Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin: Role of the autoinhibition domain

Calcineurin (CaN) contains an autoinhibitory element (residues 457-482) 43 residues COOH-terminal of the calmodulin-binding domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927) that regulates the Ca²⁺-dependent activation of its phosphatase activity. Substitution of Arg⁴⁷⁶ and Arg⁴⁷⁷ or Asp⁴⁶⁷ to Ala in the autoinhibitory peptide 457-482 significantly decreased its inhibitory potency. CaN A subunits with these residues mutated to Ala were coexpressed with the Ca²⁺-binding B subunit using the baculovirus/Sf9 cell system. Kinetic analysis showed that although the purified mutants had no activity in the absence of calcium, they were less dependent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the COOH terminus of calcineurin, the A subunit was truncated at residues 457 or 420 and co-expressed with B subunit. The V(max) values of both truncation mutants with or without Ca²⁺ were increased relative to wild-type calcineurin. The increased Ca²⁺- independent activity of CaN₄₂₀ relative to CaN₄₅₇ indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN₄₂₀ had similar high V(max) values with or without Ca²⁺, but the K(m) value for peptide substrate was increased 5-fold to 125 μM in the absence of Ca²⁺. The K(m) values of all the expressed calcineurin species were increased in the absence of Ca²⁺. The CaN or CaN A₄₂₀ subunits alone have low V(max) and high K(m) (115 μM) values even in the presence of Ca²⁺. These results indicate that 1) there are several autoinhibitory motifs between the CaM-binding domain and the COOH terminus that are relieved by Ca²⁺ binding to CaM and the B subunit, 2) Ca²⁺ binding to the B subunit also regulates enzyme activity by lowering the K(m) of the catalytic subunit for substrate, 3) binding of the B subunit is required for high V(max) values even after removal of the autoinhibitory domain. These results are consistent with synergistic activation of calcineurin by Ca²⁺ acting through both CaM and the B subunit.