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 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.
Perrino
B A
BA
Vollum Institute, Oregon Health Sciences University, Portland 97201.
Ng
L Y
LY
Soderling
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D018411:Spodoptera; D013379:Substrate Specificity
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium