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

Yasuo Watanabe, Brian A. Perrino, Thomas Soderling

    Research output: Contribution to journalArticle

    18 Citations (Scopus)

    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

    Fingerprint

    Calcineurin
    Phosphoric Monoester Hydrolases
    Chemical activation
    Calcium
    Rabies
    Phosphoprotein Phosphatases
    Calmodulin
    Assays
    Catalytic Domain

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

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

    Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit. / Watanabe, Yasuo; Perrino, Brian A.; Soderling, Thomas.

    In: Biochemistry, Vol. 35, No. 2, 16.01.1996, p. 562-566.

    Research output: Contribution to journalArticle

    Watanabe, Y, Perrino, BA & Soderling, T 1996, 'Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit', Biochemistry, vol. 35, no. 2, pp. 562-566.
    Watanabe, Yasuo ; Perrino, Brian A. ; Soderling, Thomas. / Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit. In: Biochemistry. 1996 ; Vol. 35, No. 2. pp. 562-566.
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    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.",
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    AB - 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.

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