Redox regulation of calcineurin in T-lymphocytes.
To explore whether the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin is subject to redox regulation in vivo, we used a luciferase reporter gene construct whose expression was controlled by the transcription factor NF-AT (the nuclear factor of activated T-cells) to monitor intracellular calcineurin activity following redox state perturbations. The NF-AT reporter construct was transfected into Jurkat cells, and luciferase activity was assessed following treatment with phorbol ester and ionomycin in the presence of either hydrogen peroxide or dithiothreitol (DTT). While DTT had no effect, H(2)O(2) completely abrogated NF-AT transactivation in response to stimulation. The inhibitory effect was specific for NF-AT as comparable levels of H(2)O(2) had only minor effects on constitutive transcription factors while an analogous construct under AP-1 control showed a 5-fold stimulation in transactivation in the presence of H(2)O(2). The inhibitory effect of H(2)O(2) was observed up to approximately 3 h following mitogen stimulation, a time point where NF-AT activity begins to increase under normal conditions. Protein serine/threonine phosphatase activities from Jurkat lysate indicated that calcineurin activity was inhibited not only by H(2)O(2) but also by high concentrations of DTT. These results indicate that calcineurin activity is subject to redox regulation in vivo and are discussed in the context of redox reactions involving active site metal ions.
Reiter
T A
TA
Section of Hematology Research and the Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA.
Abraham
R T
RT
Choi
M
M
Rusnak
F
F
D000199:Actins / Q000235:genetics; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019169:Jurkat Cells; D009154:Mutation; D010084:Oxidation-Reduction; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013601:T-Lymphocytes / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects
D000199:Actins; D018808:Transcription Factor AP-1; D006861:Hydrogen Peroxide; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D003545:Cysteine; D004229:Dithiothreitol