Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity.

SL Davies, A Ozawa, WD McCormick, MM Dvorak, DT Ward

    Research output: Contribution to journalArticleResearchpeer-review

    Abstract

    The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr888 contributing significantly to this effect. To determine whether CaRT888 is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaRpT888). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaRT888A, phorbol ester (PMA) treatment increased CaRpT888 immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca2+ concentration from 0.5 to 2.5 mM increased CaRT888 phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca2+ and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaRT888A did exhibit increased apparent agonist sensitivity, by converting intracellular Ca2+ (Ca2+i) oscillations to sustained plateau responses in some cells, we still observed Ca2+i oscillations in a significant number of cells. This suggests that CaRT888 contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca2+i oscillations. Finally, dephosphorylation of CaRT888 was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca2+i oscillations. In addition, calyculin/PMA cotreatment increased CaRT888 phosphorylation in bovine parathyroid cells. Therefore, CaRT888 is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.
    Original languageEnglish
    Pages (from-to)15048
    Number of pages15056
    JournalThe Journal of Biological Chemistry
    Volume282
    Early online date21 Mar 2007
    DOIs
    Publication statusPublished - May 2007

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    Calcium-Sensing Receptors
    Phosphoric Monoester Hydrolases
    Protein Kinase C
    Phosphorylation
    Phospho-Specific Antibodies
    Protein Phosphatase 1
    Protein Phosphatase 2
    HEK293 Cells
    Immunoblotting
    Fluorescent Antibody Technique
    Cell Count
    Therapeutics

    Cite this

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    title = "Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity.",
    abstract = "The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr888 contributing significantly to this effect. To determine whether CaRT888 is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaRpT888). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaRT888A, phorbol ester (PMA) treatment increased CaRpT888 immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca2+ concentration from 0.5 to 2.5 mM increased CaRT888 phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca2+ and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaRT888A did exhibit increased apparent agonist sensitivity, by converting intracellular Ca2+ (Ca2+i) oscillations to sustained plateau responses in some cells, we still observed Ca2+i oscillations in a significant number of cells. This suggests that CaRT888 contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca2+i oscillations. Finally, dephosphorylation of CaRT888 was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca2+i oscillations. In addition, calyculin/PMA cotreatment increased CaRT888 phosphorylation in bovine parathyroid cells. Therefore, CaRT888 is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.",
    author = "SL Davies and A Ozawa and WD McCormick and MM Dvorak and DT Ward",
    year = "2007",
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    doi = "10.1074/jbc.m607469200",
    language = "English",
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    Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity. / Davies, SL; Ozawa, A; McCormick, WD; Dvorak, MM; Ward, DT.

    In: The Journal of Biological Chemistry, Vol. 282, 05.2007, p. 15048.

    Research output: Contribution to journalArticleResearchpeer-review

    TY - JOUR

    T1 - Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity.

    AU - Davies, SL

    AU - Ozawa, A

    AU - McCormick, WD

    AU - Dvorak, MM

    AU - Ward, DT

    PY - 2007/5

    Y1 - 2007/5

    N2 - The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr888 contributing significantly to this effect. To determine whether CaRT888 is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaRpT888). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaRT888A, phorbol ester (PMA) treatment increased CaRpT888 immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca2+ concentration from 0.5 to 2.5 mM increased CaRT888 phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca2+ and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaRT888A did exhibit increased apparent agonist sensitivity, by converting intracellular Ca2+ (Ca2+i) oscillations to sustained plateau responses in some cells, we still observed Ca2+i oscillations in a significant number of cells. This suggests that CaRT888 contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca2+i oscillations. Finally, dephosphorylation of CaRT888 was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca2+i oscillations. In addition, calyculin/PMA cotreatment increased CaRT888 phosphorylation in bovine parathyroid cells. Therefore, CaRT888 is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.

    AB - The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr888 contributing significantly to this effect. To determine whether CaRT888 is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaRpT888). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaRT888A, phorbol ester (PMA) treatment increased CaRpT888 immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca2+ concentration from 0.5 to 2.5 mM increased CaRT888 phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca2+ and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaRT888A did exhibit increased apparent agonist sensitivity, by converting intracellular Ca2+ (Ca2+i) oscillations to sustained plateau responses in some cells, we still observed Ca2+i oscillations in a significant number of cells. This suggests that CaRT888 contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca2+i oscillations. Finally, dephosphorylation of CaRT888 was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca2+i oscillations. In addition, calyculin/PMA cotreatment increased CaRT888 phosphorylation in bovine parathyroid cells. Therefore, CaRT888 is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.

    UR - http://europepmc.org/abstract/med/17376781

    U2 - 10.1074/jbc.m607469200

    DO - 10.1074/jbc.m607469200

    M3 - Article

    VL - 282

    SP - 15048

    JO - The Journal of Biological Chemistry

    JF - The Journal of Biological Chemistry

    SN - 0021-9258

    ER -