The EMBO Journal (2004) 23, 3918–3928
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2004 European Molecular Biology Organization | All Rights Reserved 0261-4189/04
THE
EMBO
JOURNAL
Structural insights into the regulation of PDK1
by phosphoinositides and inositol phosphates
David Komander1,3, Alison Fairservice2,
Maria Deak3, Gursant S Kular3,
Alan R Prescott4, C Peter Downes2,
Stephen T Safrany2, Dario R Alessi3
and Daan MF van Aalten1,*
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, 3MRC Protein Phosphorylation Unit,
School of Life Sciences, University of Dundee, Dundee, Scotland, UK and
4
Division of Cell Biology and Immunology, School of Life Sciences,
University of Dundee, Dundee, Scotland, UK
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.
The EMBO Journal (2004) 23, 3918–3928. doi:10.1038/
sj.emboj.7600379; Published online 30 September 2004
Subject Categories: structural biology; signal transduction
Keywords: inositol phosphates; phosphoinositides; PI-3
kinase pathway; protein crystallography; protein structure
Introduction
3-phosphoinositide-dependent protein kinase-1 (PDK1) plays
an important role in insulin and growth factor signalling
cascades (Toker and Newton, 2000; Mora et al, 2004). It is
*Corresponding author. Wellcome Trust Biocentre, School of Life
Sciences, University of Dundee, MSI/WTB Complex, Dundee DD1 5EH,
UK. Tel.: þ 44 1382 344 979; Fax: þ 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
3918 The EMBO Journal VOL 23 | NO 20 | 2004
known to phosphorylate and activate at least 24 protein
kinases that belong to the AGC (cAMP-dependent, cGMPdependent, protein kinase C (PKC)) family of protein kinases.
These comprise isoforms of protein kinase B (PKB, also
known as Akt), p70 ribosomal S6-kinase (S6K), serum and
glucocorticoid responsive kinase (SGK), p90 ribosomal S6
kinase (RSK) and PKC. PDK1 activates these enzymes by
phosphorylating a Ser/Thr residue in their activation loop.
PDK1 is a 556 amino-acid protein comprising an N-terminal
Ser/Thr kinase catalytic domain (residues 71–359) and a
C-terminal pleckstrin homology (PH) domain (residues
459–550) (Alessi et al, 1997; Stephens et al, 1998). The latter
binds with high affinity to the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) (Stephens
et al, 1998; Currie et al, 1999), generated through insulinand growth factor-induced activation of PI 3-kinase
(Vanhaesebroeck et al, 2001).
Much research has shown that insulin and growth factors
control PDK1 by regulating its interaction with substrates,
rather than by directly activating PDK1. PKB, in contrast to
other PDK1 substrates, possesses a PtdIns(3,4,5)P3-binding
PH domain. The mutual ability of PKB and PDK1 to interact
with PtdIns(3,4,5)P3 via their PH domains is likely to play a
crucial role in regulating PKB activation (Anderson et al,
1998; Currie et al, 1999; Filippa et al, 2000). Consistent with
this notion, PKB was not substantially activated in a knockin
ES cell line expressing an active form of PDK1 that cannot
interact with PtdIns(3,4,5)P3 (McManus et al, 2004). Recent
structural and cellular studies have also indicated that binding of PKB to PtdIns(3,4,5)P3 induces a major conformational
change (Mora et al, 2004) that is likely to be required for PKB
activation by PDK1 (Alessi et al, 1997; Stokoe et al, 1997).
Importantly, binding of PtdIns(3,4,5)P3 to the PDK1 PH
domain does not directly affect activity of the PDK1 kinase
domain, but stimulates PKB activation by inducing the colocalisation of PKB and PDK1 (Stephens et al, 1998; Currie
et al, 1999).
The other substrates that PDK1 activates, such as S6K, RSK
and SGK, are phosphorylated by PDK1 in the cytosol, rather
than at the plasma membrane. Moreover, these substrates do
not possess PH domains or interact with phosphoinositides.
Extracellular agonists regulate activation of S6K, RSK and
SGK through PDK1, by inducing the phosphorylation of these
enzymes at a conserved C-terminal (noncatalytic) hydrophobic motif, which creates a high-affinity docking site for
interaction with a pocket located on the kinase domain of
PDK1 (reviewed in Mora et al, 2004). This has recently been
supported by a knockin cell line in which the PDK1 hydrophobic motif docking site is disrupted (Collins et al, 2003).
The PDK1 PH domain sequence is unusual compared to
other PH domains and several computer algorithms, such as
the Simple Modular Architecture Research Tool or PROSITE,
fail to classify it as a PH domain. However, lipid-binding
studies have firmly established that PDK1 binds
PtdIns(3,4,5)P3 through a C-terminal domain, and truncation
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Structure and function of the PDK1 PH domain
D Komander et al
or mutation of several conserved residues found in this
domain abolishes PtdIns(3,4,5)P3 binding (Anderson et al,
1998; Currie et al, 1999). In addition to binding
PtdIns(3,4,5)P3, PDK1 also binds with similar affinity to
PtdIns(3,4)P2, a potential signalling lipid generated through
the dephosphorylation of PtdIns(3,4,5)P3 by the SHIP
5-phosphatase (Stephens et al, 1998; Currie et al, 1999).
Surface plasmon resonance-based lipid-binding assays revealed that PDK1 also interacts with PtdIns(4,5)P2, the substrate for PI 3-kinase, albeit with a 15-fold lower affinity
compared to PtdIns(3,4,5)P3 (Currie et al, 1999). This weaker
affinity for PtdIns(4,5)P2, may be significant as the basal
cellular levels of this phospholipid are 50–200-fold higher
than those of PtdIns(3,4,5)P3/PtdIns(3,4)P2 in insulin-stimulated cells (Batty and Downes, 1996). Consistent with this
notion, quantitative electron microscopy localisation studies
revealed that in unstimulated cells B10% of the cellular pool
of PDK1 is associated with the plasma membrane in a
manner dependent on an intact PH domain, which could be
mediated through the ability of PDK1 to bind PtdIns(4,5)P2
(Currie et al, 1999). Whether insulin or growth factor stimulation induces further localisation of PDK1 at the plasma
membrane is controversial (Anderson et al, 1998; Currie et al,
1999). Nevertheless, a significant pool of PDK1 is required in
the cytoplasm to activate its other non-lipid-binding substrates such as S6K, RSK and SGK. The mechanism by
which PDK1 is anchored in the cytoplasm is not known.
