Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts calmodulin binding and calcineurin activation

Calcineurin is a Ca2+/calmodulin-activated Ser/Thr phosphatase important in cellular actions resulting in memory formation, cardiac hypertrophy, and T-cell activation. This enzyme is subject to oxidative inactivation by superoxide at low micromolar concentrations and by H2O2 at low millimolar concentrations. On the basis of the hypothesis that oxidation of Met residues in calmodulin-binding domains inhibits binding to calmodulin, purified calcineurin was used to study the susceptibility of Met residues to oxidation by H2O2. The rate for oxidation of Met(406) in the calmodulin-binding domain was determined to be 4.4 x 10(-3) M-1 s(-1), indicating a high susceptibility to oxidation. Functional repercussions of Met 06 oxidation were evaluated using native enzyme and a calcineurin mutant in which Met(406) was exchanged for Leu. Measurement of fluorescent calmodulin binding demonstrated that oxidation of Met406 results in a 3.3-fold decrease in the affinity of calmodulin for calcineurin. Calcineurin activation exhibited a loss in cooperativity with respect to calmodulin following Met(406) oxidation as shown by a reduction in the Hill slope from 1.88 to 0.86. Maximum phosphatase activity was unaffected by Met oxidation. Changes in the calcineurin-calmodulin interaction were accompanied by a 40% loss in the ability of calmodulin to stimulate binding of immunophilin/immunosuppressant to calcineurin. All effects on calmodulin binding to the native enzyme by the treatment with H2O2 could be reversed by treating the enzyme with methionine sulfoxide reductase. These results indicate that the calmodulin-binding domain of calcineurin is susceptible to oxidation at Met(406) and that oxidation disrupts calmodulin binding and enzyme activation. Oxidation-dependent decreases in the affinity of calmodulin for calcineurin can potentially modulate calmodulin-dependent signaling and calmodulin distribution.