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 language | English |
|---|---|
| Pages (from-to) | 3918-3928 |
| Number of pages | 11 |
| Journal | EMBO Journal |
| Volume | 23 |
| Issue number | 20 |
| DOIs | |
| Publication status | Published - 13 Oct 2004 |
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Keywords
- Inositol phosphates
- Phosphoinositides
- PI-3 kinase pathway
- Protein crystallography
- Protein structure
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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 journal › Article
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 - http://www.scopus.com/inward/record.url?scp=8144228588&partnerID=8YFLogxK
U2 - 10.1038/sj.emboj.7600379
DO - 10.1038/sj.emboj.7600379
M3 - Article
C2 - 15457207
AN - SCOPUS:8144228588
VL - 23
SP - 3918
EP - 3928
JO - EMBO Journal
JF - EMBO Journal
SN - 0261-4189
IS - 20
ER -