Regulation of calcineurin phosphatase activity by its autoinhibitory domain.

The Ca(2+)-dependent activation of calcineurin phosphatase activity is regulated by an autoinhibitory element (residues 457-482) located 43 residues COOH-terminal of the calmodulin-binding domain (residues 390-414). Removal of residues 457-482 does not result in full Ca(2+)/calmodulin-independent activity. Full activity in the absence of Ca(2+) requires the removal of residues 420-457. In the present study the presence of additional autoinhibitory elements within residues 420-457 was tested using two calcineurin A subunit COOH-terminal region constructs containing residues 420-511 (AI(420-511)) or 328-511 (AI(328-511)). Using recombinant, Ca(2+)/calmodulin-independent calcineurin, AI(420-511) and AI(328-511) were three- to fourfold more potent inhibitors of calcineurin phosphatase activity than the synthetic calcineurin autoinhibitory peptide(457-482). Calmodulin reversed the inhibition of calcineurin phosphatase activity by AI(328-511) but not AI(420-511). Kinetic studies indicated that AI(420-511) exhibited mixed-type inhibition and that the enzyme/substrate/inhibitor complex is partially active. These results indicate that (i) additional autoinhibitory elements are present within residues 420-457, (ii) calmodulin-binding to the autoinhibitory domain neutralizes the inhibitory function of the 420-457 autoinhibitory segment, (iii) the full-length autoinhibitory domain is a mixed-type inhibitor of calcineurin phosphatase activity, and (iv) the enzyme/substrate/inhibitor complex is partially catalytically active. Copyright 1999 Academic Press.