Structural and Biochemical Evaluation of the Interaction of the
Phosphatidylinositol 3-Kinase p85
Src Homology 2 Domains with
Phosphoinositides and Inositol Polyphosphates*
Paola Lo
Surdoab,
Matthew J.
Bottomleyab,
Alexandre
Arcarocd,
Gregg
Siegalce,
George
Panayotoucf,
Andrew
Sankarc,
Piers
R. J.
Gaffneycg,
Andrew M.
Rileyh,
Barry
V. L.
Potterhi,
Michael D.
Waterfieldac, and
Paul C.
Driscollacj
From the a Department of Biochemistry and
Molecular Biology, University College London, London WC1E 6BT,
g Department of Chemistry, University College London, London
WC1H 0AJ, h Wolfson Laboratory of Medicinal Chemistry,
Department of Pharmacy and Pharmacology, University of Bath, Bath BA2
7AY, and c Ludwig Institute for Cancer Research,
London W1P 8BT, United Kingdom
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ABSTRACT |
Src homology 2 (SH2) domains exist in many
intracellular proteins and have well characterized roles in signal
transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing
proteins. Although tyrosine phosphorylation is essential for
protein-SH2 domain interactions, the binding specificity also derives
from sequences C-terminal to the Tyr(P) residue. The high affinity and
specificity of this interaction is critical for precluding aberrant
cross-talk between signaling pathways. The p85
subunit of
phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and
it has been proposed that in competition with Tyr(P) binding they may
also mediate membrane attachment via interactions with phosphoinositide
products of PI 3-kinase. We used nuclear magnetic resonance
spectroscopy and biosensor experiments to investigate interactions
between the p85
SH2 domains and phosphoinositides or inositol
polyphosphates. We reported previously a similar approach when
demonstrating that some pleckstrin homology domains show binding
specificity for distinct phosphoinositides (Salim, K., Bottomley,
M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R.,
Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO
J. 15, 6241-6250). However, neither SH2 domain exhibited binding
specificity for phosphoinositides in phospholipid bilayers. We show
that the p85
SH2 domain Tyr(P) binding pockets indiscriminately
accommodate phosphoinositides and inositol polyphosphates. Binding of
the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these
ligands do not bind p85
SH2 domains with high affinity or
specificity. Moreover, we observed that although wortmannin blocks PI
3-kinase activity in vivo, it does not affect the ability of tyrosine-phosphorylated proteins to bind to p85
. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85
SH2 domains.
 |
INTRODUCTION |
Src homology 2 (SH2)1
domains are conserved, noncatalytic sequences of about 100 amino acids
that adopt a common three-dimensional fold. These domains are commonly
found in signal transduction proteins that regulate a variety of
cellular processes, such as phospholipid metabolism, protein
phosphorylation, and dephosphorylation, protein trafficking, and gene
expression (1). SH2 domains mediate high affinity binding to
phosphotyrosine (Tyr(P)) residues in proteins such as activated
membrane receptors and cytosolic adaptor proteins. Three to five amino
acids C-terminal to the target Tyr(P) residue bind to a groove on the
SH2 domain surface and confer the specificity of interaction that is
necessary to avoid aberrant signaling (2, 3). The role of SH2 domains
in Tyr(P)-dependent protein recruitment is critical for the
assembly of active complexes of signaling proteins (4, 5).
The p85
/p110
Class IA phosphoinositide 3-OH kinase
(PI 3-kinase) contains two SH2 domains in its regulatory p85
subunit (6). Upon cell stimulation, the SH2 domains bind to
tyrosine-phosphorylated, membrane-bound growth factor receptors. As a
result, p85
/p110
is recruited to the vicinity of its
phosphoinositide substrates (7). The p110
PI 3-kinase activity then
produces 3'-phosphorylated phosphoinositides. In this manner,
p85
/p110
mediates a dramatic increase in the basal concentration
of phosphatidylinositol 3,4,5-trisphosphate (PtdIns
(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate in the
plasma membrane shortly after cell stimulation (8, 9).
It is now clear that p85
/p110
phosphorylates phosphoinositides to
produce second messengers, which control the membrane recruitment and
activation of numerous signaling proteins, notably including regulators
of apoptosis (10-12). Many of the target proteins of these second
messengers contain pleckstrin homology (PH) domains and have been shown
to bind specifically to PtdIns (3,4,5)P3 and/or phosphatidylinositol 3,4-bisphosphate in vitro and/or
in vivo (13-19). Indeed, numerous interactions between
distinct phosphoinositides and PH domains have now been demonstrated
and appear to be essential for the function of various cytoskeletal or
signal transduction proteins (reviewed in Ref. 20).
However, a few reports have suggested that PH domains are not unique
targets of second messenger phosphoinositides produced by PI 3-kinase.
It has been proposed that PtdIns (3,4,5)P3 can also bind to
SH2 domains. These proposals followed the observation of an inverse
correlation between the amount of p85
/p110
associated with
tyrosine-phosphorylated proteins and the level of PI 3-kinase lipid
products present in the cell (21). Consequently, a model was proposed
in which the PtdIns (3,4,5)P3 produced by PI 3-kinase activation could compete for Tyr(P)-bound p85
SH2 domains and directly result in the relocalization of p85
/p110
at the plasma membrane. Similarly, the production of PtdIns (3,4,5)P3 may
regulate additional proteins such as the tyrosine kinase Src and
phospholipase C
-1 (21, 22). It has been shown that in
vitro the p85
C-terminal SH2 (C-SH2) domain can bind to PtdIns
(3,4,5)P3 (21). However, it was not demonstrated that
recombinant p85
C-SH2 domain can act as a faithful model of p85
activity. Indeed, we noted with intrigue that the reported interaction
of PtdIns (3,4,5)P3 with the p85
C-SH2 domain could be
significantly inhibited by phenyl phosphate, but that such inhibition
was not observed in the case of the reported interaction between PtdIns
(3,4,5)P3 and full-length p85
(21). The work presented
herein arose from our attempts to clarify these apparently conflicting
observations and to resolve certain issues central to these models
describing distinct phosphoinositide-SH2 domain interactions.
Prerequisites for the models above are that the SH2 domains that
interact with phosphoinositides must (a) demonstrate a
significant binding affinity for these ligands and (b)
discriminate between the numerous phosphoinositides present in the
plasma membrane. Because we had access to appropriate reagents and
assay techniques, we set out to determine whether the p85
SH2
domains indeed display clear binding specificity and affinity for
distinct phosphoinositides. We report the first high resolution
structural studies of model phosphoinositide-SH2 domain interactions,
which we performed by nuclear magnetic resonance (NMR) spectroscopy. We
also employed two sensitive biosensor assays; one to measure
interactions between proteins and phospholipid bilayers containing
phosphoinositides and another to measure directly the competition
between Tyr(P)-containing ligands and phosphoinositides for binding to
SH2 domains. In addition, we report in vivo studies in which
we sought a correlation between the association of p85
with
activated growth factor receptors or tyrosine-phosphorylated proteins
and the intracellular level of PI 3-kinase products.
 |
EXPERIMENTAL PROCEDURES |
Protein Expression and Purification
The p85
C-SH2 domain (amino acids Glu-614-Arg-724) was
expressed and purified as described previously (23). A pGEX-2T plasmid encoding glutathione S-transferase (GST) (Amersham Pharmacia
Biotech) fused to the p85
N-terminal SH2 (N-SH2) domain (amino acids
Pro-314
Tyr-431) was kindly provided by Dr. R. Stein (Ludwig Institute
for Cancer Research, London), and the protein was prepared and purified
essentially as described previously (24, 25). Transformed
Escherichia coli BL21 (DE3) cells were grown at 37 °C to
a culture density A600 ~ 0.8. Protein
expression was induced by the addition of isopropyl-1-thio-
-D-galactopyranoside to a concentration
of 0.2 mM. Cells were harvested after 4 h, resuspended
in phosphate-buffered saline (PBS), and lysed by a French press. For
NMR spectroscopy and biosensor Tyr(P) competition assays, the GST
moiety was removed by thrombin cleavage. In contrast, intact fusion
protein was used in the liposome binding assays. Further protein
purification was accomplished by gel filtration in 50 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 0.02% NaN3.
15N-isotopically enriched samples for NMR spectroscopy were
prepared as above except that cells were grown in a minimal M9 medium
using 15NH4Cl (Isotec Inc.) as the sole
nitrogen source. NMR samples were prepared in 50 mM
deuterated Tris-HCl (Cambridge Isotope Laboratories), pH 7.5, 50 mM NaCl, 2 mM dithiothreitol (for C-SH2 only),
10% (v/v) D2O.
Ligands Tested for Binding to p85
SH2 Domains
The water-soluble ligands tested included
D-myo-inositol 1,4,5-trisphosphate
(D-Ins (1,4,5)P3), L-Ins
(1,4,5)P3,
L-
-glycerophospho-D-myo-inositol 4,5-bisphosphate, and phenyl phosphate obtained from Sigma;
D-Ins (1,3,4,5)P4 and L-Ins
(1,3,4,5)P4, synthesized and purified by ion-exchange
chromatography as published (26, 27);
rac-dihexanoylphosphatidyl-D/L-myo-inositol 3,4,5-trisphosphate (di-C6-PIP3), synthesized
using published techniques (28); and the nine-residue phosphopeptide
SVDY(P)VPMLD (Y(P) is phosphotyrosine) (Genosys Ltd.). The
phospholipids tested included PtdIns, PtdIns (4)P, and PtdIns (4,
5)P2 (obtained from Lipid Products, Redhill, Surrey, UK)
and PtdIns (3,4,5)P3, which was prepared as described
previously (29) and kindly provided by Professor R. Gigg. The
additional liposome components described were purchased from Sigma.
