Intermolecular Interactions of the p85
Regulatory Subunit of
Phosphatidylinositol 3-Kinase*
Ailsa G.
Harpur
§¶,
Meredith J.
Layton
¶
,
Pamela
Das
,
Matthew J.
Bottomley**
,
George
Panayotou§§,
Paul C.
Driscoll**, and
Michael D.
Waterfield
**
From the
Ludwig Institute for Cancer Research, 91 Riding House St., London W1P 8BT, the ** Department of Biochemistry
and Molecular Biology, University College London, Gower St.,
London WC1E 6BT, United Kingdom, and the
§§ Institute of Molecular Oncology, Biomedical
Science Research Center "A. Fleming," Vari 16672, Greece
 |
ABSTRACT |
The regulatory subunit of phosphatidylinositol
3-kinase, p85, contains a number of well defined domains involved in
protein-protein interactions, including an SH3 domain and two SH2
domains. In order to investigate in detail the nature of the
interactions of these domains with each other and with other binding
partners, a series of deletion and point mutants was constructed, and
their binding characteristics and apparent molecular masses under
native conditions were analyzed. The SH3 domain and the first
proline-rich motif bound each other, and variants of p85 containing the
SH3 and BH domains and the first proline-rich motif were dimeric. Analysis of the apparent molecular mass of the deletion mutants indicated that each of these domains contributed residues to the dimerization interface, and competition experiments revealed that there
were intermolecular SH3 domain-proline-rich motif interactions and
BH-BH domain interactions mediating dimerization of p85
both in vitro and in vivo. Binding of SH2 domain
ligands did not affect the dimeric state of p85
. Recently, roles for
the p85 subunit have been postulated that do not involve the catalytic
subunit, and if p85 exists on its own we propose that it would be dimeric.
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INTRODUCTION |
The Class IA phosphatidylinositol 3-kinases
(PI3K)1 are heterodimeric
enzymes with a p110 catalytic subunit and a p85 regulatory subunit (1).
The p85 subunit is a multidomain protein comprising an amino-terminal
SH3 domain, a BCR homology (BH) domain which has homology to the GTPase
activating protein domain of the Break-point Cluster Region protein (2)
and Rho subfamily GTPases, and two SH2 domains separated by an
inter-SH2 domain through which p85 binds the catalytic subunit (3). The
BH domain is flanked by two proline-rich motifs. To date, five isoforms
of p85 have been identified. p85
has been cloned from bovine (4),
human (5), and mouse (6) cDNA libraries, whereas only bovine p85
has been identified (4). Two splice variants of p85
, termed p55 and
p50, have been identified in the human (7), the rat (8, 9), and the
mouse (10). p55
lacks the SH3 and BH domains and the first
proline-rich motif (PRM1) but retains the second proline-rich motif
(PRM2) and has an amino-terminal extension of 34 amino acids. In
p50
, this extension comprises only 6 residues. To date, no splice
variants of p85
have been identified. A variant known as p55
or
p55PIK has been cloned from
bovine2 and human (11)
cDNA libraries and is homologous to p55
, but no higher molecular
mass isoforms of this protein have yet been identified.
PI3K has been implicated in a wide range of signaling pathways
including those regulating proliferation and cell migration (12). The
modular domains in p85
possess intrinsic signaling functions, in
that they bind numerous intracellular ligands and mediate the formation
of multiprotein complexes. The proline-rich motifs bind the SH3 domains
of the Src family tyrosine kinases, Lyn and Fyn (13), whereas the small
G proteins Rac (14, 15) and Cdc42 (16) are potential PI3K regulators or
effectors that bind PI3K, presumably via the BH domain. The SH2 domains
of p85 bind phosphotyrosine (pY)-containing sequences from a range of receptor tyrosine kinases and docking proteins such as insulin receptor
substrate 1 (17-19).
The roles of the various isoforms of the adaptor subunits of the Class
IA PI3Ks are as yet undefined. Some isoforms, especially the truncated
isoforms p55
and p50
, have been shown to have restricted tissue
distributions (9) compared with the 85-kDa isoforms. In addition, the
truncated isoforms are clearly unable to interact with proline-rich
motif- or SH3 domain-containing proteins or with small G proteins, and
it has been shown that there is some selectivity in the recruitment of
p85 isoforms by receptor tyrosine kinases (20). However, it is not yet
understood whether the large number of isoforms of both subunits of
PI3K represents a functional redundancy or reflects a form of signaling specificity. We have therefore undertaken a detailed study of the
potential interactions of the individual domains of the p85 protein in
order to elucidate their roles within the adaptor subunit as a whole.
 |
MATERIALS AND METHODS |
Peptides--
Peptides were synthesized by Zinsser
Analytic or Alta Biosciences and were of the following sequences: P1,
KISPPTPKPRPPRPLPVAPGPS; P2, WNERQQPAPALPPKPPKPT; Tyr-740/Tyr-751,
GGpYMDMSKDESVDpYVPML; Tyr-740, GGpYMDMSKDESVDYVPML; Tyr-751,
GGYMDMSKDESVDpYVPML (where pY represents a phosphotyrosine residue).
