Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

David Komander, Alison Fairservice, Maria Deak, Gursant S. Kular, Alan R. Prescott, C. Peter Downes, Stephen T. Safrany, Dario R. Alessi, Daan M.F. Van Aalten

    Research output: Contribution to journalArticle

    131 Citations (Scopus)

    Abstract

    3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.

    Original languageEnglish
    Pages (from-to)3918-3928
    Number of pages11
    JournalEMBO Journal
    Volume23
    Issue number20
    DOIs
    Publication statusPublished - 13 Oct 2004

    Fingerprint

    3-Phosphoinositide-Dependent Protein Kinases
    Inositol Phosphates
    Phosphatidylinositols
    Cytosol
    90-kDa Ribosomal Protein S6 Kinases
    Phosphatidylinositol 4,5-Diphosphate
    70-kDa Ribosomal Protein S6 Kinases
    Mutagenesis
    Anchors
    Assays
    Phosphotransferases
    Crystal structure
    Binding Sites
    Ligands
    Lipids
    platelet protein P47
    Pleckstrin Homology Domains
    Substrates

    Keywords

    • Inositol phosphates
    • Phosphoinositides
    • PI-3 kinase pathway
    • Protein crystallography
    • Protein structure

    Cite this

    Komander, David ; Fairservice, Alison ; Deak, Maria ; Kular, Gursant S. ; Prescott, Alan R. ; Downes, C. Peter ; Safrany, Stephen T. ; Alessi, Dario R. ; Van Aalten, Daan M.F. / Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates. In: EMBO Journal. 2004 ; Vol. 23, No. 20. pp. 3918-3928.
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    abstract = "3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.",
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    Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates. / Komander, David; Fairservice, Alison; Deak, Maria; Kular, Gursant S.; Prescott, Alan R.; Downes, C. Peter; Safrany, Stephen T.; Alessi, Dario R.; Van Aalten, Daan M.F.

    In: EMBO Journal, Vol. 23, No. 20, 13.10.2004, p. 3918-3928.

    Research output: Contribution to journalArticle

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    T1 - Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

    AU - Komander, David

    AU - Fairservice, Alison

    AU - Deak, Maria

    AU - Kular, Gursant S.

    AU - Prescott, Alan R.

    AU - Downes, C. Peter

    AU - Safrany, Stephen T.

    AU - Alessi, Dario R.

    AU - Van Aalten, Daan M.F.

    PY - 2004/10/13

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    AB - 3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.

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    KW - PI-3 kinase pathway

    KW - Protein crystallography

    KW - Protein structure

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