NMR Spectroscopy Experiments
For NMR spectroscopy, SH2 domain samples were prepared at 0.5 mM concentration in 600 µl. Interactions were monitored
via spectra recorded during titration of the SH2 domain with 1.5-µl aliquots of test ligand (prepared at 20 mM in 20 mM Tris-HCl, pH 7.5, 50 mM NaCl). NMR
experiments were performed at 15 °C on a Varian UNITY-plus
spectrometer operating at a 1H frequency of 600 MHz.
Two-dimensional gradient enhanced sensitivity 15N-1H heteronuclear single quantum coherence
(HSQC) experiments were performed using a pulse sequence kindly
provided by Professor L. E. Kay (30). Sign discrimination in
t1 was achieved using the States-time-proportional phase
incrementation method. The HSQC spectra were acquired with 16 scans, 64 increments in t1, and sweep widths of 10000 Hz
(1H) and 2400 Hz (15N). Three-dimensional
15N-1H HSQC total correlation spectroscopy and
nuclear Overhauser effect spectroscopy experiments were recorded to
verify the published resonance assignments for the N-SH2 domain (31,
32).
NMR data were processed using NMRpipe software (33). Phase-shifted,
sine-squared shaped weighting functions and zero-filling were applied
before Fourier transformation. NMR spectra were analyzed using XEASY
(34) and AZARA software (AZARA v.II, W. Boucher, Department of
Biochemistry, University of Cambridge, UK).
Biosensor Experiments
Preparation of Liposomes for Biosensor Studies--
Large
unilamellar liposomes with a phospholipid composition approximating the
inner leaflet of the plasma membrane were prepared as described
previously (35). By weight, the liposomes contained 30%
phosphatidylcholine, 15% sphingomyelin, 20% cholesterol, 15% phosphatidylethanolamine, 10% phosphatidylserine, and 10% of the phosphoinositide to be tested. Liposomes were used in 10 mM
HEPES, pH 7.4, 80 mM KCl, 15 mM NaCl, 0.7 mM NaH2PO4, 1 mM EGTA,
0.466 mM CaCl2, 2.1 mM
MgCl2.
Liposome Binding Studies Using the Biosensor--
The basic
operating procedures of the surface plasmon resonance BIAcore biosensor
(BIACORE AB, Uppsala) have been published (36). The ability of
immobilized GST fusion SH2 domains to bind to phosphoinositides in
liposomes was examined using the method described previously (35).
Phosphoinositide Phosphotyrosine Peptide Competition Studies
Using the Biosensor--
A precoated streptavidin biosensor chip
(SA-5, BIACORE AB) was used to immobilize the N-terminal-biotinylated,
Tyr(P) peptide N-biotinyl-DMSKDESVDY(P)VPMLDMK (Y(P) is
phosphotyrosine). The Tyr(P) peptide was loaded in the buffer used
throughout the assay: 20 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20, and 4 mM dithiothreitol. Solutions of 0.5 µM N- or
C-SH2 domain were injected over the surface at a flow rate of 5 µl/min at 25 °C, and the maximum response was recorded.
Competition experiments were performed by incubating the SH2 domains
with a competitor ligand before injection. Efficacious competition
resulted in a diminished response. Between injections, protein
remaining bound to the biosensor was removed by a 5-µl pulse of
0.05% SDS solution.
Data analysis of the competition measurements was performed with the
BIAcore-2000 software package (BIACORE AB). In calculations of the
half-maximal inhibitory constants (IC50), the control
response from injection of SH2 domain over the biosensor surface
lacking the Tyr(P) peptide was subtracted from the experimental
response to yield the corrected response, R. Data was
plotted as corrected response units versus concentration of
competitor and were fitted to the following equation using a nonlinear
least-squares analysis: R = Rmax/[1 + (C/IC50)P], where
Rmax is the response for SH2 binding in the
absence of competitor, C is the concentration of competitor,
and P is the Hill coefficient.
In Vivo Assays
Cell Culture--
Mouse NIH3T3 fibroblasts were grown at
37 °C in a humidified atmosphere containing 10% CO2 in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% (v/v) heat-inactivated fetal calf serum (Life
Technologies, Inc.) and penicillin/streptomycin (Life Technologies,
Inc.). The cells were grown to confluence in 150-mm dishes and
serum-starved in Dulbecco's modified Eagle's medium containing 0.5%
(v/v) heat-inactivated fetal calf serum for 16 h.
Immunoprecipitations--
Cells grown on 150-mm dishes were
incubated for 15 min at 37 °C with wortmannin (100 nM in
Me2SO) or an equivalent volume of Me2SO and
subsequently stimulated with recombinant PDGF-
(100 nM)
for 10 min at 37 °C. The dishes were then placed on ice, washed once
in ice-cold PBS buffer (Life Technologies, Inc.), and lysed for 20 min
on ice in 1 ml of lysis buffer (20 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, 1%(w/v) Triton X-100, 2 mM EDTA,
10 mM NaF, 10 mM
Na2HPO4, 10% (w/v) glycerol, 1 mM
phenylmethylsulfonyl fluoride, 5 mM benzamidine, 7 mM diisopropylphosphofluoridate, 1 mM
1-chloro-3-tosylamido-7-amino-2-heptanone, 20 µM
leupeptin, 18 µM pepstatin, 21 µg/ml aprotinin, 2 mM dithiothreitol, 1 mM
Na3VO4, 10 mM
-glycerophosphate,
1 mM tetrasodium pyrophosphate, 1 mM sodium
molybdate). The cells were then scraped from the dishes and centrifuged
for 20 min at 15,000 × g and 4 °C. The supernatant was collected and incubated with the relevant antibody with constant agitation at 4 °C for 2 h. Protein G-Sepharose CL-4B (Amersham Pharmacia Biotech) at 10 µl of bead slurry/sample was then added, and
the incubation continued for 1 h at 4 °C on a wheel. The
immunoprecipitates were washed three times in lysis buffer and analyzed
by SDS-polyacrylamide gel electrophoresis and Western blotting or
assayed for PI 3-kinase activity.
PI 3-Kinase Assays--
PI 3-kinase activity was assayed on
immunoprecipitates resuspended in 25 µl of 2× kinase buffer (40 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2 mM dithiothreitol). PtdIns stored in CHCl3
solution was dried, sonicated for 15 min in 50 mM Tris-HCl,
pH 7.4, and added to a concentration of 0.2 mg/ml. The reactions
(50-µl final volume) were started by the addition of 40 mM ATP, 10 µCi of [
-32P ]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech), and 3.5 mM MgCl2. Kinase reactions were stopped by the addition of 100 µl of 1 M HCl. For phospholipid extraction, 200 µl of
1:1 (v/v) CHCl3/CH3OH was added. The organic
phase was collected and re-extracted with 40 µl of 1:1 (v/v) 1 N HCl/CH3OH. The samples were then dried, resuspended in 30 µl of CHCl3/ CH3OH 1:1
(v/v), and spotted onto prechanneled silica gel 60 TLC plates (Whatman)
that had been pretreated in 1% (w/v) oxalic acid, 1 mM
EDTA, H2O/CH3OH (60:40 (v/v) and baked for 15 min at 110 °C. The plates were developed in propanol, 2 M acetic acid 65:35 (v/v), and the radioactive spots were
quantified using a PhosphorImager (Molecular Dynamics).
Western Blotting--
After SDS- polyacrylamide gel
electrophoresis, polyacrylamide gels (7.5%) were transferred onto
polyvinylidene difluoride membranes (Gelman Sciences) using a semi-dry
blotter (Amersham Pharmacia Biotech). The membranes were then blocked
for 1 h in PBS buffer containing 3% (w/v) nonfat dry milk, 0.1%
(w/v) polyethylene glycol 20000. The relevant primary antibodies were
diluted in PBS buffer and 0.05% (w/v) Tween 20 (PBS/Tween) and
incubated with the membranes for 2 h. After extensive washing in
PBS/Tween, the blots were incubated for 1 h with goat anti-mouse
or anti-rabbit antibodies coupled to horseradish peroxidase (Dako) at
1:2000 dilution. The membranes were then washed in PBS/Tween, and the bands were detected using ECL (Amersham Pharmacia Biotech).
 |
RESULTS |
The Identification by NMR Spectroscopy of a Binding Site for
Phosphoinositides and Inositol Polyphosphates on the p85
SH2
Domains--
NMR spectroscopy was used to investigate the structural
details of the interactions between the p85
SH2 domains and a range of candidate phosphoinositide and inositol polyphosphate ligands. 15N-1H HSQC NMR spectra were recorded during
titrations of 15N-labeled SH2 domain with unlabeled test
ligands. It is well established that observations of chemical shift
perturbations upon titration with a ligand can be a sensitive probe of
the ligand binding site of a protein (37, 38).
During the titration, changes in 15N and 1H
chemical shift values for each residue were monitored by measuring
changes in the cross-peak positions of assigned resonances. For both
p85
SH2 domains, the introduction of any of the phosphoinositide or
inositol polyphosphate ligands tested resulted in significant chemical shift perturbations for a limited set of cross-peaks (Fig.
1). For both the p85
N- and C-SH2
domains, it was observed that the overall pattern of chemical shift
changes induced by the addition of the ligands are similar in both
direction and magnitude. The small differences in the perturbation
direction seen in the case of phenyl phosphate (Fig. 1, panels
E and I) result from additional ring-current shift
effects induced by its aromatic group. Most notably, there seem to be
very few differences when comparing the effects of D-Ins
(1,4,5)P3, D-Ins (1,3,4,5)P4,
L-Ins (1,3,4,5)P4, or
di-C6-PIP3. The results with D-Ins
(1,4,5)P3 and
L-
-glycerophospho-D-myo-inositol 4,5-bisphosphate were also qualitatively similar to the data obtained with the other inositol polyphosphates (data not shown).

