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PDBsum entry 1w1h
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References listed in PDB file
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Key reference
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Title
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Structural insights into the regulation of pdk1 by phosphoinositides and inositol phosphates.
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Authors
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D.Komander,
A.Fairservice,
M.Deak,
G.S.Kular,
A.R.Prescott,
C.Peter downes,
S.T.Safrany,
D.R.Alessi,
D.M.Van aalten.
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Ref.
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EMBO J, 2004,
23,
3918-3928.
[DOI no: ]
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PubMed id
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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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Figure 2.
Figure 2 Comparison of Ins(1,3,4,5)P[4] binding to the PH
domains of PDK1, DAPP1 and PKB  .
Interactions of Ins(1,3,4,5)P[4] (marine) with protein residues
(green in PDK1, orange in DAPP1/PKB )
in the phosphoinositide-binding site are shown in a stereo
representation. Hydrogen bonds are indicated as black dotted
lines, and conserved water molecules are shown as yellow
spheres. (A) Protein -ligand interactions of PDK1 PH domain with
Ins(1,3,4,5)P[4]. A layer of ordered water molecules (coloured
in magenta) separates the ligand from VL1, only one of which is
conserved in other PH domain structures (coloured in gold). (B)
Ins(1,3,4,5)P[4] binding to DAPP1 (pdb-id 1fao; Ferguson et al,
2000). The D5-phosphate is closely enveloped by VL1. (C)
Ins(1,3,4,5)P[4] binding to PKB (pdb-id
1h10; Thomas et al, 2001). The ligand is rotated and VL1
contacts the D1-phosphate, while the D5-phosphate is solvent
exposed. (D) Overlay of the structures of the PDK1 (green),
DAPP1 (orange) and PKB (blue)
PH domains. The Ins(1,3,4,5)P[4] ligand and the layer of ordered
water molecules of PDK1 are shown, and coloured according to (A).
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Figure 4.
Figure 4 Phosphoinositide-binding properties of wild type and
mutant PDK1. (A) Representation of the Ins(1,3,4,5)P[4]-binding
pocket on the PDK1 PH domain. The residues that make contacts
with Ins(1,3,4,5)P[4] and that are mutated in this study are
labelled. (B) The ability of the indicated wild type and mutant
forms of PDK1 to interact with phosphoinositides was analysed
using a protein-lipid overlay assay. Serial dilutions of the
indicated phosphoinositides (250, 100, 50, 25, 12.5, 6.3, 3.1
and 1.6 pmol) were spotted onto nitrocellulose membranes, which
were then incubated with the purified GST-PDK1 species. The
membranes were washed, and the GST-PDK1 bound to the membrane by
virtue of their interaction with lipid was detected using a GST
antibody (Dowler et al, 2002). A representative of at least two
separate experiments is shown. (*) indicates a long exposure of
the film to detect weak binding.
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The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
EMBO J
(2004,
23,
3918-3928)
copyright 2004.
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