Abstract

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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-independent activity of CaN420 relative to CaN457 indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN420 had similar high Vmax values with or without Ca2+, but the Km value for peptide substrate was increased 5-fold to 125 microM 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 A420 subunits alone have low Vmax and high Km (115 microM) 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.

Highlights

  • Calcineurin (CaN) contains an autoinhibitory element 43 residues COOH-terminal of the calmodulin-binding domain

  • 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 CaN A or CaN A4 20 subunits alone have low VII1ax and high K", (115 p,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 regulates enzyme activity by lowering the K", of the catalytic subunit for substrate, 3) binding of the B subunit is required for high VII1ax values even after removal of the autoinhibitory domain

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Summary

The abbreviations used are

A457, A subunit of CaN truncated at residue 457; A420, A subunit of CaN truncated at residue 420; CaM, calmodulin; CaN, calcineurin; CaN511, wild-type CaN containing the full-length A subunit; CaN., CaN containing the A457 subunit and B subunit; CaN.2o, CaN containing the A420 subunit and B subunit; Nmethyl-D-aspartate; PCR, polymerase chain reaction; PP-l, type-l tributed Ca2+/CaM-dependent Ser/Thr phosphoprotein phosphatase 2B (PP-2B) (reviewed in Ref. 1). CaN A has very low phosphatase activity by itself, but addition of Mn2+/CaM or Mn2+/B subunit gave 5- or 50-fold activations, respectively [12]. Limited proteolysis of CaN in the presence ofCa2+/CaM removes the residues COOH-terminal of the CaM-binding domain and generates a 57-kDa A subunit which still binds B subunit and CaM but which no longer requires Ca 2+ or CaM for full activity [14]. These results suggested the presence of a COOH-terminal autoinhibitory domain which was localized to residues 457-482 by use of overlapping synthetic peptides [17]. Kinetic analysis of the purified mutants allowed us to determine mechanisms by which Ca 2+ binding to its B subunit and to CaM activates CaN

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