Interactions of Calcineurin A, Calcineurin B, and Ca²⁺

Abstract

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.

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

“…The IC 50 values (10 lM-12 lM) and final extent of inhibition (90% inhibition of phosphatase activity by 90 lM CaP) obtained are similar to the previously reported values using 32 P-RII peptide as substrate [10,11]. Similar kinetics of inhibition were also obtained for CaP with CaN heterodimers containing the CnAa or CnAb catalytic subunit 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 (Fig. 3B) The structurally unrelated immunophilin/immunosuppressant complexes of FKBP12/FK506 or CypA/CsA inhibit CaN noncompetitively by binding to the CnB-binding helix, CnB, and one side of the substrate-binding cleft of the catalytic site to alter the active-site geometry [16,17,20,22].…”

“…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.…”

Calcineurin Potentiates the Activation of Procaspase-3 by Accelerating Its Proteolytic Maturation

“…12). A regulatory role of calcineurin B for phosphatase activity of calcineurin A has been well established (42,43). Tacrolimus binds to immunophilin and inhibits phosphatase activity of calcineurin (32)(33)(34)(35)(36)(37).…”