Here we define the molecular basis by which PDK1 interacts
with its phosphoinositide ligands and show that PDK1 may
be anchored in the cell cytosol through its interaction with
D6-phosphorylated inositol phosphates.
Results and discussion
Structure determination of PDK1 PH domain
Constructs containing the predicted PH domain of PDK1
designed on the basis of sequence alignments with previously
crystallised PH domains produced insoluble protein. To identify the boundaries of a soluble fragment of PDK1 containing
the PH domain, a limited proteolysis approach was performed (described in Komander et al, 2004), resulting in a
proteolytically stable fragment of PDK1 comprising residues
408–556. We cloned and expressed in Escherichia coli a
fragment of PDK1 encompassing residues 409–556, which
showed good stability after purification and was readily
crystallisable (Komander et al, 2004). The protein crystals,
grown in the presence or absence of phosphoinositides,
diffracted to below 1.50 Å on a rotating anode generator.
The single cysteine in the sequence (Cys536) was exploited
to obtain experimental phases, by derivatisation with AuCN.
The structure of the PDK1 PH domain in complex with
Ins(1,3,4,5)P4 was solved by a two-wavelength MAD experiment (Table I). The resulting map allowed building of residues 409–549, and showed well-defined electron density for
Ins(1,3,4,5)P4 (Figure 1A).
Overall structure of the ‘budded’ PDK1 PH domain
The structure of the PDK1 PH domain reveals a structural
variation of a standard PH domain fold (Lemmon and
Ferguson, 2000), with an additional ‘bud’ at the N-terminus
(Figures 1B–D). Residues 456–530 fold into two, almost
orthogonal, b sheets, one consisting of four (b1–b4) and
& 2004 European Molecular Biology Organization
one of three strands (b5–b7), constituting a barrel-like structure (Figures 1B–D). On one side, the barrel is blocked by
a C-terminal a helix (residues 531–549, Figures 1B and C).
The other side of the barrel consists of three loops (termed
variable loop (VL) 1–3 (Ferguson et al, 1994)), forming a
bowl-like structure which is lined by positively charged
residues, constituting the phosphoinositide-binding site.
N-terminal to the observed standard PH domain fold, the
PDK1 PH domain possesses an extension, here referred to as
the ‘bud’. This consists of two additional b strands, which
extend the b1–b4 sheet in an antiparallel fashion, and an
additional a helix, which packs against the newly formed sixstranded b sheet (Figures 1B and D). The bud packs against
the b1–b4 sheets of the PH domain, through several hydrophobic contacts (Figure 1D), but also through b-sheet hydrogen bonds between strands b20 and b1. Overall, the bud of the
PDK1 PH domain buries more than 30% of the surface of the
standard PH domain fold (567 Å2 of 1749 Å2), and a hydrophobic core is created between the a10 helix and residues
on one face of the six-stranded b sheet (Figure 1D). Residues
Ile416, Phe424, Leu426, Leu456, Ile457, Met460, Leu477,
Tyr485, Val487 and Leu494 from the six-stranded b sheet
interact with residues Ile412, Phe430, Leu438, Leu439,
Trp448, Phe451, Val452 and Leu456 from the a10 helix and
surrounding loops (Figure 1D). The presence of this additional hydrophobic core, which lies outside the standard PH
domain fold, suggests that the bud is an integral part of the
overall fold. This could explain the insolubility of protein
obtained from initial constructs which covered the standard
PH domain only (residue 458–556) (Komander et al, 2004). A
bud-like extension of a PH domain fold as observed in PDK1
has not been described to date in any other crystallised
PH domain containing protein. Although interactions of PH
domains with other proteins or protein domains have been
described in structures of Gbg binding to the GRK2 PH
domain (Lodowski et al, 2003), myotubularin-related protein
phosphatase MTMR2 (Begley et al, 2003), or the Rac1-Tiam1like complexes (Worthylake et al, 2000), none of these show
the antiparallel b-sheet extension observed in the PDK1 PH
domain and, therefore, do not resemble the bud on PDK1.
Interactions with the PtdIns(3,4,5)P3 head group,
Ins(1,3,4,5)P4
The structure of the PDK1 PH domain was determined from a
crystal that was grown in the presence of Ins(1,3,4,5)P4, and
well-defined ligand density could be observed in the experimental maps (Figure 1A, Table I). The PDK1 PH domain
binds Ins(1,3,4,5)P4 in a location equivalent to that described
for other PH domains (Baraldi et al, 1999; Ferguson et al,
2000; Lietzke et al, 2000; Thomas et al, 2002), that is, within
a shallow but positively charged pocket formed by VL1-3, at
the open end of the b barrel (Figure 1). The phosphoinositidebinding site is lined by positively charged residues that
contact the phosphates of Ins(1,3,4,5)P4, and a total of 11
hydrogen bonds are possible between the PDK1 PH domain
and Ins(1,3,4,5)P4 (Figure 2A). Arg472 contacts the phosphate at the D1 position (D1-phosphate) with two hydrogen
bonds. The D3-phosphate is contacted by Arg474 (two hydrogen bonds) and Lys465. Lys495 is in close proximity (4.4 Å)
to the D3-phosphate. The D4-phosphate forms up to four
hydrogen bonds to Lys465, Tyr486, and Arg521. Finally, the
D5-phosphate makes one hydrogen bond to Lys467 (Figures
The EMBO Journal
VOL 23 | NO 20 | 2004 3919
Structure and function of the PDK1 PH domain
D Komander et al
Table I Data collection and refinement statistics
PDK1 PH domain
–Ins(1,3,4,5,)P4
Beamline
Wavelength (Å)
Space group
Unit cell (Å)
Resolution (Å)
Observed reflections
Unique reflections
Redundancy
Completeness (%)
Rmerge
I/sI
Reflections in test set
Rcryst
Rfree
Phasing statistics
/FOMS
Phasing power
PDK1 PH domain
uncomplexed
PDK1 PH domain–
PtdIns(3,4,5)P3
BM-14
l1
0.861
P21
a ¼ 35.40
b ¼ 58.92
c ¼ 36.58
b ¼ 101.481
BM-14
l2
1.040
P21
a ¼ 35.42
b ¼ 58.94
c ¼ 36.58
b ¼ 101.471
Rotating anode
Rotating anode
1.54
P21
a ¼ 42.69
b ¼ 65.28
c ¼ 101.12
b ¼ 97.481
1.54
P21
a ¼ 36.26
b ¼ 58.94
c ¼ 36.44
b ¼ 102.341
25.0–1.50 (1.58–1.50)
85433
23341
3.7 (3.3)
98.6 (99.5)
0.076 (0.439)
17.7 (3.2)
617
0.150
0.204
25.0–1.80 (1.86–1.80)
33500
13556
2.5 (2.1)
95.5 (95.6)
0.043 (0.181)
17.3 (6.4)
30.0–1.45 (1.50–1.45)
422700
95063
4.5 (3.0)
97.5 (80.1)
0.048 (0.397)
13.2 (2.7)
934
0.171
0.211
30.0–1.45 (1.50–1.45)
73746
25810
2.9 (2.7)
99.7 (100)
0.054 (0.372)
8.8 (2.5)
545
0.185
0.237
0.44 (25.0–2.5 Å)
1.11 (25.0–2.5 Å)
Number of groups
Protein residues
Water
Ligand atoms
SO2
4 atoms
Glycerol atoms
Wilson B (Å2)
/BS protein (Å2)
/BS water (Å2)
/BS ligand (Å2)
143
217
28
—
6
14.5
14.6
27.6
14.6
573
715
—
50
24
19.5
18.6
35.8
—
143
163
33
—
—
23.0
30.3
45.4
36.2
R.m.s.d. from ideal geometry
Bond length (Å)
Bond angles (deg)
Main chain B (Å2)
0.020
1.8
1.6
0.015
1.6
1.5
0.016
1.7
1.7
Values between brackets are for the highest resolution shell. All measured data were included in structure refinement.
