ACTIVATION OF CALCINEURIN A-SUBUNIT PHOSPHATASE-ACTIVITY BY ITS CALCIUM-BINDING B-SUBUNIT

The protein phosphatase activity of calcineurin (CaN) is activated thr ough calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B su bnit is required for either binding to the CaN A subunit or for transd ucing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. We have previously demonstrated th at interaction of CaN B regulatory subunit with the CaN A catalytic su bunit requires hydrophobic residues within the CaN A sequence 328-390 [Watanabe Y., Perrino, B. A., Chang, B. H., & Soderling, T. R. (1995) J. Biol. Chem. 270, 456-460]. In the present study, selected hydrophob ic residues within the B subunit were mutated to Glu to Gln. CaN B sub unit mutants BE-1 (Val(115)/Leu(116) to Glu), BE-2 (Val(156/157/168/16 9) to Glu), and BQ-2 (Val(156/157/168/169) to Gln) were expressed and purified. The three mutant B subunits bound Ca-45(2+) normally. Mutant s BE-2 and BQ-2 interacted with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390), and th ey stimulated the phosphatase activity of the CaN A subunit in an in v itro reconstitution assay. Mutant BE-1 had a 3-fold reduced affinity f or binding CaN A, and this mutant, even at saturating concentrations, gave very little stimulation of CaN A phosphatase activity. We conclud e that residues Val(115)/Leu(116) in the B subunit participate in high -affinity binding to the A subunit and are required for transducing th e effects [i.e., decrease K-m and increase V-max; Perrino, B. A., Ng, L. Y., & Soderling, T. R. (1995) J. Biol. Chem. 270, 340-346] of B sub unit Ca2+ binding to stimulation of CaN A phosphatase activity.