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Fig. 1.
NMR investigation of PI 3-kinase
p85 subunit SH2 domains with inositol
polyphosphate and phosphoinositide ligands. Superimposed contour
plots of highlighted regions of two-dimensional
1H-15N HSQC NMR spectra obtained from
titrations of 0.5 mM 15N-labeled samples of the
p85 N-SH2 domain (A-E) and C-SH2 domain
(F-I) with increasing ligand concentration (0-1.0
mM). The ligands used for each experiment are
D-InsP3 (A and F),
D-InsP4 (B and G),
L-InsP4 (C),
di-C6-PIP3 (D and H), and
phenyl phosphate (E and I). Cross-peaks represent
correlations between the 15N and 1H resonances
of polypeptide backbone amide groups. A single contour level is plotted
per step in the titration. Only a subset of cross-peaks change position
during the titrations. For each set of titrations, the same spectral
region is shown, revealing that di-C6-PIP3 and
the inositol polyphosphates induce similar selective chemical shift
perturbations in all titrations.
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By mapping of the chemical shift perturbation data onto the
three-dimensional protein structures, the ligand binding sites of the
p85
N- and C-SH2 domains were determined. In all cases, it was seen
that the SH2 domain residues affected by the ligands are similar and
are localized to a discrete region of the protein structure (Fig.
2). The results obtained for
di-C6-PIP3, the inositol polyphosphates, and
phenyl phosphate show that all these ligands bind to the region
corresponding to the Tyr(P) binding pocket seen in the high resolution
structures of p85
SH2 domains (39, 40). From quantitative analysis
of the chemical shift variation in the NMR studies, the equilibrium
dissociation constants (KD) of the interactions of
the SH2 domains with di-C6-PIP3 and inositol polyphosphates were found to be between 0.5 and 1 mM.
However, these averaged values contain ranging contributions from
different residues involved in the binding and therefore are best
considered as approximation estimates. The absence of chemical shift
perturbations for the majority of SH2 domain resonances suggests that
the ligands tested neither induced long range conformational changes
nor resulted in local or global unfolding of the protein structure.

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Fig. 2.
The ligand binding sites of
p85 SH2 domain revealed in NMR
titrations. Aligned structures of the p85 N-SH2 domain
(A) and C-SH2 domain (B), made using MOLSCRIPT
(47), Raster3D (48), and GRASP (49), are shown. Secondary structure
elements are represented in schematic form. The blue balls
represent N-H groups, which displayed large chemical shift changes
(modular vector sum 1H and 15N shift changes
>40 Hz) upon the addition of the ligand. In all cases, the ligand
binds between -helix A (23, 39) and the opposing face of the
-sheet. In the upper left of each panel, the SH2 domain
surface is colored by electrostatic potential to highlight the
positively charged ligand binding site (blue); the
red color corresponds to negatively charged surface. This
conserved binding pocket is partly formed by a number of Arg and Lys
residues, including the invariant Arg at the B5 position (23).
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Phosphoinositides and Inositol Polyphosphates Compete Poorly for
the Binding of the p85
SH2 Domains to Tyrosine-phosphorylated
Proteins--
Because the results from NMR spectroscopy showed that
phosphoinositides and inositol polyphosphates can all bind to the
Tyr(P) binding pockets of both p85
SH2 domains, an assay was
performed to assess whether these interactions were sufficiently strong to displace Tyr(P)-containing ligands. A biosensor-based competition assay was used to measure the binding of p85
SH2 domains to an immobilized Tyr(P) peptide with the sequence DMSKDESVDY(P)VPMLDMK (Y(P)
is phosphotyrosine). This phosphopeptide corresponds to the
autophosphorylation site at Tyr751 of the PDGF-
receptor
and is known to bind to both p85
SH2 domains with high affinity
(KD ~ 200 nM) (3). The biosensor
results revealed that when present in relatively high concentrations,
di-C6-PIP3 or any of the inositol polyphosphate ligands tested can compete for the interaction between the p85
SH2
domains and the immobilized Tyr(P) peptide ligand. However, it was
readily apparent that free Tyr(P) peptide was a much more effective
competitor than any of the other ligands tested, by a factor ~1000
(Fig. 3).

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Fig. 3.
Biosensor competition assay. Biosensor
results showing the ability of ligand compounds to compete for the
binding of p85 C-SH2 (panels A and B) and
N-SH2 (panels C and D) domains to a
tyrosine-phosphorylated ligand corresponding to the Tyr-751
(pY751) site of the cytoplasmic domain of the PDGF receptor.
IC50 values were determined by data fitting as described
under "Experimental Procedures" and are listed in Table I.