2A and 4A). Neither the D2- nor the D6-hydroxy groups show
any direct hydrogen bonds to the protein (Figure 2A).
Figure 2 shows a comparison of the structures of the PDK1,
DAPP1 (Ferguson et al, 2000) and PKBa (Thomas et al, 2002)
PH domains in complex with Ins(1,3,4,5)P4. DAPP1 binds
Ins(1,3,4,5)P4 in a similar fashion to GRP1 (Ferguson et al,
2000; Lietzke et al, 2000) and Btk (Baraldi et al, 1999). The
orientation of Ins(1,3,4,5)P4 in PDK1 is identical to that found
in DAPP1/Btk/GRP1 (Figure 2A and B), while in PKBa
the Ins(1,3,4,5)P4 molecule is rotated by 601 (Figure 2C)
(Thomas et al, 2001). However, in all PH domain
Ins(1,3,4,5)P4 complex structures described to date,
Ins(1,3,4,5)P4 interacts with both protein side chains as
well as the backbone, whereas in PDK1 Ins(1,3,4,5)P4 interacts with protein side chains only. This results in a significantly reduced number of protein–ligand hydrogen bonds in
the PDK1 PH domain Ins(1,3,4,5)P4 complex (a total of 11),
compared to the PKBa (15 hydrogen bonds), DAPP1 (15
hydrogen bonds), Grp1 (16 hydrogen bonds) and Btk (16
hydrogen bonds) PH domain–Ins(1,3,4,5)P4 complexes
(Figure 2). A major difference between the PH domain–
Ins(1,3,4,5)P4 complexes is the position of VL1 (Figure 2D).
In the structures of the DAPP1/GRP1/Btk PH domains, VL1
envelopes the D5-phosphate group, which in the case of
3920 The EMBO Journal VOL 23 | NO 20 | 2004
DAPP1 forms four backbone hydrogen bonds with VL1. In
the case of the PDK1 PH domain, the D5-phosphate forms
only a single hydrogen bond to Lys467, the residue preceding
VL1, and no further direct interactions between VL1 and the
inositol headgroup are observed (Figure 2A). In the case of
PKBa, due to different orientation of Ins(1,3,4,5)P4, VL1
interacts with the D1-phosphate, leaving the D5-phosphate
solvent exposed (Figure 2C). For DAPP1/GRP1/Btk and
PKBa, backbone hydrogen bonds between the D2-hydroxyl
groups are observed. In the case of PDK1, there is no direct
interaction of the D2-hydroxyl group with the protein; instead, we observe it binding to the protein via an ordered
water molecule. Strikingly, in the PDK1 Ins(1,3,4,5)P4-binding pocket, a layer of five-ordered water molecules (B factors
10.7–23.4 Å2) separate Ins(1,3,4,5)P4 from the protein.
The water molecules mediate a number of hydrogen bonds
from Ins(1,3,4,5)P4 to protein side chains and backbone
(Figure 2A). One of the water molecules is consistently
found in the other PH domain–Ins(1,3,4,5)P4 complexes, in
a similar position and contacting the D3-phosphate (coloured
yellow, Figure 2). The presence of the four additional ordered
water molecules is surprising, as this would appear to be
entropically unfavourable. Overall, the PDK1 PH domain
phosphoinositide pocket is significantly more spacious than
& 2004 European Molecular Biology Organization
Structure and function of the PDK1 PH domain
D Komander et al
A
B
Arg521
Lys467
β1'
α1'
β2'
Tyr486
α1
β1
Lys465
β2
β5
β3
Lys495
Arg474
β6
β4
Arg472
β7
VL2
VL3
C
D
α1
C
N
VL1
β5
β1'
α1'
β6
β1'
α1
VL3
β2'
β7
β1
VL1
α1'
β2'
β2
β3
α1'
β1
β2
β2
β4
β3
VL2
β4
β3
β4
Figure 1 (A) Electron density maps calculated from experimental phases after solvent flattening, contoured at 0.6s. Blue electron density
covers the protein, the density for the phosphoinositide ligand is shown in green, for clarity. The refined, final model is shown as stick
representation. (B) Ribbon representation of the PDK1 PH domain (residues 409–549). The standard PH domain fold is coloured green and red
(b strands and the C-terminal a helix, respectively). The extension on the N-terminus is coloured blue. The Ins(1,3,4,5)P4 ligand is shown as a
stick representation. A coloured, semitransparent surface covers the protein molecule. (C) Topology diagram of the PH domain of PDK1,
coloured according to B. (D) Hydrophobic interactions of the six-stranded b sheet with the N-terminal a10 helix. Hydrophobic residues on the
six-stranded b sheet and on the a10 helix are shown as a stick representation, as is the Ins(1,3,4,5)P4 ligand.
that of other Ins(1,3,4,5)P4-binding PH domains. Perhaps,
this more spacious binding pocket explains the observed
ability of the PDK1 PH domain to bind different stereoisomers
of phosphoinositides (Stephens et al, 1998).
