Oxidative stress inhibits MEKK1 by site-specific glutathionylation in the ATP-binding domain.
Many intracellular signalling events are accompanied by generation of reactive oxygen species in cells. Oxidation of protein thiol groups is an emerging theme in signal-transduction research. We have found that MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1], an upstream activator of the SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, is directly inhibited by cysteine alkylation using NEM (N-ethylmaleimide). The related kinase, ASK1 (apoptosis signal-regulating kinase 1), was not inhibited, but was instead activated by NEM. Inhibition of MEKK1 requires a single unique cysteine residue (Cys1238) in the ATP-binding domain of MEKK1. Oxidative stress induced by menadione (2-methyl-1,4-naphthoquinone) also inhibited MEKK1, but activated ASK1, in cells. MEKK1 inhibition by menadione also required Cys1238. Oxidant-inhibited MEKK1 was re-activated by dithiothreitol and glutathione, supporting reversible cysteine oxidation as a mechanism. Using various chemical probes, we excluded modification by S-nitrosylation or oxidation of cysteine to sulphenic acid. Oxidant-inhibited MEKK1 migrated normally on non-reducing gels, excluding the possibility of intra- or inter-molecular disulphide bond formation. MEKK1 was inhibited by glutathionylation in vitro, and MEKK1 isolated from menadione-treated cells was shown by MS to be modified by glutathione on Cys1238. Our results support a model whereby the redox environment within the cell selectively regulates stress signalling through MEKK1 versus ASK1, and may thereby participate in the induction of apoptosis by oxidative stress.
Cross
Janet V
JV
Department of Pathology, University of Virginia Medical School, PO Box 800214, Charlottesville, VA 22908-0214, U.S.A.
Templeton
Dennis J
DJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000478:Alkylation; D000595:Amino Acid Sequence / Q000235:genetics / Q000502:physiology; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D020134:Catalytic Domain / Q000187:drug effects; D045744:Cell Line, Tumor; D003545:Cysteine / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008198:Lymph Nodes / Q000201:enzymology / Q000473:pathology; D048728:MAP Kinase Kinase Kinase 1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D016877:Oxidants / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000473:pathology; D017434:Protein Structure, Tertiary; D014633:Valine / Q000378:metabolism
D004791:Enzyme Inhibitors; D016877:Oxidants; D010455:Peptides; D000255:Adenosine Triphosphate; D048728:MAP Kinase Kinase Kinase 1; D005978:Glutathione; D014633:Valine; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol