Enzymology
Calcium Regulation of Calcineurin Phosphatase Activity by Its B Subunit and Calmodulin: ROLE OF THE AUTOINHIBITORY DOMAIN (∗)

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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 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 A or CaN A 420 subunits alone have low Vmax 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 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.

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This work was supported in part by National Institutes of Health Grants DK17808 and GM41292. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supported by Neuroendocrinology Training Grant DK07680.