Interactions of the PDK1 PH domain
with diC4-PtdIns(3,4,5)P3
It is possible that some of the water molecules close to the
D1-phosphate are replaced by the glycerol backbone upon
PtdIns(3,4,5)P3 binding. To investigate this, the PDK1 PH
domain was co-crystallised with a PtdIns(3,4,5)P3 analogue,
containing two C4 acyl chains (diC4-PtdIns(3,4,5)P3).
& 2004 European Molecular Biology Organization
Crystals were obtained under similar conditions as for the
Ins(1,3,4,5)P4 complex, and were of the same space group
with similar unit cell dimensions (Table I). After refinement
of the protein model, well defined |Fo|–|Fc| density was
visible for the inositol ring, the phosphates and the glycerol
backbone (Figure 3A). A partial model for diC4PtdIns(3,4,5)P3 was then included in the refinement
(Table I). Analysis of the resulting model shows that while
Arg472 coordinates the free oxygen atoms on the D1-phosphate, the oxygen atom involved in the ester bond to the
glycerol does not make any significant contact with the
protein. Of the lipid moiety, only the glycerol backbone is
The EMBO Journal
VOL 23 | NO 20 | 2004 3921
Structure and function of the PDK1 PH domain
D Komander et al
A
Arg474
Arg474
Lys465
Arg472
D3
Lys465
Arg472
D4
D3
D4
D5
D5
D1
D1
Lys467
Lys467
VL1
VL1
PDK1
B
Arg184
Arg184
Lys173
D4
D3
D3
Lys173
D4
D5
D5
D1
D1
VL1
VL1
DAPP1
C
Arg25
Arg25
Lys14
D4
D3
Lys14
D4
D5
D3
D1
D1
VL1
VL1
D5
PKBα
D
DAPP1
DAPP1
PKBα
PDK1
PKBα
PDK1
Figure 2 Comparison of Ins(1,3,4,5)P4 binding to the PH domains
of PDK1, DAPP1 and PKBa. Interactions of Ins(1,3,4,5)P4 (marine)
with protein residues (green in PDK1, orange in DAPP1/PKBa) 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)P4. 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)P4 binding to DAPP1 (pdb-id 1fao; Ferguson et al, 2000).
The D5-phosphate is closely enveloped by VL1. (C) Ins(1,3,4,5)P4
binding to PKBa (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 PKBa (blue) PH domains. The
Ins(1,3,4,5)P4 ligand and the layer of ordered water molecules of
PDK1 are shown, and coloured according to (A).
ordered in the crystal structure (Figure 3A). The glycerol
backbone projects away from the surface of the protein and
does not display any other interactions.
Currently, the only other example of a protein domain in
complex with an ordered phosphoinositide is the PtdIns(3)Pbinding phox homology (PX) domain of the p40phox subunit
3922 The EMBO Journal VOL 23 | NO 20 | 2004
of NADPH oxidase (pdb-id 1h6h; Bravo et al, 2001). In
Figure 3, the structures of the PDK1 PH domain–diC4–
PtdIns(3,4,5)P3 and the PX domain–diC4–PtdIns(3)P complexes are compared after superposition on the inositol
ring. Despite possessing completely different folds, the binding arrangements by which these proteins interact with the
D1-phosphodiester are strikingly similar. The two oxygen
atoms not involved in the two phosphoester linkages will
carry most of the 1e negative charge, and it is these oxygen
atoms that interact with basic residues in the underlying
binding pocket on the surface of the proteins. In the PDK1
PH domain, the guanidinium group of Arg472 hydrogen
bonds both these oxygen atoms, while in the PX domain
each oxygen atom interacts separately with a basic residue
(Arg60/Lys92). Consequently, the oxygen atom involved in
the phosphoester bond to the glycerol projects away from the
binding pocket and in neither structure interacts with the
protein. However, in contrast to the PDK1 PH domain diC4–
PtdIns(3,4,5)P3 complex, the glycerol backbone in the PX
domain–diC4–PtdIns(3)P complex displays hydrophobic interactions with an exposed tyrosine side chain (Tyr94), which
is not observed in the PDK1 PtdIns(3,4,5)P3 complex (compare Figure 3A and B).
To investigate whether the PDK1 PH domain undergoes
ligand-induced conformational change when it binds
PtdIns(3,4,5)P3, the PDK1 PH domain structure was solved
in the absence of any ligand (Table I). However, all four
independent molecules in the asymmetric unit show one
molecule of sulphate, presumably originating from the
0.2 M ammonium sulphate containing crystallisation buffer,
at the position equivalent to the D3-phosphate in the
Ins(1,3,4,5)P4 structure (Supplementary Figure 1). Crystal
structures of other noncomplexed PH domains, including
Grp1 (Ferguson et al, 2000) and PKBa (Milburn et al,
2003), also contain a phosphate/sulphate at equivalent positions. The uncomplexed structures of PDK1 superimpose well
onto the Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 complexes (pairwise with an average RMSD of up to 0.6 Å, Supplementary
Figure 1). No significant conformational changes are visible
even in the VLs that are involved in ligand binding, with the
exception of Lys467, which occupies the position of the
inositol ring in the absence of ligand but rotates to a position
to contact the D5-phosphate upon ligand binding. This
suggests that the PDK1 PH domain, like most other PH
domains analysed to date, assumes the same conformation
in the presence or absence of phosphoinositides. In contrast,
there is evidence that the PKBa PH domain undergoes a
conformational change when it binds to PtdIns(3,4,5)P3
(Mora et al, 2004), which is thought to enhance the rate at
which PKB can be activated by PDK1 (Vanhaesebroeck and
Alessi, 2000).