Construction, Expression, and Purification of Recombinant
Proteins--
Subcloning of cDNAs encoding p85
and p85
into
the pAcC4 baculovirus transfer vector has been previously described
(21). p55
is the bovine homologue of human p55PIK (11)
and rat p55
(8). p49
is the bovine homologue of rat p50
(9)
but with the unique 6-residue amino-terminal sequence deleted.
cDNAs encoding p110
(22), p85
SH3, p85
BH,
p85
PRM1, p85
PRM2, p85
PRM1:PRM2, and p55
(Table I) were subcloned into the
baculovirus transfer vector, pVL1393 (Invitrogen). A sequence encoding
a hexa-histidine tag was added to the 3' end of the p49
cDNA,
which was then subcloned into the baculovirus transfer vector,
pBlueBac4 (Invitrogen). cDNAs encoding p85
SH3-BH-SH2, p85
SH3-BH, p85
SH3-PRM1, p85
SH3, p85
cSH2, and p85
BH
(Table I) were subcloned into the bacterial expression vector
pGEX-2T (Amersham Pharmacia Biotech). Myc epitope-tagged (23) p85
, hexa-histidine-tagged p49
and p55
were also subcloned into pMT-SM (24) for expression in mammalian cells.
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Table I
Amino acid sequence specifications for p85 deletion and
substitution mutants
The residues derived from the amino acid sequence of full-length p85
(1-742) that comprise each mutant are listed. Single amino acid
substitutions are denoted as P96A, indicating that the proline at
position 96 was mutated to arginine. At the bottom is a schematic
representation of the domain structure of p85 . The positions of the
designated domain boundaries are numbered according to the sequence of
p85 .
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All cDNAs in pGEX-2T were expressed as glutathione
S-transferase fusion proteins and purified by glutathione
affinity chromatography according to the manufacturer's instructions.
cDNAs in the baculovirus transfer plasmid pVL1393 were
co-transfected into Sf9 cells with BaculoGold-linearized
baculovirus DNA (PharMingen), whereas those in the transfer plasmid
pBlueBac4 were co-transfected into Sf9 cells with Bac-N-Blue
baculovirus DNA (Invitrogen). The recombinant baculovirus was
plaque-purified and amplified as described previously (25).
Exponentially growing Sf9 cells at a density of 1.5-2 × 106/ml were infected with recombinant baculoviruses at an
multiplicity of infection between 10 and 20 and harvested 2-3 days
post-infection. SV40-transformed monkey kidney cells (Cos7) were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum, at 37 °C and 10% CO2. Transient
transfections were performed using DEAE-Dextran (Sigma). Cells (50%
confluent) were transfected with 15 µg of plasmid DNA per 15-cm
tissue culture dish and harvested 48 h post-transfection.
Sf9 cells expressing recombinant p85 variants were lysed in
Triton X-100 lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 5 mM dithiothreitol
(DTT), and a range of protease inhibitors (50 µg/ml
4-(2-aminoethyl)-benzenesulphenylfluoride hydrochloride, 16 µg/ml
benzamidine, 10 µg/ml 1,10-phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 µg/ml pepstatin A)). p85 variants were
purified by affinity chromatography using a phosphotyrosine-affinity
column that was synthesized by immobilizing 2 mg of phosphotyrosine
(Sigma) per ml of Actigel (Sterogene) according to the manufacturer's
instructions. Briefly, up to 50 ml of cell lysate was applied to a
14 × 1-cm column of phosphotyrosine-Actigel equilibrated in
Buffer A (20 mM Tris, pH 8.0, containing 5 mM DTT) and eluted with 50 ml of Buffer A followed by 50 ml of Buffer A
containing 150 mM NaCl and 50 ml of Buffer A containing 2 M NaCl. Fractions of 2.5 ml were collected at a flow rate
of 2.5 ml/min and exchanged into 3.5 ml of Buffer A using pre-packed Sephadex G-25 columns (PD10, Amersham Pharmacia Biotech). If a second
purification step was required, fractions containing p85 variants were
pooled and applied to a 0.46 × 10-cm POROS 20 HQ column
(Perspective Biosystems) equilibrated in Buffer A. Elution was carried
out using 17 ml of Buffer A followed by a 75-ml linear gradient to 1 M NaCl in the same buffer. Fractions of 2.5 ml were collected at a flow rate of 2.5 ml/min.
High Performance-Size Exclusion Chromatography
(HP-SEC)--
Transfected or wild type Cos7 cells were harvested by
trypsinization and Dounce-homogenized in hypotonic lysis buffer (5 mM Tris, pH 7.5, 2.5 mM KCl, 1 mM
DTT, 1 mM EDTA, 1 mM phenylmethylsufonyl fluoride, 10 µM leupeptin, 10 µM pepstatin
A, 1 mM 1,10-phenanthroline, 1 mM sodium
orthovanadate). The cytoplasmic fraction was clarified by
centrifugation at 100,000 × g at 4 °C for 45 min
and filtered through a 0.22-µm membrane (Ultrafree-MC, Millipore).
Aliquots of 200 µl or less of molecular mass standards (Bio-Rad),
recombinant proteins, or cell lysates were applied to a Superose 12/30
HR column (Amersham Pharmacia Biotech) equilibrated in TBS (20 mM Tris, pH 8.0, 150 mM NaCl). Samples were
eluted isocratically with TBS at a flow rate of 0.3 ml/min and 0.3- or
0.15-ml fractions were collected where necessary.
Sedimentation Equilibrium-Analytical Ulracentrifugation
(SE-AUC)--
All SE-AUC experiments were carried out using an
Optima XL-A analytical ultracentrifuge (Beckman) equipped with
absorbance optics and an An60Ti rotor. Protein samples were buffer
exchanged into SE-AUC buffer (50 mM Tris, pH 7.4, 50 mM NaCl, 7 mM
-mercaptoethanol, and 0.02%
NaN3) using either pre-packed Sephadex G-25 columns (NAP5,
Amersham Pharmacia Biotech) or by dialysis overnight. Three samples of
110 µl of protein at approximately 0.6, 0.3 and 0.15 mg/ml were
analyzed, and the reference cells contained 125 µl of SE-AUC buffer.
Experiments were performed at 4 °C at an optimized range of speeds
between 4000 and 28,000 rpm. Equilibrium data were collected at 280 nm
in step scan mode with a radial increment of 0.001 cm between data
points. Five readings were averaged each scan, from which a base-line
scan taken at 360 nm was subtracted in order to correct for optical
imperfections. Readings were taken at 8-h intervals until no difference
could be detected between consecutive scans. The equilibrium
distributions from three different loading concentrations and up to
three rotor speeds were analyzed both individually and
simultaneously using the Nonlin curve-fitting algorithm supplied with
the ultracentrifuge (Beckman).
Binding Studies Using an Optical Biosensor--
The procedure
for measuring interactions between domains and peptides using the
BIAcore biosensor (Biacore AB) has been previously described (26).
Briefly, biotinylated peptides were captured on immobilized avidin, and
then sample was injected in running buffer (20 mM Hepes, pH
7.4, 150 mM NaCl, 3.4 mM EDTA, 4 mM
DTT, 0.005% Tween 20), and the response at equilibrium was recorded. For competition experiments, the response at equilibrium was compared with that obtained upon preincubation of the protein or isolated domain
solution with increasing amounts of free peptide. In order to calculate
IC50 values, the results were plotted as resonance units
versus peptide concentration, and the curve obtained was fitted with the equation R = (Rmax/1 + (C/IC50)P), where R is the
response, Rmax is the response obtained in the absence of competitor, C is the concentration of competitor,
and P is the Hill coefficient.
Immunoprecipitations and Western Blotting--
Transfected Cos7
cells were lysed on ice in Nonidet P-40 lysis buffer (1% Nonidet P-40,
20 mM Tris, pH 7.4, 50 mM NaCl, 50 mM NaF, 1 mM EDTA, 500 µM sodium
orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 1 mg/ml
leupeptin, and 1 mM aprotinin), and cell debris and nuclei
were removed by centrifugation at 10,000 × g for 10 min at 4 °C.
Immunoprecipitations from Cos7 cell lysates were performed for 2 h
using p85
monoclonal antibodies (U9, U13, and U14 (27)) or an
anti-Myc epitope monoclonal antibody (23) immobilized using
protein G-Sepharose Fast Flow (Amersham Pharmacia Biotech) or a
metal-chelate affinity matrix (Talon, CLONTECH).
Precipitated proteins were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting
onto polyvinylidene difluoride membrane (Gelman Sciences), probing with
an appropriate mouse monoclonal antibody, and detection of bound
horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad)
using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
Protein in fractions from HP-SEC was precipitated using 10%
trichloroacetic acid and analyzed by SDS-PAGE and Western blotting as
described above. Proteins were precipitated from Sf9 cell
lysates using GST fusion proteins captured on glutathione-Sepharose CL 4B (Amersham Pharmacia Biotech) and analyzed for lipid kinase assay as
described below.
Phosphatidylinositol 3-Kinase Assays--
PI3K assays of
glutathione-Sepharose CL 4B-precipitated proteins were carried out
essentially as described previously (28). Lipid kinase assays contained
2 mM MgCl2, 1 mM ATP, 20 µCi of [
-32P]ATP, and 200 µg/ml phosphatidylinositol.
Extracted phospholipids were analyzed by thin layer chromatography in
65% 1-propanol, 0.7 M acetic acid, 50 mM
phosphoric acid.
 |
RESULTS |
Interactions of the SH3 Domain and the Proline-rich Motifs of
p85
--
It has previously been reported that both proline-rich
motifs in p85
conform to the consensus ligand for the SH3 domain of p85
(29). In order to determine which of the two proline-rich motifs
was the preferred ligand for the isolated p85
SH3 domain (p85
SH3), we compared its ability to bind to peptides derived from
the sequences of the first (P1) or second (P2) proline-rich motifs of
p85
, using an optical biosensor (Fig.
1A). Binding of p85
SH3 to
immobilized P1 was observed in this system, producing a response of
greater than 400 resonance units (Fig. 1A), whereas little
or no p85
SH3 injected at the same concentration bound to P2 (Fig.
1A), suggesting that P1 is the preferred ligand for p85
SH3.

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Fig. 1.
Interactions of the p85
SH3 domain and the proline-rich motifs of
p85 . A, comparison of binding
of p85 SH3 to immobilized peptides corresponding to the first
proline-rich motif, P1 (---), or the second proline-rich motif, P2
(- - -), from p85 using an optical biosensor. B,
comparison of binding of equal concentrations of p85
(-· · ·-) and p85 variants with point mutations in the
first, p85 PRM1 (---), second, p85 PRM2 (- - -) or both,
p85 PRM1:PRM2 (- -) proline-rich motifs to immobilized P1
peptide. C, precipitation of phosphatidylinositol kinase
activity associated with p110 /p85 or p110 /p85 SH3 from
Sf9 cell lysates using GST-p85 SH3 or antibodies to
p85 .
