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Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates
Author(s) -
Komander David,
Fairservice Alison,
Deak Maria,
Kular Gursant S,
Prescott Alan R,
Peter Downes C,
Safrany Stephen T,
Alessi Dario R,
van Aalten Daan MF
Publication year - 2004
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/sj.emboj.7600379
Subject(s) - biology , inositol , biochemistry , microbiology and biotechnology , receptor
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)P 3 /PtdIns(3,4)P 2 and with lower affinity to PtdIns(4,5)P 2 . We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P 3 and Ins(1,3,4,5)P 4 . 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)P 5 and InsP 6 , 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)P 5 /InsP 6 , 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.