Mutations of the lipid-binding site: functional analysis
Figure 4A shows a summary of the ligand–protein hydrogen
bonds between PtdIns(3,4,5)P3 and residues of the PDK1 PH
domain. To determine the contribution of the interactions
to the affinity for phosphoinositides, these residues were
mutated in full-length PDK1 and expressed as GST-fusion
proteins in HEK-293 cells. The ability of these mutants to
bind PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 was first
tested with a qualitative protein-phospholipid overlay assay
(Dowler et al, 2002) (Figure 4B). As reported previously,
& 2004 European Molecular Biology Organization
Structure and function of the PDK1 PH domain
D Komander et al
A
C3
C1
O1
C3
C2
O1
Arg472
Arg474
C2
PDK1
Arg472
Arg474
B
C1
Tyr94
O1 C1
Tyr94
C3
Acyl-1
O1 C1
C2
C3
Acyl-1
C2
Lys92
L ys92
Acyl-2
Arg58
Acyl-2
Arg58
Arg60
p40
Arg60
phox
Figure 3 Analogy of PH and PX domain binding to phosphoinositides. The protein and ligand molecules are coloured as in Figure 2. Under the
semitransparent surface, the conserved Arg residues contacting the D1- and the D3-phosphates are drawn as stick representation. (A) Stereo
representation of the PH domain of PDK1 interacting with diC4-PtdIns(3,4,5)P3. A |Fo|–|Fc| simulated annealing omit map, contoured at 2.25s,
is shown in purple, covering the ligand molecule. (B) PtdIns(3)P binding to the PX-domain of p40phox (pdb-id 1h6h; Bravo et al, 2001). Lys94
and Arg60 contacting the D1-phosphate are drawn as stick representation, as is Tyr94 from the membrane attachment loop.
wild-type PDK1 interacts strongly with PtdIns(3,4,5)P3 and
PtdIns(3,4)P2, and weakly with PtdIns(4,5)P2 (Currie et al,
1999) (Figure 4B). Only two of the mutants (PDK1[K465E] and
PDK1[R472A/R474A]) failed to bind phosphoinositides
(Figure 4B). Lys465 and Arg474 of PDK1 are equivalent to
Lys14 and Arg25 of PKBa, and Lys173 and Arg184 of DAPP1,
where mutation of these residues was previously shown
to inhibit the binding to PtdIns(3,4,5)P3 (Dowler et al,
2000; Thomas et al, 2002) (Figure 2). Three mutations
(PDK1[R474A], PDK1[R474A/K467A] and PDK1[K465A]) significantly reduced binding (Figure 4B). The individual mutation of Arg472, Tyr486, Lys 495 or Arg521 had little or
moderate effects on phosphoinositide binding.
Of particular interest was the effect of mutation of Lys467
on the ability of PDK1 to bind D5-phosphorylated phosphoinositides, as Lys467 is the only residue to contact the
D5-phosphate (Figures 2 and 4). In order to assess accurately
the contribution of Lys467 to ligand binding, we employed a
novel time-resolved fluorescence resonance energy transfer
(TR-FRET) assay described in Materials and methods, which
quantitatively measures binding of PDK1 to phosphoinositides (Figure 5). In agreement with previous surface plasmon
resonance (SPR) studies (Currie et al, 1999), in the TR-FRET
assays, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were found to bind
PDK1 with significantly higher affinity than PtdIns(4,5)P2
(Figure 5). Affinities obtained are in close agreement for
TR-FRET and SPR for both PtdIns(3,4,5)P3 (4.0 and 1.6 nM),
and PtdIns(3,4)P2 (23 and 5.2 nM), respectively. In contrast,
for PtdIns(4,5)P2, a lower affinity of 700 nM was measured by
TR-FRET compared to 24 nM that was calculated from previous SPR studies (Currie et al, 1999). We next used the TRFRET assay to investigate how mutation of Lys467 affected
& 2004 European Molecular Biology Organization
binding to PtdIns(4,5)P2. Mutation of Lys467 to Ala resulted
in an Beight-fold reduction in affinity for PtdIns(4,5)P2,
whereas the more conservative replacement of Lys467 to
Arg maintained high-affinity PtdIns(4,5)P2 binding.
Interestingly, in agreement with the structural data, the
PDK1[K467A] mutant interacted with six- to eight-fold
lower affinity to all D5-phosphorylated inositides including
PtdIns(3,4,5)P3 (Figure 5). In comparison, affinities for
PtdIns(3,4)P2 were reduced by two-fold.
Binding of PDK1 to inositol phosphates
The structures of the PDK1 PH domain show that the
phosphoinositide-binding site of PDK1 is unusually spacious,
and, in contrast to other PtdIns(3,4,5)P3-binding PH domains,
additional space is present around the D2- and D6-hydroxyl
groups, which potentially could accommodate further phosphate groups (Figure 3). Physiologically, only inositol
phosphates but not phosphoinositides are known to be
phosphorylated at the D2 and/or D6 position (Michell,
1997; Irvine and Schell, 2001). Ins(1,3,4,5,6)P5 and InsP6
are present at high micromolar levels within the cell, at
higher levels than Ins(1,3,4,5)P4 or Ins(1,4,5)P3 (Orchiston
et al, 2004). These observations prompted us to analyse the
binding of inositol phosphates to full-length PDK1 employing
the TR-FRET assay, which can be used for this purpose.
Interestingly, Ins(1,3,4,5,6)P5 and InsP6, as well as
Ins(1,3,4,5)P4, were found to bind PDK1 with high affinity,
with Ki values of 20–60 nM (Figure 6A). In contrast,
Ins(1,4,5)P3 binding to PDK1 could not be detected. The
hydrogen bonds to the D1-phosphate (Figures 2–4) appear
to play a crucial role in mediating binding of inositol phosphates to PDK1, as Ins(3,4,5,6)P4 had a five-fold lower
The EMBO Journal
VOL 23 | NO 20 | 2004 3923
Structure and function of the PDK1 PH domain
D Komander et al
120
A
K495A
R521A
Y486F
R474A
% of control bound
A
D4
D3
K465A
K465E
D5
80
60
40
20
0
0
D1
R472A/R474A
double
100
K467A
K467R
K467A/R474A
double
200
300
400
500
[bPIP3] (nM)
125
B
% of control bound
R472A
B
wt PDK1
PDK1-PH
PDK1 K467R
PDK1 K467A
100
250 100 50 25 12.5 6.3 3.1 nmol
PtdIns(3,4,5)P3
PtdIns(3,4)P2
PtdIns(3,4,5)P3
PtdIns(3,4)P2
PtdIns(4,5)P2
PtdIns(3,5)P2
PtdIns(5)P
100
75
50
25
0
−3
PtdIns(4,5)P2
−2
−1
0
1
2
3
log[competing ligand] (µM)
PtdIns(3,5)P2
Wild-type
R521A
C
K D for bPIP3 (µM)
R472A
K495A
*
R474A
Y486F
*
R472A / R474A
*
R474A / K467A
*
K465A
*
K465E
Figure 4 Phosphoinositide-binding properties of wild type and
mutant PDK1. (A) Representation of the Ins(1,3,4,5)P4-binding
pocket on the PDK1 PH domain. The residues that make contacts
with Ins(1,3,4,5)P4 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 proteinlipid 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.
affinity compared to Ins(1,3,4,5,6)P5 (Figure 6A). This is also
consistent with our notion of Arg472 interacting with the
delocalised negative charge on the D1-phosphate (Figure 3).
