Identification in the calcineurin A subunit of the domain that binds the regulatory B subunit.
Calcineurin (CaN) is the serine/threonine protein phosphatase (phosphatase 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 residues 328-390 of the A subunit 1) binds CaN B subunit, and 2) inhibits (IC50 = 0.1 microM) the in vitro stimulation of CaN A phosphatase activity by purified CaN B subunit. A synthetic peptide corresponding to residues 341-360 blocked the binding of CaN B to residues 328-390 in the fusion protein, so 4 hydrophobic residues within this region (Val349-Phe350 and Phe356-Val357) were mutated to either Glu (E mutant) or Gln (Q mutant). The wild-type and mutant A subunits were expressed individually or coexpressed with B subunit in Sf9 cells, purified and characterized. The mutant A subunits were similar to wild-type A subunit in terms of basal phosphatase activity (1-3 nmol/min/mg) and activation by Mn2+/CaM. Addition of purified B subunit to purified wild-type A subunit at a 1:1 molar ratio gave a 40-fold increase in phosphatase activity whereas addition of B subunit to either of the mutant A subunits had no effect on phosphatase activity, even at a 3:1 molar excess of B subunit. Furthermore, when wild-type or mutant A subunits were coexpressed with B subunit and purified on CaM-Sepharose, the B subunit 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 subunit bind B subunit and that the mutated hydrophobic residues are essential.
Watanabe
Y
Y
Vollum Institute, Oregon Health Sciences University, Portland 97201.
Perrino
B A
BA
Chang
B H
BH
Soderling
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D007313:Insecta; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats
D002148:Calmodulin-Binding Proteins; D009838:Oligodeoxyribonucleotides; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases