IDENTIFICATION IN THE CALCINEURIN A SUBUNIT OF THE DOMAIN THAT BINDS THE REGULATORY B-SUBUNIT

Calcineurin (CaN) is the serine/threonine protein phosphatase (phospha tase 2B) that is activated by binding of Ca2+ to its B subunit and to calmodulin (CaM). This paper identifies residues between the catalytic region and the CaM-binding domain of the A subunit as the domain that binds the regulatory B subunit.A purified fusion protein containing r esidues 328-390 of the A subunit 1) binds CaN B subunit, and 2) inhibi ts (IC50 = 0.1 mu M) the in vitro stimulation of CaN A phosphatase act ivity by purified CaN B subunit. A synthetic peptide corresponding to residues 341-360 blocked the binding of CaN B to residues 328-390 in t he fusion protein, so 4 hydrophobic residues within this region (Val(3 49)-Phe(350) and Phe(356)-Val(357)) were mutated to either Glu (E muta nt) or Gin (Q mutant). The wild-type and mutant A subunits were expres sed individually or coexpressed with B subunit in Sf9 cells, purified and characterized. The mutant A subunits were similar to wildtype A su bunit in terms of basal phosphatase activity (1-3 nmol/min/mg) and act ivation by Mn2+/CaM. Addition of purified B subunit to purified wild-t ype A subunit at a 1:1 molar ratio gave a 40-fold increase in phosphat ase activity whereas addition of B subunit to either of the mutant A s ubunits had no effect on phosphatase activity, even at a 3:1 molar exc ess of B subunit. Furthermore, when wild-type or mutant A subunits wer e coexpressed with B subunit and purified on CaM-Sepharose, the B subu nit co-eluted with the wild-type A subunit but not with either mutant A subunit. These results demonstrate that residues 328-390 in the A su bunit bind B subunit and that the mutated hydrophobic residues are ess ential.