title: Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit.
abstract: The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subunit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. We have previously demonstrated that interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobic residues within the CaN A sequence 328-390 [Watanabe Y., Perrino, B.A., Chang, B.H., & Soderling, T.R. (1995) J. Biol. Chem. 270, 456-460]. In the present study, selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bound 45Ca2+ normally. Mutants BE-2 and BQ-2 interacted with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390), and they stimulated the phosphatase activity of the CaN A subunit in an in vitro reconstitution assay. Mutant BE-1 had a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gave very little stimulation of CaN A phosphatase activity. We conclude that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects [i.e., decrease Km and increase Vmax; Perrino, B.A., Ng, L.Y., & Soderling, T.R. (1995) J. Biol. Chem. 270, 340-346] of B subunit Ca2+ binding to stimulation of CaN A phosphatase activity.
authors.affiliation: Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Watanabe
authors.forename: B A
authors.initials: BA
authors.lastname: Perrino
authors.forename: T R
authors.initials: TR
authors.lastname: Soderling
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D051381:Rats; D017386:Sequence Homology, Amino Acid
chemical_list: D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium