14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase

Mercedes Pozuelo-Rubio, Mark Peggie, Barry H. C. Wong, Nick Morrice, Carol MacKintosh

    Research output: Contribution to journalArticle

    68 Citations (Scopus)

    Abstract

    The cardiac isoform of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2), regulator of the glycolysis-stimulating fructose-2,6-bisphosphate, was among human HeLa cell proteins that were eluted from a 14-3-3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho-specific antibodies showed that Ser466 and Ser483 of 14-3-3-affinity-purified PFK-2 were phosphorylated. 14-3-3 binding was abolished by selectively dephosphorylating Ser483, and 14-3-3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS483VGS blocked binding of PFK-2 to 14-3-3s. These data indicate that 14-3-3s bind to phosphorylated Ser483. When HeLa cells expressing HA-tagged PFK-2 were co-transfected with active PKB or stimulated with IGF-1, HA-PFK-2 was phosphorylated and bound to 14-3-3s. The response to IGF-1 was abolished by PI 3-kinase inhibitors. In addition, IGF-1 promoted the binding of endogenous PFK-2 to 14-3-3s. When cells were transduced with penetratin-linked AARAApSAPA, we found that this reagent bound specifically to 14-3-3s, blocked the IGF-1-induced binding of HA-PFK-2 to 14-3-3s, and completely inhibited the IGF-1-induced increase in cellular fructose-2,6-bisphosphate. These findings suggest that PKB-dependent binding of 14-3-3s to phospho-Ser483 of cardiac PFK-2 mediates the stimulation of glycolysis by growth factor.
    Original languageEnglish
    Pages (from-to)3514-3523
    Number of pages10
    JournalThe EMBO Journal
    Volume22
    Issue number14
    DOIs
    Publication statusPublished - 15 Jul 2003

    Fingerprint

    Insulin-Like Growth Factor I
    Phosphoric Monoester Hydrolases
    Phosphotransferases
    Phosphopeptides
    Glycolysis
    HeLa Cells
    Phosphofructokinase-2
    Phospho-Specific Antibodies
    AMP-Activated Protein Kinases
    Phosphatidylinositol 3-Kinases
    Intercellular Signaling Peptides and Proteins
    Protein Isoforms
    Cells
    fructose 2,6-diphosphate
    Proteins

    Keywords

    • Glycolysis
    • Growth factors
    • Pasteur effect
    • Penetratin
    • Warburg effect

    Cite this

    Pozuelo-Rubio, Mercedes ; Peggie, Mark ; Wong, Barry H. C. ; Morrice, Nick ; MacKintosh, Carol. / 14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase. In: The EMBO Journal. 2003 ; Vol. 22, No. 14. pp. 3514-3523.
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    abstract = "The cardiac isoform of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2), regulator of the glycolysis-stimulating fructose-2,6-bisphosphate, was among human HeLa cell proteins that were eluted from a 14-3-3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho-specific antibodies showed that Ser466 and Ser483 of 14-3-3-affinity-purified PFK-2 were phosphorylated. 14-3-3 binding was abolished by selectively dephosphorylating Ser483, and 14-3-3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS483VGS blocked binding of PFK-2 to 14-3-3s. These data indicate that 14-3-3s bind to phosphorylated Ser483. When HeLa cells expressing HA-tagged PFK-2 were co-transfected with active PKB or stimulated with IGF-1, HA-PFK-2 was phosphorylated and bound to 14-3-3s. The response to IGF-1 was abolished by PI 3-kinase inhibitors. In addition, IGF-1 promoted the binding of endogenous PFK-2 to 14-3-3s. When cells were transduced with penetratin-linked AARAApSAPA, we found that this reagent bound specifically to 14-3-3s, blocked the IGF-1-induced binding of HA-PFK-2 to 14-3-3s, and completely inhibited the IGF-1-induced increase in cellular fructose-2,6-bisphosphate. These findings suggest that PKB-dependent binding of 14-3-3s to phospho-Ser483 of cardiac PFK-2 mediates the stimulation of glycolysis by growth factor.",
    keywords = "Glycolysis, Growth factors, Pasteur effect, Penetratin, Warburg effect",
    author = "Mercedes Pozuelo-Rubio and Mark Peggie and Wong, {Barry H. C.} and Nick Morrice and Carol MacKintosh",
    note = "dc.publisher: Nature Publishing Group dc.description.sponsorship: We thank Agnieszka Kieloch and Richard Grier for culturing cells, colleagues at the DSTT for supplies, Maria Deak for PKB constructs, Grahame Hardie for AMPK, and Alan Prescott for help with cell imaging. We also thank the European Community programme 'Quality of Life and Management of Living Resources' for a Marie Curie Fellowship to M.P.R. under contract No. QLK1-CT-2000-51184, the Wellcome Trust for a studentship to B.W., the BBSRC and MRC for grants to C.M., and the pharmaceutical companies that support DSTT (AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Novo-Nordisk and Pfizer).",
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    14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase. / Pozuelo-Rubio, Mercedes; Peggie, Mark; Wong, Barry H. C.; Morrice, Nick; MacKintosh, Carol.

    In: The EMBO Journal, Vol. 22, No. 14, 15.07.2003, p. 3514-3523.

    Research output: Contribution to journalArticle

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    AU - Pozuelo-Rubio, Mercedes

    AU - Peggie, Mark

    AU - Wong, Barry H. C.

    AU - Morrice, Nick

    AU - MacKintosh, Carol

    N1 - dc.publisher: Nature Publishing Group dc.description.sponsorship: We thank Agnieszka Kieloch and Richard Grier for culturing cells, colleagues at the DSTT for supplies, Maria Deak for PKB constructs, Grahame Hardie for AMPK, and Alan Prescott for help with cell imaging. We also thank the European Community programme 'Quality of Life and Management of Living Resources' for a Marie Curie Fellowship to M.P.R. under contract No. QLK1-CT-2000-51184, the Wellcome Trust for a studentship to B.W., the BBSRC and MRC for grants to C.M., and the pharmaceutical companies that support DSTT (AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Novo-Nordisk and Pfizer).

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    KW - Glycolysis

    KW - Growth factors

    KW - Pasteur effect

    KW - Penetratin

    KW - Warburg effect

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