title: 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+].
abstract: Signal transduction mechanisms activated during the early stages of necrotic cell death are poorly characterized. We have recently identified the Sterile 20 (Ste20)-like oxidant stress response kinase-1, 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 by exposure of Madin-Darby canine kidney or LLC-PK1 renal tubular epithelial cells to 2-deoxyglucose (2-DG) and any one of three inhibitors of the electron transport chain, cyanide (CN), rotenone, or antimycin A. Since oxidant stress activates SOK-1, we postulated that reactive oxygen species (ROS), which are produced by the electron transport chain during chemical anoxia, might be responsible for SOK-1 activation. The time course of CN/2-DG-induced SOK-1 activation and of production of ROS, measured in cells loaded with dichlorofluorescein, were compatible with a role for ROS in SOK-1 activation. Furthermore, preincubation of LLC-PK1 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 increase in cytosolic free [Ca2+] ([Ca2+]i) was necessary but not sufficient for CN/2-DG-induced activation of SOK-1. Preincubation of cells with BAPTA-AM prevented activation of SOK-1. Incubation of cells with thapsigargin or the calcium ionophore, A23187, had no effect on SOK-1 activity, 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-dependent mechanism that is both critically dependent upon and markedly amplified by an increase in [Ca2+]i. This requirement for dual inputs of oxidant stress and an increase in [Ca2+]i may prevent inappropriate 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 necrotic cell death.
authors.affiliation: Cardiac, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02129, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Pombo
authors.forename: T
authors.initials: T
authors.lastname: Tsujita
authors.forename: J M
authors.initials: JM
authors.lastname: Kyriakis
authors.forename: J V
authors.initials: JV
authors.lastname: Bonventre
authors.forename: T
authors.initials: T
authors.lastname: Force
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death; D003600:Cytosol / Q000378:metabolism*; D004285:Dogs; D004789:Enzyme Activation; D000860:Hypoxia / Q000201:enzymology*; D047908:Intracellular Signaling Peptides and Proteins; D018374:LLC-PK1 Cells; D020930:MAP Kinase Kinase Kinases; D018384:Oxidative Stress; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013552:Swine
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae; D002118:Calcium