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In contrast, wild type p85
, which contains the SH3 domain, did not
bind immobilized P1 to a significant extent (Fig. 1B). The
small amount of binding observed was equivalent to that observed for
binding of the same concentration of p85
to a surface on which P1
was not immobilized (data not shown). A mutant of p85
, in which two
proline residues in PRM1 that have been shown to be important for
p85
SH3 domain binding (30) were changed to alanine (p85
PRM1),
was able to bind P1; however, a variant of p85
with mutations at the
equivalent residues in PRM2 (p85
PRM2) was not. A variant of
p85
in which both proline-rich motifs contained mutations
(p85
PRM1:PRM2) bound to P1 to a similar extent as p85
PRM1
(Fig. 1B). Mutations in PRM1 therefore allowed p85
SH3 to
bind exogenous P1 peptide, whereas the SH3 domain in the context of
wild type p85
could not bind to exogenous P1. Mutations in PRM2 did
not affect binding to P1 peptide, confirming that the first
proline-rich motif was the preferred ligand for the p85
SH3 domain.
Thus, mutation of key residues in PRM1 of p85
not only affected this
site but also the binding characteristics of the SH3 domain. These
mutations increased the ability of the SH3 domain to bind exogenous
ligands, indicating that in wild type p85
the SH3 domain interacts
with PRM1 and that when this interaction is disrupted the SH3 domain is
free to bind exogenous peptide.
Similarly, deletion of the SH3 domain of p85
would be expected to
free PRM1 and allow it to bind exogenous SH3 domains. When wild type
p85
was co-expressed with the p110 catalytic subunit in Sf9
cells, a p85
SH3 GST fusion protein (GST-p85
SH3) immobilized on
glutathione-Sepharose CL4B was poorly able to co-precipitate PI3K lipid
kinase activity from Sf9 cell lysates. However, a monoclonal antibody directed against the SH2 domain of p85
was able to
immunoprecipitate PI3K activity; thus both p85
and p110
were
expressed in these cells. The same antibody was able to
immunoprecipitate PI3K activity from lysates of Sf9 cells
co-infected with p110
and a mutant of p85
in which the SH3 domain
had been deleted (p85
SH3). Immobilized GST-p85
SH3 was able to
co-precipitate the p110
-p85
SH3 complex to a much greater
extent than the p110
-p85
complex (Fig. 1C); thus
deletion of the SH3 domain increased the ability of an exogenous SH3
domain to bind PRM1, confirming that the SH3 domain and PRM1 are bound
to each other in both p85
and the p110
-p85
complex.
Intermolecular Interactions of p85 Isoforms--
Examination of
purified, recombinant p85
and p85
by HP-SEC under native
conditions showed that they had apparent molecular masses that were
approximately double (162 ± 14 and 151 ± 12 kDa, respectively) that expected from their amino acid sequences (Fig. 2, A and B). In
contrast, the apparent molecular masses of the truncated p85
isoforms, p55
and p49
(82 ± 4 kDa and 52 ± 2 kDa),
were much closer to their predicted molecular masses (55 and 49 kDa;
Fig. 2, A and B).

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Fig. 2.
Analysis of naturally occurring
p85 isoforms by HP-SEC. A,
high performance size-exclusion chromatography of recombinant p85
(---), p85 (- -), p55 (- - -) or p49 (-···-). The
Superose 12/30 column was equilibrated and run in TBS at 0.3 ml/min.
The retention times of the molecular mass standards are indicated (in
kDa), as is the void volume (Vo). B,
summary of data from HP-SEC of recombinant p85 isoforms and p85
deletion mutants and a diagrammatic representation of the domains in
each p85 isoform is indicated. The predicted molecular mass for each
isoform (in kDa) was calculated from its amino acid sequence. Molecular
masses determined by HP-SEC were calculated from the retention time
(min) of the maximum height of the peak for each recombinant protein.
The retention time was converted to an apparent molecular mass by
comparison to a plot of log molecular mass (kDa) versus the
retention time (min) of the maximum height of the peak for a range of
standard globular proteins chromatographed using the same buffer
conditions. Apparent molecular masses are denoted as the mean of at
least three measurements ± 1 S.D. Where only one measurement was
taken, an approximate molecular mass is indicated. For HP-SEC of
transfected Cos7 cytosolic extracts, a range of apparent molecular
masses is calculated by converting the fraction numbers that are
positive for the appropriate band by Western blotting into retention
time (min) and therefore into apparent molecular mass (kDa) as
described above.
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There are several mechanisms by which a protein under native conditions
can have an apparent molecular mass different from that predicted from
its amino acid sequence. HP-SEC measures the hydrodynamic volume of a
protein, which is defined as the spherical volume occupied by that
protein as it tumbles rapidly in solution. Non-spherical proteins often
elute with an abnormally high apparent molecular mass compared with the
globular proteins used to calibrate the column. Proteins can also
interact nonspecifically with the column matrix, impeding their
progress through the column and resulting in a later elution time,
leading to an underestimate of the molecular mass of a protein. High
apparent molecular masses may also result from dimerization or
oligomerization of the protein; thus we investigated whether the higher
than expected molecular mass of p85
was due to dimerization by
determining the molecular masses of p85
and p49
under different conditions.
