Regulation of Calcineurin Phosphatase Activity by Its Autoinhibitory Domain

The Ca2+-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 Ca2+/calmodulin-independent activity. Full activity in the absence of Ca2+ 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 (AI420–511) or 328–511 (AI328–511). Using recombinant, Ca2+/calmodulin-independent calcineurin, AI420–511 and AI328–511 were three- to fourfold more potent inhibitors of calcineurin phosphatase activity than the synthetic calcineurin autoinhibitory peptide457–482. Calmodulin reversed the inhibition of calcineurin phosphatase activity by AI328–511 but not AI420–511. Kinetic studies indicated that AI420–511 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.