Phe, phenyl
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|
By curve-fitting the data to obtain IC50 values for the
interactions, it was observed that for each SH2 domain the
phosphoinositide and inositol polyphosphates tested were similarly
competitive. Thus, no preference for ligand binding for either SH2
domain was observed. For example, for the C-SH2 domain, approximately
the same IC50 values were obtained for
di-C6-PIP3, D-Ins
(1,3,4,5)P4, L-Ins (1,3,4,5)P4, and
D-Ins (1,4,5)P3 (Fig. 3, panels A
and B and Table I). Although
the N-SH2 domain exhibited a pattern of interactions similar to that of
the C-SH2 domain, the N-SH2 domain generally interacted even more
weakly with all the compounds tested (Fig. 3, panels C and
D). The IC50 values obtained for the phenyl phosphate interactions were slightly smaller than for the
phosphoinositide or inositol polyphosphate ligands but considerably
larger than for the free Tyr(P) peptide (Fig. 3 and Table I). The
latter result reflects that residues C-terminal to the Tyr(P) are also essential for the known physiological high affinity interaction.
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Table I
Inhibition of binding of phosphoinositide 3-kinase p85 subunit SH2
domains to an immobilized phosphotyrosine peptide in a biosensor assay
N-Biotinyl-DMSKDESVDY(P)VPMLDMK (corresponding to the
Tyr-751 autophosphorylation site of the cytoplasmic domain of the
platelet-derived growth factor receptor) was fixed to a precoated
streptavidin biosensor chip. Solutions of 0.5 µM p85
N- or C-SH2 domain were injected over the surface at a flow rate of 5 µl/min at 25 °C, and the maximum response was recorded.
Competition experiments were performed by incubating the SH2 domains
with a competitor ligand before injection of the protein solution.
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The p85
C-SH2 Domain Does Not Display Distinct Binding
Specificity for Phosphoinositides in Phospholipid Bilayers--
The
results presented above did not suggest that the p85
SH2 domains
undergo specific interactions with water-soluble phosphoinositide or
inositol polyphosphate ligands. However, to eliminate the possibility that the previous assays were not representative of interactions with
phosphoinositides available in vivo, a second biosensor
assay was performed. Using this alternative assay, it was shown
previously that the Btk PH domain binds to phospholipid bilayers
containing PtdIns (3,4,5)P3 but not to those containing
other negatively charged phosphoinositides (35). Subsequently, this
result has been supported by numerous reports of a high affinity
interaction between the Btk PH domain and PtdIns (3,4,5)P3
or D-Ins (1,3,4,5)P4 (15, 41, 42).
Thus, a second biosensor assay was performed to establish whether the
p85
C-SH2 domain could bind to specific phosphoinositides when
presented in large unilamellar liposomes (solely the C-SH2 domain was
tested because the previous assay yielded IC50 values that
were smaller for the C-SH2 domain than for the N-SH2 domain). In brief,
GST fusion SH2 domain was immobilized on an anti-GST antibody-coated
surface, as described previously (35). Solutions of liposomes with
differing phosphoinositide compositions were then injected over the
surface, and the responses were observed. The experimental conditions
used were exactly the same as those for the previously reported study
of phosphoinositide interactions with the Btk PH domain (35). However,
in contrast with the results obtained for the Btk PH domain, the p85
C-SH2 domain did not bind significantly to any of the
phosphoinositide-containing liposomes tested. When the liposome
injection dosages were increased to more than 50 times the quantity
sufficient to give clear binding signals when assaying PH domains (35),
it was possible to observe a low level of nonspecific binding between
the SH2 domain and the entire array of liposomes tested. However, the
C-SH2 domain did not display a preference for binding to liposomes
containing PtdIns (3,4,5)P3 (Fig.
4). Furthermore, it was observed that
under these rather extreme conditions, even a control GST protein
exhibited a basal level of nonspecific binding to all the liposomes
tested (data not shown).

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|
Fig. 4.
Biosensor assay of
p85 SH2 domain interactions with
phosphoinositide-containing liposomes. Biosensor measurements for
interactions between liposomes and the p85 C-SH2 domain. The
specific phospholipid tested in the liposome composition is indicated
next to each sensorgram. PC, phosphatidylcholine
|
|
However, in contrast with the results obtained for the Btk PH domain,
it was observed that the p85
C-SH2 domain did not display a
preference for binding to liposomes containing PtdIns
(3,4,5)P3 (Fig. 4). Indeed, the C-SH2 domain shows only a
low level of binding to an array of liposomes with different
phosphoinositide compositions, with no clear preference emerging for
any of the phosphoinositides tested.
The Inhibition of PI 3-Kinase Activity Does Not Increase the Levels
of Phosphotyrosine-bound p85
in Vivo--
After the in
vitro assays reported above, an in vivo experiment
based on that reported previously (21) was performed to search for a
correlation between the intracellular levels of Tyr(P)-bound p85
and
PI 3-kinase products. This involved measuring the levels of p85
bound to the PDGF receptor and/or tyrosine-phosphorylated proteins
after cell stimulation in the presence or absence of wortmannin.
Because wortmannin inhibits PI 3-kinase activity (43), these
experiments are taken to be in the presence or absence of 3'-phosphorylated phosphoinositides.