HP-SEC was carried out in buffers containing either 5 mM
DTT or 8 M urea in order to determine whether the putative
p85
dimeric interaction could be disrupted. The apparent molecular
masses of p85
and p55
were unaltered in the presence of 5 mM DTT (Fig. 2B), suggesting that dimerization
of p85
was not due to the presence of an intermolecular disulfide
bond. In contrast, p85
had an apparent molecular mass of 89 ± 5 kDa in the presence of 8 M urea. High concentrations of
urea disrupt non-covalent, but not covalent, bonds, indicating that
p85
forms a dimer via a non-covalent interaction. The apparent
molecular mass of p49
was relatively unaffected in the presence of 8 M urea however (Fig. 2B), suggesting that it is
monomeric under native conditions.
Sedimentation Equilibrium-Analytical Ultracentrifugation (SE-AUC) was
employed to determine the apparent molecular masses of a number of
p85
variants, as the equilibrium distribution of a solute in a
gravitational field is dependent on the molecular mass of the solute,
but independent of its shape, whereas HP-SEC is dependent on both mass
and shape. Analysis of the equilibrium distribution of the
concentration of protein with respect to radial position in the
centrifuge demonstrated the occurrence of self-association in some
samples. Self-association was defined as a lack of adherence to the
Lamm equation, which describes the distribution of ideal, non-associating solute particles in a gravitational field. The occurrence of self-association manifests as a non-random distribution of residuals to the fit of the experimental (absorbance
versus radius) data to a derivative of the Lamm equation.
The equilibrium distribution of p85
did not fit that of an ideal,
non-associating solute, as the residuals were non-random (Fig.
3D). The apparent molecular
mass of p85
was dependent on protein concentration and ranged
between 80 and 110 kDa (Fig. 3D), suggesting that p85
exists as an equilibrium of monomers and dimers under these conditions.
In contrast, p49
was monomeric under the conditions examined. The
residuals of the fit for an ideal, non-associating solute were
distributed randomly around zero, and p49
had an apparent molecular
mass of approximately 47 kDa (Fig. 3C). Thus, p85
, which
was apparently dimeric by HP-SEC, fitted a model for self-association,
although the equilibrium dissociation constant for p85
dimer
formation (estimated from the plot of apparent molecular mass
versus protein concentration; Fig. 3D) was in the
micromolar range, which is a lower affinity than that expected for a
protein that was determined to be constitutively dimeric by HP-SEC.
Given that p49
was shown to be monomeric using both techniques, this
suggested that the dimerization of p85 was mediated by domains that are
not present in p49
.

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Fig. 3.
Sedimentation equilibrium-analytical
ultracentrifugation analysis of domains of
p85 . The data ( ) are fitted to a curve
describing the concentration distribution of single, ideal species
using the Nonlin curve-fitting algorithm (Beckman). A plot of the
residuals for this fit is shown in the upper panel. The
dependence of apparent molecular mass on protein concentration is shown
in the lower panel for p85 BH (A),
p85 SH3-PRM1 (B), p49 (C), and p85
(D).
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An Intermolecular Interaction between the SH3 Domain and PRM1
Contributes to the Dimerization of p85
--
The observation that
p85
and p85
were dimeric under native conditions, but p55
and
p49
were not, suggested that the amino-terminal half of the higher
molecular mass regulatory subunits was involved in intermolecular
interactions that caused them to dimerize. Given that the SH3 domain
and the first proline-rich motif reside in this region of the protein,
and interact with each other (Fig. 1), we investigated whether this
interaction was involved in the dimerization of p85
.
Indeed, expression of the amino-terminal half of p85
on its own,
either with or without one of the SH2 domains (p85
SH3-BH-SH2 and
p85
SH3-BH; Fig. 2B), resulted in proteins with apparent
molecular masses by HP-SEC (101 ± 10 and 53 ± 4 kDa,
respectively) that were still approximately double those predicted (50 and 38 kDa, respectively). This confirms that the amino-terminal half
of p85
contains all the amino acid residues required for dimerization.
When the SH3 domain of p85 was deleted, the apparent molecular mass of
the resulting protein (p85
SH3) determined by HP-SEC under native
conditions was 139 ± 7 kDa, which was approximately double that
predicted from its amino acid sequence (74 kDa) (Fig. 2B).
This suggested that removal of the SH3 domain did not disrupt the dimer
interface to a significant extent. Similarly, mutation of two proline
residues (Pro-96 and Pro-99) in the first proline-rich motif to alanine
(p85
PRM1, Fig. 2B) did not convert p85
to a
monomeric species. Deletion of the remaining domain within the amino-terminal portion of p85
, the BH domain (p85
BH), also did
not disrupt dimerization, as its molecular mass by HP-SEC (145 ± 11 kDa) was still approximately double that predicted (64 kDa). In
contrast, deletion of both the SH3 and BH domains, as is the case for
both p55
and p49
, had already been shown to result in a monomeric
protein. Therefore, the dimerization interface resides in the
amino-terminal half of the molecule, either in the SH3 or BH domains or
the intervening regions.
