Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts calmodulin binding and calcineurin activation.
Calcineurin is a Ca (2+)/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 H 2O 2 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 H 2O 2. 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 406 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 Met 406 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 H 2O 2 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.
Carruthers
Nicholas J
NJ
Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan 48201, USA.
Stemmer
Paul M
PM
D019703:Calcineurin / Q000737:chemistry* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002942:Circular Dichroism; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005454:Fluorescence Polarization; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D002147:Calmodulin; D008715:Methionine; D006861:Hydrogen Peroxide; D019703:Calcineurin; D004229:Dithiothreitol