Ca²⁺ Binding Site 2 in Calcineurin-B Modulates Calmodulin-Dependent Calcineurin Phosphatase Activity

Abstract

Calcineurin is the Ca(2+)- and calmodulin-dependent Ser/Thr phosphatase. Human calcineurin-Aalpha 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 Ca(2+) binding site replaced by a Lys, resulting in the loss of Ca(2+) binding to that site. Phosphatase activities of the enzymes toward a (32)P-labeled phosphopeptide substrate were measured. Inactivating Ca(2+) binding sites 1, 2, or 3 in calcineurin-B reduced Ca(2+)-dependent phosphatase activity of the enzymes in the absence of calmodulin with the site 2 mutation being most effective. Inactivating Ca(2+) binding site 4 did not change enzyme activity or sensitivity to Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) concentrations. These data support a role for Ca(2+) binding site 3 in the carboxyl half of calcineurin-B in transmitting the Ca(2+) signal to calcineurin-A and indicate that site 2 in the amino half of calcineurin-B is critical for enzyme activation.

Regulatory Subunit Myristoylation Antagonizes Calcineurin Phosphatase Activation in Yeast

“…Cnb1, like its mammalian counterpart CNB, contains four EF hand Ca 2ϩ binding domains. Mutation of either the first or the second Ca 2ϩ binding EF hand domain of mammalian CNB inhibits calcineurin activation in vitro (37). We have recently obtained similar results when EF hand mutations are introduced into yeast Cnb1.…”

Methionine Oxidation in the Calmodulin-Binding Domain of Calcineurin Disrupts Calmodulin Binding and Calcineurin Activation

“…This discrepancy is likely a reflection of the limitations of surface area calculations from a static crystal structure that does not take into account molecular dynamics (4). It is possible that the N-terminal half of calcineurin-B, which has a lower calcium affinity and a more dynamic role in calcineurin regulation than the C-terminal half (24), may be flexible in solution and have a greater exposure to the solvent than suggested by the crystal structure.…”

Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the α or β catalytic subunit

“…Phospho-RII peptide, CaP, and BioMol Green reagent were purchased from BioMol. Phospho-DARPP-32 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) [LDPRQVE-MIRRRRPT(PO 4 )PAML] was purchased from American Peptide Company. Human CnAb cDNA was generously provided by M. M. Lai and S. Snyder (The Johns Hopkins University School of Medicine [9]).…”

“…The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the a or b catalytic subunit were compared using in vitro phosphatase assays. CaN containing the a isoform (CnAa) has lower K m and higher V max values than CaN containing the b isoform (CnAb) toward the PO 4 -RII, PO 4 -DARPP-32 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the a or b catalytic subunit isoform displayed identical calmodulin dissociation rates.…”

“…At low concentrations of FKBP12/FK506, CaN containing the a isoform is more sensitive to inhibition than CaN containing the b isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of b CaN using PO 4 -DARPP-32 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) as substrate. The functional differences conferred upon CaN by the a or b catalytic subunit isoforms suggest that the a:b and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types.…”