A construct comprising just the SH3 domain and PRM1 (p85
SH3-PRM1,
Fig. 2B) had an apparent molecular mass by HP-SEC (23 ± 2 kDa) approximately double that predicted (12 kDa), suggesting it
was dimeric. Like p85
, the equilibrium sedimentation characteristics of p85
SH3-PRM1 were consistent with that of a protein undergoing self-association (Fig. 3B). The residuals of the fit for an
ideal, non-associating solute were non-random, and the apparent
molecular mass of p85
SH3-PRM1 was dependent on protein concentration
and ranged between 16 and 22 kDa, with an equilibrium dissociation constant for dimer formation in the micromolar range (Fig.
3B). In contrast, the SH3 domain alone was unable to
dimerize, as it had a similar apparent molecular mass by HP-SEC to that
predicted from its amino acid sequence (13 ± 1 and 9.7 kDa, respectively).
The apparent molecular mass of the isolated BH domain was determined by
HP-SEC (32 ± 2 kDa) and was approximately 1.5 times that
predicted (22 kDa). The p85
BH also appeared to self-associate by
SE-AUC, although to a lesser degree than for p85
SH3-PRM1 (Fig. 3A). The estimated equilibrium dissociation constant for
dimer formation was in the millimolar range.
The interaction surface for dimerization therefore does not reside in a
single domain of the amino terminus of p85
, as neither the SH3 nor
BH domain alone was observed to dimerize fully. Moreover, deletion of
either domain or mutation of PRM1 did not generate monomeric p85
(Fig. 2B). The interaction surface involved in dimerization
must therefore involve residues that are widely distributed in the
primary structure of p85
, so that no single domain self-associates to a significant degree, but are positioned in three-dimensional space
in the folded protein so that they cooperate to form a single binding
surface. In contrast, p85
SH3-PRM1 was able to self-associate, and
the estimated equilibrium dissociation constants for dimerization of
p85
SH3-PRM1 and p85
were similar, suggesting that SH3-PRM1 binding was intermolecular and contributed to dimerization. However, the contribution of the BH domain to dimerization of full-length p85
did not allow us to discriminate between an inter- or intramolecular SH3-PRM1 interactions in the whole protein.
If the interaction between the p85
SH3 domain and the first
proline-rich motif was intermolecular and contributed to dimerization, exogenous P1 peptide should compete for this interaction and result in
the formation of monomeric p85
. The affinities for self-association as determined by SE-AUC were lower than the apparent affinities suggested by the molecular masses of the p85
variants as determined by HP-SEC, thus competition experiments were performed using SE-AUC. In
the presence of a 20-fold molar excess of P1 peptide, both p85
SH3-PRM1 and wild type p85
behaved as ideal, non-associating solutes, and their apparent molecular masses were not dependent on
protein concentration (Fig. 4,
A and B), suggesting that P1 peptide competed for
binding in a site involved in dimer formation. However, the apparent
molecular mass of p85
bound to P1 peptide was greater than expected,
suggesting that either a small degree of self-association was mediated
by the BH domain or that the high concentration of P1 in the analyte
solution increased its viscosity, resulting in an artificially high
estimation of apparent molecular mass. An intermolecular interaction
between the p85
SH3 domain and the p85
PRM1 therefore seems to
contribute to p85
dimerization, although the BH domain also
contributes residues to this interface.

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Fig. 4.
Sedimentation equilibrium-analytical
ultracentrifugation analysis of p85 and
p85 SH3-PRM1 in the presence of competing P1
peptide. p85 SH3-PRM1 (A) and p85 (B)
were incubated with a 20-fold molar excess of P1 peptide and analyzed
as described in Fig. 3.
|
|
Dimerization of p85
in Vivo--
In order to confirm whether
p85
dimerization observed in vitro also occurred in
vivo, p85 isoforms were transfected into Cos7 cells and Western
blot analysis of HP-SEC fractions of whole cell lysates revealed
immunoreactive bands in fractions covering a relatively broad range of
molecular masses. p85
was detected in fractions 36-38, which
corresponded to a molecular mass range of 93-176 kDa (Figs.
5A and 2B).
Although this was a broad range, the majority of the p85
eluted at a
time that corresponded to a molecular mass greater than the monomeric
molecular mass for p85
(83 kDa), and there was a population at a
dimeric molecular mass (176 kDa). p85
therefore is dimeric both as a
homogeneous, purified recombinant protein and within the context of a
cellular environment, when it is at a relatively low concentration and in a protein-rich environment, which are conditions that would be
expected to reduce nonspecific self-association.

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Fig. 5.
p85 from transfected
Cos7 cells behaves as a dimeric complex upon size-exclusion
chromatography and co-precipitates with
p85 BH but not with
p49 . A-C, cytosolic extracts
prepared from unstimulated Cos7 cells, transfected with p85
(A), p55 (B), or p49 (C), were
subjected to size-exclusion chromatography. Aliquots of each fraction
were analyzed by Western blotting with an appropriate mouse monoclonal
antibody (lower strip of each panel A, B, and
C). Absorbance at 220 nm (---) and the corresponding elution
volumes of the molecular mass standards (kDa) are indicated, as is the
void volume (Vo). The Superose-12 column was
equilibrated and run in TBS at 0.3 ml/min, and 0.3-ml fractions were
collected and analyzed by SDS-PAGE followed by Western blotting with
the appropriate antibody. Unfractionated cytosolic extracts were
analyzed in parallel (Lys) with the relevant antibody. D and
E, Nonidet P-40 lysates were prepared from untreated Cos7
cells transfected with Myc epitope-tagged p85 and/or p85 BH
(D) or Myc epitope-tagged p85 and/or p49
(E). Myc epitope-tagged p85 was immunoprecipitated using
an anti-Myc tag monoclonal antibody (9E10) and immunoblotted for
co-precipitated p85 BH or p49 using U14, a mouse monoclonal
antibody directed against the SH2 domain of p85 which recognizes
p85 , p85 BH, or p49 . The relative migration positions of the
various isoforms on SDS-PAGE are indicated.
