Is calcineurin a peroxide-specific sensor in T-lymphocytes?
Calcineurin activity in T-lymphocytes has been shown to be sensitive to H(2)O(2). In this report, we investigate the effects of H(2)O(2)and other physiological oxidants on calcineurin activity both in vivo and in vitro. Intracellular calcineurin activity, as determined by NF-AT phosphorylation state and activity, is inhibited by H(2)O(2) but is insensitive to superoxide and NaOCl. Similarly, treatment of T-lymphocytes with NaOCl and paraquat does not drastically alter the activity of calcineurin in crude cell lysate, while H(2)O(2) causes significant inhibition. Sensitivity to H(2)O(2) and NaOCl in vivo correlates with the half-life of each species in cell medium. The intracellular redox potential is unaffected by H(2)O(2) (100 micro M) or NaOCl (600 micro M), indicating that H(2)O(2) inhibits calcineurin via a direct mechanism that does not involve a change in the cytosolic redox potential. In contrast, calcineurin activity in cell lysate is inhibited by all three oxidants. H(2)O(2) inactivation of calcineurin is rapid, with inactivation occurring in <or=10 s. H(2)O(2) inactivation of calcineurin is reversible in T-lymphocyte lysate using dithiothreitol (DTT) alone, while in rat brain lysate, reversibility requires both DTT and Fe(II), implicating that the enzyme may exist in different metal/redox-sensitive states in different tissues.
Section of Hematology Research and the Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, 200 First Street, S.W., Rochester, MN 55905, USA.
Tiffany A
TA
Reiter
Frank
F
Rusnak
D000818:Animals; D015153:Blotting, Western; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D017930:Genes, Reporter; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D051379:Mice; D016877:Oxidants / Q000502:physiology; D010084:Oxidation-Reduction; D010545:Peroxides / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012973:Sodium Hypochlorite / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D014158:Transcription, Genetic
D065095:Calcineurin Inhibitors; D016877:Oxidants; D010545:Peroxides; D013481:Superoxides; D006861:Hydrogen Peroxide; D012973:Sodium Hypochlorite; D008156:Luciferases; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases