Calcium Regulation of Calcineurin Phosphatase Activity by Its B Subunit and Calmodulin

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 co-expressed 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 Vmax values of both truncation mutants with or without Ca2+ were increased relative to wild-type calcineurin. The increased Ca2+-independent activity of CaN 420 relative to CaN 457 indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN 420 had similar high Vmax values with or without Ca2+, but the Km value for peptide substrate was increased 5-fold to 125 μM in the absence of Ca2+. The Km values of all the expressed calcineurin species were increased in the absence of Ca2+. The CaN A or CaN A 420 subunits alone have low Vmax and high Km (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 Km of the catalytic subunit for substrate, 3) binding of the B subunit is required for high Vmax 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.