Irvine and colleagues have suggested that binding studies
using InsP6 should be performed in the presence of Mg2 þ
as the counter ion as the physiological form of InsP6 is
complexed to Mg2 þ (RF Irvine, unpublished observation).
3924 The EMBO Journal VOL 23 | NO 20 | 2004
Phosphoinositides
PtdIns(3,4,5)P3
PtdIns(3,4)P2
PtdIns(4,5)P2
PtdIns(3,5)P2
PtdIns(3)P
PtdIns(5)P
wt PDK1
0.012
PDK1 [K467A]
0.196
PDK1 [K467R]
0.009
K i (µM)
K i (µM)
K i (µM)
0.003
0.019
0.59
0.84
2.6
4.0
0.023
0.046
5.9
6.7
21.8
42.4
0.001
0.017
0.41
0.54
1.9
3.4
Figure 5 TR-FRET measurements of phosphoinositide binding to
PDK1. Affinities of wild-type and mutant PDK1 for binding to
phosphoinositides was measured by the TR-FRET assay, as described in Materials and methods. (A) Saturation-binding isotherms
of the indicated forms of PDK1 binding to biotinylatedPtdIns(3,4,5)P3 (bPIP3). (B) The PDK1-bPIP3 complexes formed
at 80 nM bPIP3 were incubated with nonlabelled phosphoinositides
and the displacement of bound bPIP3 measured. The data in (A)
and (B) are presented as an average of three independent experiments7s.d. (C) The KD values were calculated from (A). IC50 values
obtained from (B) were used to calculate Ki values using the Cheng
and Prusoff equation (Cheng and Prusoff, 1973). The KD value for
bPIP3 for the wild type and indicated mutant of PDK1 is also stated.
Binding of inositol lipids and inositol phosphates to PDK1
was not affected by the inclusion of 1 mM Mg2 þ (data not
shown).
Evidence for physiological interaction of PDK1
with D6-phosphorylated inositol phosphates
Initially, we attempted to design a mutant of PDK1 that
inhibited binding of D6-phosphorylated inositol phosphates
without affecting binding of phosphoinositides. Unfortunately, all the point mutants of PDK1 that we generated,
which reduced binding of InsP6, also affected binding to
PtdIns(3,4,5)P3 and PtdIns(4,5)P2. We also attempted to cocrystallise the PDK1 PH domain with Ins(1,3,4,5,6)P5 or
InsP6, without success. This prompted us to compare the
affinities of the isolated PH domain and full-length PDK1 for
phosphoinositides and D6-phosphorylated inositol phosphates.
& 2004 European Molecular Biology Organization
Structure and function of the PDK1 PH domain
D Komander et al
Figure 6 Interaction of PDK1 with inositol phosphates. (A) Affinities of wild-type PDK1 and its isolated PH domain (residues 409–556), for the
indicated phosphoinositides and inositol phosphates were measured by the TR-FRET assay using the approach described in Figure 5A and B.
The KD value for biotinylated-PtdIns(3,4,5)P3 (bPIP3) is also stated. (B) HEK-293 cells were transiently transfected with DNA constructs
encoding the GFP-tagged PKBa PH domain (residues 1–123, panels 1–3), wild-type PDK1 (panels 4–6), the isolated PDK1 PH domain (residues
409–556, panels 7–9) and the non-PtdIns(3,4,5)P3-binding isolated PDK1[K465E] PH domain (residues 409–556, panels 10–12). At 10 h posttransfection, the cells were serum starved for 16 h and either left unstimulated, stimulated with IGF1 for 10 min, or treated with 100 nM
wortmannin for 20 min and then stimulated with IGF1 for 10 min. The cells were then fixed and imaged using a Zeiss LSM 510 META confocal
microscope with an optical section thickness of 0.5 mm. The cells shown are representative images obtained in three separate experiments. (C)
Wild-type GST PKBa was incubated with PDK1, magnesium, g[32P]ATP in the presence ( þ ) or absence of 100 mM phosphatidylserine (PS),
100 mM phosphatidylcholine (PC) vesicles containing 10 mM PtdIns(3,4,5)P3 and/or 300 mM of the indicated inositol phosphates, and the
activation of PKBa was assessed as described previously (Alessi et al, 1997). In samples in which PtdIns(3,4,5)P3 was omitted, the PC/PS
vesicles were still added. Phosphorylation of the PKB was determined following electrophoresis on a 4–12% gradient polyacrylamide gel, either
by immunoblotting with the phosphospecific Thr308 antibody or by autoradiography of the Coomassie Blue-stained PKBa band. PDK1 activity
was assayed as above, employing the PDKtide peptide substrate rather than by measuring PKB activation and phosphorylation. Experiments
were performed in quadruplicate and similar results obtained in two to four separate experiments.
Surprisingly, we observed that the isolated PH domain of
PDK1, although it still interacted with PtdIns(3,4,5)P3 and
PtdIns(4,5)P2 with high affinity, possessed 40-fold reduced
& 2004 European Molecular Biology Organization
affinity for InsP6 and five-fold lower affinity for
Ins(1,3,4,5,6)P5 (Figure 6A). Inspection of the structure
of the PH domain–Ins(1,3,4,5)P4 complex suggests that a
The EMBO Journal
VOL 23 | NO 20 | 2004 3925
Structure and function of the PDK1 PH domain
D Komander et al
D6-phosphate would be solvent exposed and unable to interact with the PH domain. However, in the context of the fulllength PDK1, the D6-phosphate may well form interactions
with non-PH domain regions of the protein, explaining the
observed higher affinity of full-length PDK1 for D6-phosphorylated inositol phosphates. Clearly, further structural
studies are required to investigate this specific interaction
and to understand the alignment of the PH domain and the
kinase domain in context of the full-length PDK1 protein.
