Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit

Yasuo Watanabe, Brian A. Perrino, Thomas R. Soderling

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    18 Scopus citations

    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 subnit 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 hydrophobia 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.

    Original languageEnglish (US)
    Pages (from-to)562-566
    Number of pages5
    JournalBiochemistry
    Volume35
    Issue number2
    StatePublished - Jan 16 1996

    ASJC Scopus subject areas

    • Biochemistry

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  • Cite this

    Watanabe, Y., Perrino, B. A., & Soderling, T. R. (1996). Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit. Biochemistry, 35(2), 562-566.