Substrate recognition by the cell surface palmitoyl transferase DHHC5

Jacqueline Howie, Louise Reilly, Niall J. Fraser, Julia Vlachaki Walker, Krzysztof J. Wypijewski, Michael L. J. Ashford, Sarah Calaghan, Heather McClafferty, Lijun Tian, Michael J. Shipston, Andrii Boguslavskyi, Michael J. Shattock, Will Fuller (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    33 Citations (Scopus)

    Abstract

    The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discslarge/ ZO-1 homology (PDZ) binding motif, but requires a ?120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipids interactions that inhibit the Na pump.

    Original languageEnglish
    Pages (from-to)17534-17539
    Number of pages6
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume111
    Issue number49
    Early online date24 Nov 2014
    DOIs
    Publication statusPublished - 9 Dec 2014

    Fingerprint

    Transferases
    Lipoylation
    Acyltransferases
    Cysteine
    phospholemman
    Caveolins
    Sodium-Potassium-Exchanging ATPase
    Phosphoproteins
    Sulfhydryl Compounds
    Muscle Cells
    Phospholipids
    Protein Isoforms
    Fatty Acids
    Carbon
    Amino Acids
    Muscles
    Enzymes

    Keywords

    • DHHC
    • Ion transport
    • Palmitoylation
    • Phospholemman
    • Sodium pump

    Cite this

    Howie, Jacqueline ; Reilly, Louise ; Fraser, Niall J. ; Vlachaki Walker, Julia ; Wypijewski, Krzysztof J. ; Ashford, Michael L. J. ; Calaghan, Sarah ; McClafferty, Heather ; Tian, Lijun ; Shipston, Michael J. ; Boguslavskyi, Andrii ; Shattock, Michael J. ; Fuller, Will. / Substrate recognition by the cell surface palmitoyl transferase DHHC5. In: Proceedings of the National Academy of Sciences of the United States of America. 2014 ; Vol. 111, No. 49. pp. 17534-17539.
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    abstract = "The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discslarge/ ZO-1 homology (PDZ) binding motif, but requires a ?120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipids interactions that inhibit the Na pump.",
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    author = "Jacqueline Howie and Louise Reilly and Fraser, {Niall J.} and {Vlachaki Walker}, Julia and Wypijewski, {Krzysztof J.} and Ashford, {Michael L. J.} and Sarah Calaghan and Heather McClafferty and Lijun Tian and Shipston, {Michael J.} and Andrii Boguslavskyi and Shattock, {Michael J.} and Will Fuller",
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    Howie, J, Reilly, L, Fraser, NJ, Vlachaki Walker, J, Wypijewski, KJ, Ashford, MLJ, Calaghan, S, McClafferty, H, Tian, L, Shipston, MJ, Boguslavskyi, A, Shattock, MJ & Fuller, W 2014, 'Substrate recognition by the cell surface palmitoyl transferase DHHC5', Proceedings of the National Academy of Sciences of the United States of America, vol. 111, no. 49, pp. 17534-17539. https://doi.org/10.1073/pnas.1413627111

    Substrate recognition by the cell surface palmitoyl transferase DHHC5. / Howie, Jacqueline; Reilly, Louise; Fraser, Niall J.; Vlachaki Walker, Julia ; Wypijewski, Krzysztof J.; Ashford, Michael L. J.; Calaghan, Sarah; McClafferty, Heather; Tian, Lijun; Shipston, Michael J.; Boguslavskyi, Andrii; Shattock, Michael J.; Fuller, Will (Lead / Corresponding author).

    In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 111, No. 49, 09.12.2014, p. 17534-17539.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Substrate recognition by the cell surface palmitoyl transferase DHHC5

    AU - Howie, Jacqueline

    AU - Reilly, Louise

    AU - Fraser, Niall J.

    AU - Vlachaki Walker, Julia

    AU - Wypijewski, Krzysztof J.

    AU - Ashford, Michael L. J.

    AU - Calaghan, Sarah

    AU - McClafferty, Heather

    AU - Tian, Lijun

    AU - Shipston, Michael J.

    AU - Boguslavskyi, Andrii

    AU - Shattock, Michael J.

    AU - Fuller, Will

    PY - 2014/12/9

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    N2 - The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discslarge/ ZO-1 homology (PDZ) binding motif, but requires a ?120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipids interactions that inhibit the Na pump.

    AB - The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discslarge/ ZO-1 homology (PDZ) binding motif, but requires a ?120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipids interactions that inhibit the Na pump.

    KW - DHHC

    KW - Ion transport

    KW - Palmitoylation

    KW - Phospholemman

    KW - Sodium pump

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