Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the alpha or beta catalytic subunit.
The calcineurin (CaN) alpha and beta catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the alpha or beta catalytic subunit were compared using in vitro phosphatase assays. CaN containing the alpha isoform (CnA alpha) has lower K(m) and higher V(max) values than CaN containing the beta isoform (CnA beta) toward the PO4-RII, PO4-DARPP-32(20-38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the alpha or beta catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at K(m) concentrations, except for a five- to ninefold higher IC50 value measured for CaN containing the beta isoform with p-nitrophenylphosphate as substrate. No difference in stimulation of phosphatase activity toward p-nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the alpha isoform is more sensitive to inhibition than CaN containing the beta isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of beta CaN using PO4-DARPP-32(20-38) as substrate. The functional differences conferred upon CaN by the alpha or beta catalytic subunit isoforms suggest that the alpha:beta and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the alpha and beta catalytic subunit isoforms give rise to substrate-dependent differences in sensitivity toward FKBP12/FK506.
Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA. perrino@physio.unr.edu
Brian A
BA
Perrino
Andrew J
AJ
Wilson
Patricia
P
Ellison
Lucie H
LH
Clapp
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D019703:Calcineurin / Q000737:chemistry; D065095:Calcineurin Inhibitors; D002147:Calmodulin / Q000378:metabolism; D002352:Carrier Proteins / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology*; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D055503:Protein Multimerization; D021122:Protein Subunits; D051381:Rats; D013379:Substrate Specificity; D016559:Tacrolimus / Q000494:pharmacology*; D022061:Tacrolimus Binding Protein 1A / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; C113573:Cabin1 protein, rat; D065095:Calcineurin Inhibitors; D002147:Calmodulin; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D021122:Protein Subunits; C008644:nitrophenylphosphate; D016572:Cyclosporine; D019703:Calcineurin; D022061:Tacrolimus Binding Protein 1A; D016559:Tacrolimus