency for JNK activation. BAPTA-AM stimulates Ca2+ influx across the plasma membrane, and the resulting local Ca2+ increases are probably involved in activation of JNK because Ca2+ influx inhibitors (SKF-96365, nifedipine) and lowering of the free extracellular Ca2+ concentration with EGTA reduce the BAPTA-induced JNK activation.