Ca2+ binding site 2 in calcineurin-B modulates calmodulin-dependent calcineurin phosphatase activity

Calcineurin is the Ca2+- and calmodulin-dependent Ser/Thr phosphatase. Human calcineurin-A alpha and wild-type or mutated calcineurin-Bs were coexpressed in Escherichia coli and purified by calmodulin-Sepharose affinity chromatography. Four calcineurin-B mutants were studied. Each had a single conserved Glu in the 12th position of one EF-hand Ca2+ binding site replaced by a Lys, resulting in the loss of Ca2+ binding to that site. Phosphatase activities of the enzymes toward a P-32-labeled phosphopeptide substrate were measured. Inactivating Ca2+ binding sites 1, 2, or 3 in calcineurin-B reduced Ca2+-dependent phosphatase activity of the enzymes in the absence of calmodulin with the site 2 mutation being most effective. Inactivating Ca2+ binding site 4 did not change enzyme activity or sensitivity to Ca2+ in either the absence or presence of calmodulin. The calmodulin-dependent phosphatase activity of the enzymes containing site 1, 2, or 3 mutations in calcineurin-B was also decreased compared to enzyme with wild-type calcineurin-B. Of these enzymes, the one with the site 2 mutation was most profoundly affected as determined by the magnitude of the shift in Ca2+ concentration dependence. Binding of a fluorescein-labeled calmodulin to the wild-type and the site 2 mutant enzymes was examined using fluorescence polarization measurements. The decrease in Ca2+ sensitivity for the enzyme with calcineurin-B site 2 inactivated is apparently due to a decrease in the affinity of that enzyme for calmodulin at low Ca2+ concentrations. These data support a role for Ca2+ binding site 3 in the carboxyl half of calcineurin-B in transmitting the Ca2+ signal to calcineurin-A and indicate that site 2 in the amino half of calcineurin-B is critical for enzyme activation.