We next studied the cellular location of green fluorescent
protein (GFP)-tagged full-length PDK1 compared to the isolated PH domain of PDK1. As observed previously (Currie
et al, 1999), full-length PDK1 was mainly cytosolic, with only
a small fraction being at the membrane, and was excluded
from the nucleus in unstimulated HEK-293 cells. PDK1 did
not markedly translocate to the plasma membrane following
stimulation of cells with the PI 3-kinase agonist IGF1.
Similarly, localisation of full-length PDK1 was not affected
by the PI 3-kinase inhibitor wortmannin (Figure 6B, panels
4–6). In parallel experiments, we observed IGF1-induced
plasma membrane translocation of the PtdIns(3,4,5)P3-binding PH domain of PKBa, which was inhibited by wortmannin
(Figure 6B, panels 1–3). The isolated PDK1 PH domain was
localised in the cytoplasm and nucleus of unstimulated cells,
and, in contrast to full-length PDK1, IGF1 induced a marked
translocation to the plasma membrane that was inhibited by
wortmannin (Figure 6B, panels 7–9). A non-phosphoinositide-binding isolated PDK1[K465E] PH domain mutant was
localised throughout the cell and did not translocate to the
plasma membrane upon IGF1 stimulation (Figure 6B, panels
10–12). Control Western blotting demonstrated the absence
of proteolytic degradation of the expressed constructs (data
not shown). Taken together, these findings are consistent
with a model in which full-length PDK1 is anchored in the
cytosol through high-affinity binding to D6-phosphorylated
inositol phosphates. Similar observations have been made in
Dictyostelium discoideum, in which Luo et al (2003) followed
the localisation of the PH domain of Crac (PHCrac). Crac
shows high affinity for diphosphoinositol pentakisphosphate
(PP-InsP5, the isomer was not stated), and appears mostly
cytosolic, as is PDK1 in our study. Following cAMP stimulation, which causes PtdIns(3,4,5)P3 accumulation in D. discoideum, the movement of PHCrac is potentiated in mutants
lacking inositol hexakisphosphate kinase, the enzyme responsible for PP-InsP5 synthesis. It should be noted that
levels of inositol diphosphates in D. discoideum are about
100 times those of mammalian tissues, but still only about
half those of InsP6 (Luo et al, 2003). PDK1 was found to
interact with 5PP-InsP5, with a significantly lower affinity (Ki
400 nM) than Ins(1,3,4,5,6)P5 and InsP6 (Figure 6A). As the
cellular concentration of 5PP-InsP5 is much lower than
Ins(1,3,4,5,6)P5 and InsP6, it is unlikely that 5PP-InsP5 is a
physiological ligand for PDK1.
The PKBa PH domain shows rapid translocation to the
plasma membrane (Figure 6B, panels 1–3) upon IGF1 stimulation. PKBa PH domain and PDK1 show similar affinities for
phosphoinositides, but PKBa PH domain shows significantly
lower affinity towards higher inositol phosphates (Frech
et al, 1997). Indeed, InsP6 binds PDK1 with 80-fold higher
affinity than PKBa PH domain. As such, the competition
between cytosol-localised inositol phosphates and membrane-localised phosphoinositides greatly favours phosphoi3926 The EMBO Journal VOL 23 | NO 20 | 2004
nositide binding in the case of the PKBa PH domain, resulting
in a marked translocation of PKBa PH domain to the plasma
membrane in IGF1 treated cells (Figure 6B, panels 1–3).
Consistent with the notion that high-affinity binding to
inositol phosphates maintains PDK1 located in the cytoplasm, the isolated PH domain of PDK1, which displays
high-affinity interactions with phosphoinositides, but has
an affinity (4.1 mM) similar to that of PKBa for InsP6
(8.7 mM, (Frech et al, 1997)), is seen to translocate in a
fashion analogous to the PKBa PH domain. Our data do not
rule out the possibility that other ligands/factors could interact with full-length PDK1, contributing towards its cytosolic
or membrane localisation. In this regard, it has recently been
reported that PDK1 can be stabilised at the cell membrane by
interaction with Grb14, which binds the insulin receptor
through an SH2 domain (King and Newton, 2004).
In many studies of PH domain-containing proteins, the
lipid-binding and localisation properties of the PH domain are
assessed employing the isolated PH domain rather than the
full-length protein (Irvine, 2004). Our data emphasise a pitfall
of this approach, as the isolated PH domain of PDK1 has quite
distinct inositol phosphate-binding properties and localisation pattern, compared to the full-length protein. Recently, it
was shown that the isolated PH domain of phospholipase Cd4
(PLCd4) was localised at the plasma membrane, whereas the
full-length protein localises at intracellular membranes, and
also showed differences in PtdIns(4,5)P2 versus Ins(1,4,5)P3
binding (Lee et al, 2004). Thus, in order to determine the key
localisation signals of lipid-binding proteins, we would recommend comparing the lipid-binding and localisation properties of the isolated lipid-binding domain with that of the
full-length protein.
We next investigated whether Ins(1,3,4,5,6)P5 and InsP6
could compete with PtdIns(3,4,5)P3 in regulating the activation of PKB by PDK1 in an in vitro assay. Addition of 300 mM
InsP6 or Ins(1,3,4,5,6)P5 inhibited the activation and phosphorylation of PKB in the presence of PtdIns(3,4,5)P3, by
over 80% (Figure 6C). InsP6 or Ins(1,3,4,5,6)P5 did not
directly inhibit PDK1 catalytic activity, as the phosphorylation of the PDKtide peptide substrate by PDK1 was not
affected by D6-phosphorylated inositol phosphates
(Figure 6C). It has also been reported that increasing extracellular Ins(1,3,4,5,6)P5 in cells lowered PKB activation,
through an undefined mechanism (Piccolo et al, 2004). The
data presented here indicate that these effects could be
mediated, at least in part, through binding of PDK1 to
Ins(1,3,4,5,6)P5 and anchoring it in the cytosol. It is also
possible that Ins(1,3,4,5,6)P5 might bind to the PH domain of
PKB in vivo, and thus affect its translocation to the plasma
membrane.
Several other PtdIns(3,4,5)P3-binding PH domains including Grp1 and Btk have been shown to interact in vitro with
InsP6 and Ins(1,3,4,5,6)P5. Btk interacts with both InsP6 and
Ins(1,3,4,5,6)P5 with Ki of 90 nM (Kojima et al, 1997),
whereas Grp1 binds to InsP6 and Ins(1,3,4,5,6)P5 with KD’s
of 6 and 0.3 mM, respectively (Kavran et al, 1998).