The stimulation of NIH3T3 fibroblasts by PDGF induced both the
association of p85
with the PDGF receptor (Fig.
5A) and the appearance of
p85
in anti-phosphotyrosine immunoprecipitates (Fig. 5B),
as judged by the anti-p85
immunoblotting of anti-PDGF receptor and
anti-phosphotyrosine immunoprecipitates, respectively. These
experiments were then repeated, the only difference being the
pretreatment of the fibroblasts with 100 nM wortmannin. The wortmannin treatment did not significantly affect the amount of p85
present in either anti-PDGF receptor immunoprecipitates (Fig. 5A) or in anti-phosphotyrosine immunoprecipitates (Fig.
5B). For control purposes, the efficacy of wortmannin with
respect to PI 3-kinase inhibition was confirmed by the total inhibition
of PI 3-kinase activity present in anti-phosphotyrosine
immunoprecipitates after PDGF stimulation (Fig. 5C).

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Fig. 5.
Lack of correlation of p85-phosphotyrosine
interactions with wortmannin treatment. Wortmannin, a PI 3-kinase
catalytic domain inhibitor, does not promote the association of p85
with the PDGF receptor or, more generally, with tyrosine-phosphorylated
proteins. Serum-starved NIH3T3 fibroblasts were treated with wortmannin
(Wort, 100 nM) or Me2SO (vehicle)
for 15 min and stimulated with PDGF (100 nM) for 10 min,
and cell lysates were immunoprecipitated with anti-PDGF receptor
(Ippt -PDGFR, A) or anti-phosphotyrosine
(Ippt -PY, B and C)
antibodies and protein G-Sepharose. A and B,
anti-phosphotyrosine immunoprecipitates were analyzed by
SDS-polyacrylamide gel electrophoresis and anti-p85 immunoblotting.
C, the immunoprecipitates were assayed for PI 3-kinase
activity. Radioactive lipids were analyzed by TLC. The results are
representative of three independent experiments.
|
|
 |
DISCUSSION |
We sought to verify whether the products of PI 3-kinase activity,
3'-phosphorylated phosphoinositides, can interact with the SH2 domains
derived from the p85
regulatory subunit of PI 3-kinase itself. Such
interactions have been proposed as competitors of the association of PI
3-kinase with tyrosine-phosphorylated proteins and as regulators of
other SH2 domain-containing proteins, e.g. Src and
phospholipase C
-1 (21, 22). Although the SH2 domain-mediated phosphoinositide-dependent regulation of p85
/p110
,
Src, or phospholipase C activity has interesting implications, it is a
model yet to be clearly established.
Therefore, our primary experimental aim was to discover whether the
p85
N- and C-SH2 domains can bind to distinct phosphoinositides with
high affinity, specificity, and stereoselectivity. Furthermore, we
investigated whether the well defined interactions between the p85
SH2 domains and Tyr(P)-containing ligands could be competed by
phosphoinositides or inositol polyphosphates. We tested both phosphoinositides and inositol polyphosphates as potential ligands of
SH2 domains. Inositol polyphosphates were in part used as conveniently water-soluble analogues of the head groups of phosphoinositides but may
also represent physiological ligands. In our opinion this usage of
inositol polyphosphates is valid because protein-phosphoinositide interactions appear to be predominantly governed by the charge status,
phosphorylation positions, and stereochemistry of the inositol ring
(42, 44, 45). For example, it has been demonstrated that
D-Ins (1,4,5)P3 can functionally replace PtdIns
(4, 5)P2 in the activation of the dynamin GTPase (35) and
that the Btk PH domain binds specifically to both D-Ins
(1,3,4,5)P4 and PtdIns (3,4,5)P3 (35, 41)
in vitro and to PtdIns (3,4,5)P3 in
vivo (15).
In search of specific phosphoinositide binding preferences of the
p85
SH2 domains, we chose to compare their interactions with
D-Ins (1,4,5)P3 and D-Ins
(1,3,4,5)P4 or PtdIns (4,5)P2 and PtdIns
(3,4,5)P3. This choice was based on the knowledge that PtdIns (4,5)P2 is abundant in the plasma membrane of
resting cells, whereas PtdIns (3,4,5)P3 is only present in
appreciable quantities after cell stimulation (8, 9). We also compared
the binding of the p85
SH2 domains to the physiological
D- and nonphysiological L-enantiomers of the
inositol polyphosphates, because stereoselectivity should be exhibited
in the case of true, biological interactions.
We observed that numerous different phosphoinositides and inositol
polyphosphates can bind to the p85
SH2 domains, albeit weakly. Using
NMR spectroscopy, we found that di-C6-PIP3 and
all the inositol polyphosphates tested bound to the SH2 domains in the
Tyr(P) binding pockets that accommodate protein ligands. However, the
SH2 domains failed to display clear preferences for distinct phosphoinositides or inositol polyphosphates presented in solution. Similarly, the C-SH2 domain did not demonstrate a distinct binding specificity for phosphoinositides presented in phospholipid bilayers. Surface representations of the SH2 domain structures that display their
calculated electrostatic potentials show that the Tyr(P) binding
pockets of the N- and particularly of the C-SH2 domains are highly
positively charged. Thus, the lack of binding specificity or
stereoselectivity shown by the SH2 domains for the test ligands may
reflect the likelihood that their interaction is largely based on
electrostatic interactions that have little dependence on distinct structural features. Such interactions are thus very different from
those of high specificity observed between SH2 domains and physiological Tyr(P)-containing ligands.
