CALCINEURIN SUBUNIT INTERACTIONS - MAPPING THE CALCINEURIN-B BINDING DOMAIN ON CALCINEURIN-A

Recombinant forms of the A and B subunits of the protein phosphatase c alcineurin were produced in Escherichia coli, reconstituted into a het erodimer and purified to homogeneity. The reconstituted heterodimer ex hibited properties like that of bovine brain calcineurin. This include d calmodulin-stimulated activity and a subunit stoichiometry and Stoke s radius consistent with native-like structure. In order to map the re gion on the A subunit where calcineurin B binds, a series of overlappi ng 20-residue peptides corresponding to this putative domain were synt hesized. Using isolated calcineurin A and B subunits, an assay that re lied upon peptide inhibition of calcineurin B stimulation of calcineur in A activity was developed. All five peptides, but not a control pept ide, inhibited calcineurin B-dependent stimulation of calcineurin A al though with different potencies. The three most effective inhibitory p eptides spanned calcineurin A residues 338-377. These three peptides a lso altered the electrophoretic mobility of the isolated calcineurin B subunit during native polyacrylamide gel electrophoresis indicating a direct interaction between these peptides and calcineurin B. The pept ide corresponding to residues 348-367 was also able to block binding o f calcineurin B to the catalytic subunit.