Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn²⁺. Calmodulin increased the Mn²⁺ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The K(m) of calcineurin A for ³²P-R(II) pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 μM with or without calmodulin, and calmodulin increased the V(max) about 4-fold. The K(m) of reconstituted calcineurin A plus B for ³²P-R(II) pep was 20 μM, and calmodulin increased the V(max) 18-fold without affecting the K(m). CaN A_467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn²⁺/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC₅₀'s of 25 μM and 90 μM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.