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

TY - JOUR

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

Y1 - 2004/10/13

N2 - 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.

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.

KW - Inositol phosphates

KW - Phosphoinositides

KW - PI-3 kinase pathway

KW - Protein crystallography

KW - Protein structure

UR - https://www.scopus.com/pages/publications/8144228588

U2 - 10.1038/sj.emboj.7600379

DO - 10.1038/sj.emboj.7600379

M3 - Article

C2 - 15457207

AN - SCOPUS:8144228588

SN - 0261-4189

VL - 23

SP - 3918

EP - 3928

JO - EMBO Journal

JF - EMBO Journal

IS - 20

ER -