Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

H. K. Liu, S. Perrier, C. Lipina, D. Finlay, H. McLauchlan, C. J. Hastie, H. S. Hundal, C Sutherland, C. Sutherland

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    Abstract

    Background: Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBP alpha) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBP alpha is a link between GSK3 and these gene promoters.

    Results: C/EBP alpha represses the IGFBP1 thymine-rich insulin response element (TIRE), but mutation of T222 or T226 of C/EBP alpha to non-phosphorylatable alanines has no effect on C/EBP alpha activity in liver cells (towards the TIRE or a consensus C/EBP binding sequence). Phosphorylation of T222/ T226 is decreased by GSK3 inhibition, suggesting GSK3 does phosphorylate T222/226 in intact cells. However, phosphorylation was not altered by treatment of liver cells with insulin. Meanwhile C/EBP alpha activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/ T226 and/ or S230 to alanine residues. Finally, we demonstrate that C/EBP alpha is a very poor substrate for GSK3 in vitro and in cells.

    Conclusion: The work demonstrates an important role for this domain in the regulation of C/EBP alpha activity in adipocytes but not hepatocytes, however GSK3 phosphorylation of these residues does not mediate regulation of this C/EBP activity. In short, we find no evidence that C/EBP alpha activity is regulated by direct phosphorylation by GSK3.

    Original languageEnglish
    Article number14
    Pages (from-to)-
    Number of pages12
    JournalBMC Molecular Biology
    Volume7
    DOIs
    Publication statusPublished - 6 Apr 2006

    Cite this

    @article{f3c238971277425c9bb8a56749bbdac0,
    title = "Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226",
    abstract = "Background: Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBP alpha) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBP alpha is a link between GSK3 and these gene promoters.Results: C/EBP alpha represses the IGFBP1 thymine-rich insulin response element (TIRE), but mutation of T222 or T226 of C/EBP alpha to non-phosphorylatable alanines has no effect on C/EBP alpha activity in liver cells (towards the TIRE or a consensus C/EBP binding sequence). Phosphorylation of T222/ T226 is decreased by GSK3 inhibition, suggesting GSK3 does phosphorylate T222/226 in intact cells. However, phosphorylation was not altered by treatment of liver cells with insulin. Meanwhile C/EBP alpha activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/ T226 and/ or S230 to alanine residues. Finally, we demonstrate that C/EBP alpha is a very poor substrate for GSK3 in vitro and in cells.Conclusion: The work demonstrates an important role for this domain in the regulation of C/EBP alpha activity in adipocytes but not hepatocytes, however GSK3 phosphorylation of these residues does not mediate regulation of this C/EBP activity. In short, we find no evidence that C/EBP alpha activity is regulated by direct phosphorylation by GSK3.",
    author = "Liu, {H. K.} and S. Perrier and C. Lipina and D. Finlay and H. McLauchlan and Hastie, {C. J.} and Hundal, {H. S.} and C Sutherland and C. Sutherland",
    year = "2006",
    month = "4",
    day = "6",
    doi = "10.1186/1471-2199-7-14",
    language = "English",
    volume = "7",
    pages = "--",
    journal = "BMC Molecular Biology",
    issn = "1471-2199",
    publisher = "Springer Verlag",

    }

    TY - JOUR

    T1 - Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

    AU - Liu, H. K.

    AU - Perrier, S.

    AU - Lipina, C.

    AU - Finlay, D.

    AU - McLauchlan, H.

    AU - Hastie, C. J.

    AU - Hundal, H. S.

    AU - Sutherland, C

    AU - Sutherland, C.

    PY - 2006/4/6

    Y1 - 2006/4/6

    N2 - Background: Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBP alpha) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBP alpha is a link between GSK3 and these gene promoters.Results: C/EBP alpha represses the IGFBP1 thymine-rich insulin response element (TIRE), but mutation of T222 or T226 of C/EBP alpha to non-phosphorylatable alanines has no effect on C/EBP alpha activity in liver cells (towards the TIRE or a consensus C/EBP binding sequence). Phosphorylation of T222/ T226 is decreased by GSK3 inhibition, suggesting GSK3 does phosphorylate T222/226 in intact cells. However, phosphorylation was not altered by treatment of liver cells with insulin. Meanwhile C/EBP alpha activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/ T226 and/ or S230 to alanine residues. Finally, we demonstrate that C/EBP alpha is a very poor substrate for GSK3 in vitro and in cells.Conclusion: The work demonstrates an important role for this domain in the regulation of C/EBP alpha activity in adipocytes but not hepatocytes, however GSK3 phosphorylation of these residues does not mediate regulation of this C/EBP activity. In short, we find no evidence that C/EBP alpha activity is regulated by direct phosphorylation by GSK3.

    AB - Background: Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBP alpha) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBP alpha is a link between GSK3 and these gene promoters.Results: C/EBP alpha represses the IGFBP1 thymine-rich insulin response element (TIRE), but mutation of T222 or T226 of C/EBP alpha to non-phosphorylatable alanines has no effect on C/EBP alpha activity in liver cells (towards the TIRE or a consensus C/EBP binding sequence). Phosphorylation of T222/ T226 is decreased by GSK3 inhibition, suggesting GSK3 does phosphorylate T222/226 in intact cells. However, phosphorylation was not altered by treatment of liver cells with insulin. Meanwhile C/EBP alpha activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/ T226 and/ or S230 to alanine residues. Finally, we demonstrate that C/EBP alpha is a very poor substrate for GSK3 in vitro and in cells.Conclusion: The work demonstrates an important role for this domain in the regulation of C/EBP alpha activity in adipocytes but not hepatocytes, however GSK3 phosphorylation of these residues does not mediate regulation of this C/EBP activity. In short, we find no evidence that C/EBP alpha activity is regulated by direct phosphorylation by GSK3.

    U2 - 10.1186/1471-2199-7-14

    DO - 10.1186/1471-2199-7-14

    M3 - Article

    C2 - 16600022

    VL - 7

    SP - -

    JO - BMC Molecular Biology

    JF - BMC Molecular Biology

    SN - 1471-2199

    M1 - 14

    ER -