Identification of a redox-sensitive switch within the JAK2 catalytic domain

• Published in: Free radical biology & medicine Vol. 52; no. 6; pp. 1101 - 1110
• Main Authors: Smith, John K., Patil, Chetan N., Patlolla, Srikant, Gunter, Barak W., Booz, George W., Duhé, Roy J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-03-2012
• Subjects: active sites; Animals; catalytic activity; Catalytic Domain - genetics; Cell Line; Cysteine; Cysteine - genetics; Cytokines; Diabetes; Diabetes Mellitus - metabolism; Diabetes Mellitus - pathology; Disease Models, Animal; dithiothreitol; Dithiothreitol - pharmacology; Enzyme Activation - drug effects; Free radicals; Hydrogen Peroxide - pharmacology; Insulin-Secreting Cells - drug effects; Insulin-Secreting Cells - metabolism; Insulin-Secreting Cells - pathology; Janus kinase; Janus Kinase 2 - genetics; Janus Kinase 2 - metabolism; Mice; Mutagenesis, Site-Directed; mutants; Mutation - genetics; non-specific protein-tyrosine kinase; Oxidation-Reduction; Oxidative Stress; Phosphorylation - drug effects; reactive oxygen species; Redox; site-directed mutagenesis; STAT5 Transcription Factor - metabolism; β-Islet; Cysteine; Free radicals; Cytokines; DTT; Redox; GST; SDS–PAGE; o-IBZ; STAT; JAK; Sf21; ROS; rJAK2; Janus kinase; Diabetes; β-Islet
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2011.12.025

Four cysteine residues (Cys866, Cys917, Cys1094, and Cys1105) have direct roles in cooperatively regulating Janus kinase 2 (JAK2) catalytic activity. Additional site-directed mutagenesis experiments now provide evidence that two of these residues (Cys866 and Cys917) act together as a redox-sensitive switch, allowing JAK2's catalytic activity to be directly regulated by the redox state of the cell. We created several variants of the truncated JAK2 (GST/(NΔ661)rJAK2), which incorporated cysteine-to-serine or cysteine-to-alanine mutations. The catalytic activities of these mutant enzymes were evaluated by in vitro autokinase assays and by in situ autophosphorylation and transphosphorylation assays. Cysteine-to-alanine mutagenesis revealed that the mechanistic role of Cys866 and Cys917 is functionally distinct from that of Cys1094 and Cys1105. Most notable is the observation that the robust activity of the CC866,917AA mutant is unaltered by pretreatment with dithiothreitol or o-iodosobenzoate, unlike all other JAK2 variants previously examined. This work provides the first direct evidence for a cysteine-based redox-sensitive switch that regulates JAK2 catalytic activity. The presence of this redox-sensitive switch predicts that reactive oxygen species can impair the cell's response to JAK-coupled cytokines under conditions of oxidative stress, which we confirm in a murine pancreatic β-islet cell line.
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► Cys866 and Cys917 function as a redox sensor switch in JAK2. ► Converting these cysteines to alanines creates a redox-refractive enzyme. ► This switch is conserved as Cys892 and Cys944 in JAK1. ► It provides a biochemical basis for direct redox regulation of JAK activity. ► This switch also explains H2O2 inhibition of JAK2-dependent STAT5 phosphorylation in βTC-6 cells.