CALCIUM REGULATION OF CALCINEURIN PHOSPHATASE-ACTIVITY BY ITS B-SUBUNIT AND CALMODULIN - ROLE OF THE AUTOINHIBITORY 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 Arg(476) and Arg(477) or Asp(467) 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 mu M in the absence of Ca2+. The K-m 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 V-max and high K-m (115 mu M) 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 sub-unit 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.