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| Subject Categories: Structural Biology | Signal Transduction |
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| The EMBO Journal
(2004) 23, 3918–3928, doi:10.1038/sj.emboj.7600379 Published online 30 September 2004 |
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| Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates |
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| David Komander1, 3, Alison Fairservice2, Maria Deak3, Gursant S Kular3, Alan R Prescott4, C Peter Downes2, Stephen T Safrany2, Dario R Alessi3 and Daan M F van Aalten1 |
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1 Division of Biological Chemistry & Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, Scotland, UK 2 Division of Cell Signalling, School of Life Sciences, University of Dundee, Dundee, Scotland, UK 3 MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee, Scotland, UK 4 Division of Cell Biology and Immunology, School of Life Sciences, University of Dundee, Dundee, Scotland, UK To whom correspondence should be addressed Daan M F van Aalten, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, MSI/WTB Complex, Dundee DD1 5EH, UK. Tel.: +44 1382 344 979; Fax: +44 1382 345 764; E-mail: dava@davapc1.bioch.dundee.ac.uk Received 2 June 2004; Accepted 29 July 2004; Published online 30 September 2004. |
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| Abstract |
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| 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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| Keywords: inositol phosphates, phosphoinositides, PI-3 kinase pathway, protein crystallography, protein structure |
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