The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation

Edward J. McManus, Barry J. Collins, Peter R. Ashby, Alan R. Prescott, Victoria Murray-Tait, Laura J. Armit, J. Simon C. Arthur, Dario R. Alessi

    Research output: Contribution to journalArticle

    103 Citations (Scopus)

    Abstract

    We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that PtdIns(3,4,5)P3 binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that PtdIns(3,4,5)P 3 stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the PtdIns(3,4,5)P3 binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.

    Original languageEnglish
    Pages (from-to)2071-2082
    Number of pages12
    JournalEMBO Journal
    Volume23
    Issue number10
    DOIs
    Publication statusPublished - 19 May 2004

    Fingerprint

    3-Phosphoinositide-Dependent Protein Kinases
    Proto-Oncogene Proteins c-akt
    Mutation
    Ribosomal Protein S6 Kinases
    Embryonic Structures
    Chemical activation
    Phosphorylation
    Proteins
    Phosphatidylinositols
    Cell Size
    Protein Kinase C
    Catalyst activity
    Protein Isoforms
    Stabilization
    Cells
    phosphatidylinositol 3,4,5-triphosphate
    Pleckstrin Homology Domains
    platelet protein P47
    Technology
    Phenotype

    Keywords

    • mTOR
    • PI 3-kinase
    • PKB/Akt
    • PKC
    • RSK

    Cite this

    McManus, Edward J. ; Collins, Barry J. ; Ashby, Peter R. ; Prescott, Alan R. ; Murray-Tait, Victoria ; Armit, Laura J. ; Arthur, J. Simon C. ; Alessi, Dario R. / The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation. In: EMBO Journal. 2004 ; Vol. 23, No. 10. pp. 2071-2082.
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    abstract = "We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that PtdIns(3,4,5)P3 binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that PtdIns(3,4,5)P 3 stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the PtdIns(3,4,5)P3 binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.",
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    The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation. / McManus, Edward J.; Collins, Barry J.; Ashby, Peter R.; Prescott, Alan R.; Murray-Tait, Victoria; Armit, Laura J.; Arthur, J. Simon C.; Alessi, Dario R.

    In: EMBO Journal, Vol. 23, No. 10, 19.05.2004, p. 2071-2082.

    Research output: Contribution to journalArticle

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    AU - McManus, Edward J.

    AU - Collins, Barry J.

    AU - Ashby, Peter R.

    AU - Prescott, Alan R.

    AU - Murray-Tait, Victoria

    AU - Armit, Laura J.

    AU - Arthur, J. Simon C.

    AU - Alessi, Dario R.

    PY - 2004/5/19

    Y1 - 2004/5/19

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    AB - We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that PtdIns(3,4,5)P3 binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that PtdIns(3,4,5)P 3 stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the PtdIns(3,4,5)P3 binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.

    KW - mTOR

    KW - PI 3-kinase

    KW - PKB/Akt

    KW - PKC

    KW - RSK

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