PtdIns(3,4,5)P3 recruits Grp1 and Btk to the plasma membrane, where they are thought to exert their physiological
effects. Thus, if it occurred in vivo, inositol phosphate binding to these PH domain-containing proteins would inhibit
their function. In the case of PDK1, cytosolic localisation is
important in enabling PDK1 to activate substrates that do not
& 2004 European Molecular Biology Organization
Structure and function of the PDK1 PH domain
D Komander et al
bind lipids, such as SGK or S6K. Our data indicate that the
micromolar levels of Ins(1,3,4,5,6)P5 and InsP6 found in cells
would be sufficient to act as physiological ligands of PDK1,
and could compete with PtdIns(3,4,5)P3, PtdIns(3,4)P2 and
PtdIns(4,5)P2 for binding to PDK1. Such a mechanism could
ensure that sufficient PDK1 was distributed in both the
cytosol and plasma membrane to enable PDK1 to fulfil its
cellular functions. Thus, where the binding of Btk and Grp1
to inositol phosphates would act as a brake in signalling, for
PDK1 this binding would accelerate activation of cytoplasmic
substrates. In this respect, PDK1 is unique among PH
domain-containing proteins described so far.
Conclusions
Here we describe the unusual structure of the PDK1 PH
domain. It possesses several unique features not previously
observed in other PH domains, including the N-terminal bud
which forms an integral part of the overall PH domain
structure. The PDK1 PH domain in complex with a
PtdIns(3,4,5)P3 analogue shows a surprising analogy in the
phosphoinositide-binding mode of the PDK1 PH domain and
the PX domain of p40phox. We define the relative importance
of the residues in the ligand-binding site for enabling PDK1 to
bind phosphoinositides. In particular, we identify Lys467 as
the residue in PDK1 responsible for binding the D5-phosphorylated ligands and thus permitting interaction with
PtdIns(4,5)P2. Moreover, the PDK1 PH domain possesses a
much more spacious ligand-binding site, which we demonstrate enables PDK1 to interact with Ins(1,3,4,5,6)P5 and
InsP6 with high affinity. We perform studies that support
the notion that these ligands could function to anchor PDK1
in the cytosol, where it is needed to activate its nonlipidbinding substrates. Overall, our results provide a molecular
explanation for how PDK1 could be localised both at the
membrane and the cytosol of cells.
Materials and methods
Protein expression, purification, crystallisation
and data collection
PDK1 PH domain (residues 409–556) was expressed in Escherichia
coli, purified and crystallised as described previously (Komander
et al, 2004). Further details on crystallisation conditions, data
collection, as well as structure determination and analysis can be
found in the Supplementary data. The refinement statistics can be
found in Table I.
Analysis of PDK1 phosphoinositide and inositol phosphate
interactions
For qualitative protein lipid overlay assays, wild type and mutants
of full-length PDK1 were expressed as GST fusion proteins (pEBG
vector) in human embryonic kidney HEK-293 cells, as described
previously (Alessi et al, 1997). The phospholipid overlay assays
were performed as described previously (Dowler et al, 2002), using
di-palmitoyl-phosphoinositides from Cell Signals Inc. (Columbus,
USA). The quantitative TR-FRET analysis of binding was performed
as described by Gray et al (2003). To determine KD values for
biotinylated diC8-PtdIns(3,4,5)P3, increasing amounts (0–500 nM)
of biotinylated diC8-PtdIns(3,4,5)P3 were incubated with GSTtagged wild type and mutant full-length PDK1 or wild-type PH
domain of PDK1 (20 nM) in the presence of excess europiumlabeled goat anti-GST antibody and streptavidin-conjugated allophycocyanin (APC). The binding of the biotinylated lipid to the
protein allows FRET to occur between europium (donor) and APC
(acceptor). Fluorescence is monitored at 665 and 505 nm; the ratio
of these signals allows determination of relative amount of binding.
Saturation-binding isotherms are presented in Figure 5A. Competition assays were performed with a fixed amount (80 nM) of
biotinylated lipid and increasing amounts of competing agent (diC8
phosphoinositides or inositol phosphates). IC50 values were
determined, from which Ki values were calculated, according to
the equation below (Cheng and Prusoff, 1973). All curve fitting was
performed using Prism (GraphPad).
Ki ¼
IC50
1 þ ½L=KD
Localisation studies
Cells were fixed in 4% (w/v) paraformaldehyde in PHEM buffer
(60 mM Pipes, 25 mM Hepes, 10 mM EGTA and 2 mM MgSO4, pH
7.0). The cells were washed three times with PBS and mounted on
slides with Hydromount. The cells were imaged to monitor GFP
fluorescence and confocal sections collected, using a Zeiss LSM 510
META confocal microscope.
A detailed description of all other methods employed in this
study can be found in the supplementary data of this paper.
Supplementary data
Supplementary data are available at The EMBO Journal Online.
Acknowledgements
We thank the European Synchrotron Radiation Facility for beam
time at BM14, and Martin Walsh for assistance in data collection,
Mike Hodgkinson for assistance with in-house data collection, the
School of Life Sciences DNA Sequencing Service and Robin Irvine
for helpful discussion and sharing unpublished data. 5PP-InsP5 was
kindly provided by JR Falck, University of Texas Southwestern
Medical Center, USA. An MRC Predoctoral Fellowship funds DK,
and AF is supported by a BBSRC Committee Studentship. STS is a
Royal Society University Research Fellow, and thanks The Royal
Society and Tenovus-Scotland for financial support. DMFvA is
supported by a Wellcome Trust Senior Research Fellowship and
an EMBO Young Investigator Award. CPD is supported by a
Programme Grant from the MRC. DRA thanks the Association for
International Cancer Research, Diabetes UK, the Medical Research
Council, the Moffat Charitable Trust, as well as the pharmaceutical
companies that support the Division of Signal Transduction Therapy
(AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck
& Co. Inc., Merck KGaA and Pfizer). The coordinates and structure
factors have been deposited with the PDB (entries 1w1d (PDK1 PH
domain–Ins(1,3,4,5)P4 complex), 1w1g (PDK1 PH domain–
PtdIns(3,4,5)P3 complex) and 1w1h (uncomplexed PDK1 PH
domain)). All Figures were created with PyMol (www.pymol.org).
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