In addition, in a competition assay we observed that despite an overlap
of binding sites, phosphoinositides and inositol polyphosphates only
poorly displaced SH2 domains from a Tyr(P) peptide ligand. Furthermore,
among the ligands tested, there was no significant variation in the
efficacy of competition. From this assay, it also emerged that the
N-SH2 domain bound to di-C6-PIP3 and inositol polyphosphates similarly to, but even more weakly than, the C-SH2 domain. This observation may perhaps be explained by two factors. First, the Tyr(P) binding pocket produces a greater density of positive
charge on the surface of the C-SH2 domain compared with the surface of
the N-SH2 domain (see Fig. 2), thus favoring interactions of the former
with negatively charged ligands. Second, it has been observed that the
unoccupied Tyr(P) binding pocket of the C-SH2 domain is relatively
exposed, whereas that of the N-SH2 domain is not fully formed in the
absence of a peptide ligand (39) and may therefore be less accessible
to phosphoinositides.
However, the similar patterns of ligand binding observed for both
p85
SH2 domains suggest that all the ligands contact the SH2 domains
in the same, rather nonspecific manner. We conclude that although
in vitro both p85
SH2 domains may interact weakly with
PtdIns (3,4,5)P3 and inositol polyphosphates, the lack of specificity of these interactions and their inability to compete effectively with Tyr(P) peptide ligands suggest that they do not represent physiologically significant interactions. Rather, it seems
that in vitro the SH2 domain Tyr(P) binding pocket has a tendency to bind somewhat indiscriminately to negatively charged ligands. This promiscuity is further witnessed in a crystal form of the
p85
C-SH2 domain in which the Tyr(P) binding pocket accommodates an
aspartate side chain.2
Although the mode of interaction is highly reminiscent of Tyr(P) binding (coordination of the aspartate carboxylate group with both R36
and R18), the aspartate side chain is clearly not a consensus ligand.
Finally, we demonstrated that although in vivo, wortmannin
blocks the activity of PI 3-kinase, it does not affect the ability of
activated PDGF receptors (or other tyrosine-phosphorylated proteins) to
bind the p85
regulatory subunit. These results are in agreement with
characterizations of wortmannin activity (46) but contrast with
previous reports of an inverse correlation between the level of
3'-phosphorylated phosphoinositides in the cell and the association of
PI 3-kinase with tyrosine-phosphorylated proteins (insulin receptor and
insulin receptor substrate) (21). Therefore we suggest the levels of PI
3-kinase products in the plasma membrane are unlikely to regulate
signal transduction events through interactions with SH2 domains.
Rather, we consider that the immediate targets of PI 3-kinase activity
are represented by those proteins that display high affinity, distinct
binding specificity, and stereoselectivity for 3'-phosphorylated
phosphoinositides, such as the PH domain-containing proteins Akt, Btk,
PDK-1, and phospholipase C
-1.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
b
Present address: Structural Biology Programme, EMBL,
Meyerhofstrasse 1, Heidelberg 69117, Germany.
e
Present address: Leiden Institute of Chemistry, Leiden
University, Einsteinweg 55, 2300-RA Leiden, The Netherlands.
d
Present address: Ludwig Institute for Cancer Research,
Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland.
f
Present address: Institute of Molecular Oncology,
B.S.R.C. "A. Fleming", 14-16 Fleming St., Vari 16672, Greece.
i
Supported by Wellcome Trust Programme Grant 045491.
j
Supported by the Royal Society. To whom correspondence
should be addressed: Dept. of Biochemistry and Molecular Biology,
University College London, Gower St., London WC1E 6BT, UK. Tel.: 44-171 380 7035; Fax: 44-171 380 7193; E-mail:
driscoll{at}biochem.ucl.ac.uk.
2
F. Hoedemaker, G. Siegal, S. M. Roe, P. C. Driscoll, and J. P. Abrahams, manuscript submitted.
 |
ABBREVIATIONS |
The abbreviations used are:
SH2, Src homology 2;
di-C6-PIP3, rac-dihexanoylphosphatidyl-D/L-myo-inositol
3,4,5-trisphosphate;
HSQC, heteronuclear single quantum coherence;
GST, glutathione S-transferase;
Ins, D-myo-inositol;
PDGF, platelet-derived growth
factor;
PH, pleckstrin homology;
PI, phosphoinositide;
Ptd, phosphatidyl;
Tyr(P), phosphotyrosine;
PBS, phosphate-buffered
saline.
 |
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