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 (Hashimot o, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927) that regulates the Ca2+-dependent activation of its phosph atase activity, Substitution of Arg(476) and Arg(477) or Asp(467) to A la in the autoinhibitory peptide 457-482 significantly decreased its i nhibitory potency. CaN A subunits with these residues mutated to Ala w ere coexpressed with the Ca2+-binding B subunit using the baculovirus/ Sf9 cell system. Kinetic analysis showed that although the purified mu tants had no activity in the absence of calcium, they were less depend ent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the C OOH terminus of calcineurin, the A subunit was truncated at residues 4 57 or 420 and co expressed with B subunit. The V-max values of both tr uncation mutants with or without Ca2+ were increased relative to wild- type calcineurin. The increased Ca2+-independent activity of CaN420 re lative to CaN457 indicates the presence of additional autoinhibitory e lement(s) within residues 420-457. CaN420 had similar high V-max value s with or without Ca2+, but the K-m value for peptide substrate was in creased 5-fold to 125 mu M in the absence of Ca2+. The K-m values of a ll 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 indic ate that 1) there are several autoinhibitory motifs between the CaM-bi nding domain and the COOH terminus that are relieved by Ca2+ binding t o CaM and the B subunit, 2) Ca2+ binding to the B subunit also regulat es enzyme activity by lowering the K-m of the catalytic subunit for su bstrate, 3) binding of the B sub-unit is required for high V-max value s even after removal of the autoinhibitory domain. These results are c onsistent with synergistic activation of calcineurin by Ca2+ acting th rough both CaM and the B subunit.