ACTIVATION OF THE STE20-LIKE OXIDANT STRESS-RESPONSE KINASE-1 DURING THE INITIAL-STAGES OF CHEMICAL ANOXIA-INDUCED NECROTIC CELL-DEATH - REQUIREMENT FOR DUAL INPUTS OF OXIDANT STRESS AND INCREASED CYTOSOLIC [CA2+]

Signal transduction mechanisms activated during the early stages of ne crotic cell death are poorly characterized, We have recently identifie d the Sterile 20 (Ste20)-like oxidant stress response kinase-l, SOK-1, which is a member of the Ste20 kinase family, We report that SOK-1 is markedly activated as early as 20 min after chemical anoxia induced b y exposure of Madin-Darby canine kidney or LLC-PK1 renal tubular epith elial cells to 2-deoxyglucose (2-DG) and any one of three inhibitors o f the electron transport chain, cyanide (CN), rotenone, or antimycin A . Since oxidant stress activates SOK-I, we postulated that reactive ox ygen species (ROS), which are produced by the electron transport chain during chemical anoxia, might be responsible for SOR-I activation, Th e time course of CN/2-DG-induced SOK-1 activation and of production of ROS, measured in cells loaded with dichlorofluorescein, were compatib le with a role for ROS in SOH-l activation, Furthermore, preincubation of LLC-PK, cells with three unrelated scavengers of ROS, pyrrolidine dithiocarbamate, pyruvate, or nordihydroguaiaretic acid, reduced both cellular oxidant stress and activation of SOK-1 by CN/2-DG. An increas e in cytosolic free [Ca2+] ([Ca2+](i)) was necessary but not sufficien t for CN/2-DG-induced activation of SOK-1, Preincubation of cells with BAPTA-AM prevented activation of SOK-1. Incubation of cells with thap sigargin or the calcium ionophore, A23187, had no effect on SOK-1 acti vity, but preincubation of cells with either of these agents markedly enhanced CN/2-DG-induced activation of SOK-1 (20-fold versus 7-fold). In summary, chemical anoxia activates SOK-1 via an oxidant stress depe ndent mechanism that is both critically dependent upon and markedly am plified by an increase in [Ca2+](i). This requirement for dual inputs of oxidant stress and an increase in [Ca2+](i) may prevent inappropria te activation of the kinase by milder degrees of oxidant stress, which are insufficient to generate an increase in [Ca2+](i). The activation of SOK-1 may be one of the cell's earliest responses to inducers of n ecrotic cell death.