|
|
In contrast, p55
and p49
were detected in fractions that
corresponded to molecular mass ranges of 40-61 and 26-49 kDa,
respectively, in this system (Figs. 5, B and C,
and 2B). The highest molecular mass populations for both
p55
and p49
were not greater than their predicted monomeric
molecular masses; therefore they are apparently monomeric within a
cellular context. Interestingly, p55
and p49
seemed to bind to
other intracellular proteins from Cos7 cells and form high molecular
mass (>300 kDa) complexes. In addition, two variants of p55
were
observed, but the origin of these was not investigated further.
cDNAs encoding an amino-terminally Myc epitope-tagged version of
p85
, untagged p85
BH, or hexa-histidine-tagged p49
were transiently transfected into Cos7 cells, alone or in combination. Precipitation with appropriate antibodies immobilized on protein G-Sepharose or affinity matrices showed that each protein was expressed
in Cos7 cells transfected with a single cDNA (Fig. 5D, lanes 2 and 3 and Fig. 5E, lane 2).
When Myc-tagged p85
and p85
BH, a deletion mutant of p85 that
still had the ability to dimerize in vitro (Fig.
2B), were co-transfected, immunoprecipitation with a
monoclonal antibody directed against the Myc epitope was able to
co-immunoprecipitate untagged p85
BH (Fig. 5D, lane 1).
The difference in molecular mass between these two forms of p85
allowed them to be resolved by SDS-PAGE. In contrast, p49
, a
deletion mutant of p85 that did not have the ability to dimerize
in vitro (Fig. 2B), was unable to
co-immunoprecipitate with Myc-tagged p85
(Fig. 5E,
lane 1); therefore, forms of p85
that have the ability to
dimerize in vitro can form mixed heterodimers in
vivo, whereas monomeric forms cannot.
Binding of Phosphopeptides to the SH2 Domains Does Not Affect the
Oligomerization States of p85
Variants--
Binding of a
diphosphotyrosine-containing peptide that mimics an activated
platelet-derived growth factor
-R (Tyr-740/Tyr-751) to the SH2
domains of p85
affects the oligomerization state of the
p110
-p85
complex3;
therefore, we investigated the effect of Tyr-740/Tyr-751 binding on
dimerization of p85
isoforms. The IC50 for competition
of either Tyr-740/Tyr-751 or peptides with the same amino acid sequence but with only one phosphotyrosine residue (Tyr-740 or Tyr-751) with p85
variants for binding to immobilized Tyr-751 was compared. The
IC50 for the competition of Tyr-740, Tyr-751, or
Tyr-740/Tyr-751 with the isolated carboxyl-terminal SH2 domain of
p85
(p85
cSH2) for binding to immobilized Tyr-751 was similar,
although Tyr-740 had a slightly lower affinity for p85
cSH2 compared
with the other peptides (Fig.
6A). The binding
characteristics of p85
and p49
in this system were very similar
(Fig. 6, B and C). Both p85
and p49
had
lower IC50 values for the competition of Tyr-740/Tyr-751 for binding to Tyr-751 compared with either mono-phosphopeptide, suggesting that both SH2 domains engage both phosphotyrosine residues within this peptide (Fig. 6, B and C).

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Fig. 6.
The role tyrosine phosphopeptide-binding in
the dimerization of p85 . Inhibition of
p85 cSH2 (A), p49 (B), or p85
(C) binding to immobilized Tyr-751 by increasing
concentrations of Tyr-740 ( ), Tyr-751 ( ), and Tyr-740/Tyr-751
( ). D, apparent molecular mass of p85
(circles) and p49 (triangles) at a range of
concentrations in the absence (open symbols) or presence
(closed symbols) of a constant 10-fold molar excess of
Tyr-740/Tyr-751. Molecular masses were determined by HP-SEC and were
calculated from the retention time (min) of the maximum peak height of
p85 or p49 . The retention time was converted to apparent
molecular mass by comparison to a plot of log molecular mass (kDa)
versus retention time (min) of the maximum height of the
peak for a range of standard proteins (Bio-Rad) chromatographed using
the same buffer conditions as in Fig. 2A.
|
|
The apparent molecular masses of p85
and p49
were determined at a
range of concentrations by HP-SEC (Fig. 6D) and agreed with
those previously described (Fig. 2B). There appeared to be a
slight increase in apparent molecular mass of p85
with increasing protein concentration (Fig. 6D), supporting the idea that
p85
can undergo self-association, whereas p49
cannot. Although
Tyr-740/751 was shown to bind p85
or p49
with high affinity (Fig.
6, B and C), addition of Tyr-740/751 did not
affect the apparent molecular mass of either p85
or p49
,
regardless of protein concentration (Fig. 6D); therefore the
engagement of the SH2 domains of the regulatory subunits of PI3K
does not affect their oligomerization state.
 |
DISCUSSION |
The p85 subunit of PI3K may represent yet another intracellular
signal transduction protein which utilizes dimerization as a regulatory
mechanism, in a similar manner to cell-surface receptors, which are
often activated by ligand-induced dimerization or oligomerization (31).
Several other intracellular signaling proteins, such as the STAT
transcription factors (32) and c-Raf (33, 34), have also been shown to
be regulated by dimerization.
In this study, a number of techniques have been used to demonstrate
that the amino-terminal portion of the p85
subunit of PI3K binds to
itself in an intermolecular manner (Fig.
7), which leads to dimerization of p85
both in vitro and in vivo. Additionally, we
determined that the isolated SH3 domain of p85
binds only one of its
endogenous proline-rich motifs, PRM1, and the same interaction occurs
within the whole protein. In order to determine whether these two
interactions were related, we further investigated the binding
properties of each of the domains of p85
. However, it became clear
that dimerization was not mediated by a single domain, and the
involvement of the BH domain in dimerization made interpretation of
this data more complicated. However, the ability of the P1 peptide to
disrupt both p85
SH3-PRM1 and p85
dimers (Fig. 4) indicated that
the SH3 domain of one p85
molecule binds the PRM1 of a second p85
(Fig. 7).

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Fig. 7.
Proposed model of intermolecular interactions
of the p85 regulatory subunit of phosphatidylinositol 3-kinase.
As indicated with the arrows, the N-terminal domains of
p85 , the SH3, BH, and PRM1 domains, interact in an intermolecular
manner leading to the formation of a p85 dimer (as discussed in the
text).
|
|
Previous studies have also demonstrated that various fragments of
p85
are dimeric. A fragment of p85
encompassing residues 1-101
(similar to p85
SH3-PRM1 in this study) (35) and a fragment similar
to p85
SH3-BH (36) have been shown to be dimers. Crystals of the BH
domain of p85
have previously been shown to contain two monomers per
asymmetric unit (36); however, the hydrophobic dimerization interface
was very small, involving only four residues of each monomer. In
comparison, the three-dimensional structure of the p85
SH3 domain has
been determined by both x-ray crystallography and NMR, and no evidence
for self-association of the isolated SH3 domain of p85
has been
described (30, 37).
The suggestion from the crystal structure of the BH domain that the
4-residue interface represents a small portion of a larger interaction
(36) is confirmed by the contribution of the BH domain to the overall
dimeric interface of p85
(Fig. 2B). The size of the
interface shown in the crystal structure suggests that the affinity of
dimerization would be low. If monomeric and dimeric BH domain existed
in equilibrium, and the interconversion rate was fast, the low affinity
for self-association would result in only a small proportion of dimeric
BH domain and thus may explain the slightly higher average molecular
mass observed by both HP-SEC and SE-AUC (Figs. 2B and
3A).
Interactions between SH3 domains and proline-rich motifs have been
reported to regulate several intracellular signaling molecules. In
p85
, this interaction not only participates in the dimerization interface but may also block the binding of exogenous ligands for the
p85
SH3 domain and PRM1. Stimulation of the B cell receptor has been
shown to lead to the binding of the SH3 domains of the Src family
tyrosine kinases, Lyn and Fyn, to the p85
PRM1 and the up-regulation
PI3K activity (13). The endogenous, intermolecular interaction may
therefore also regulate the binding of exogenous ligands to the SH3
domain and PRM1. A similar mechanism has been demonstrated in Itk, a
member of the Tec family of cytoplasmic tyrosine kinases (38), although
the SH3 domain proline-rich motif interaction was intramolecular in
this protein. Interaction of the SH3 domain with an adjacent
proline-rich motif in Itk prevented the binding of the SH3 domain to
proline-rich motifs in Sam-68 and Grb2. Tyrosine phosphorylation within
the SH3 domain of another Tec family tyrosine kinase, Btk, was shown to
disrupt an intramolecular SH3 domain proline-rich motif interaction and
release the Btk SH3 domain to allow it to recruit substrates of the Btk
kinase domain (39). Intramolecular SH3 domain binding to proline-rich motifs may therefore be a general mechanism for the regulation of SH3
domain function, although there seems to be a number of ways in which
the SH3 domain can be released to bind its exogenous ligands. The
interaction of the p85
SH3 domain and p85
PRM1 seems to be a
special case of this general mechanism in which an intermolecular
SH3-PRM1 interaction occurs between the two units of a dimer.
Recent evidence has suggested that the p85 subunit may have functions
that are independent of the p110 catalytic subunit. A truncated form of
p85
, lacking the carboxyl-terminal SH2 domain and 52 residues of the
inter-SH2 region, was identified as a potential oncogene in a number of
murine lymphomas (40). Overexpression of this mutant protein (p65)
caused transformation of NIH-3T3 fibroblasts, and p65 and v-Raf had
synergistic transforming activities. Murine embryonic fibroblasts in
which the gene for p85
was deleted by homologous recombination had a
defect in a p53-mediated apoptotic pathway (41). Apoptosis caused
by oxidative stress was reduced in cells lacking p85
. Apoptosis was
not inhibited by the PI3K inhibitor, wortmannin, suggesting that the
lipid kinase activity of the p110 subunit is not a requisite component
of the p53- and p85
-mediated pathways.
One of the best defined functions of the regulatory subunit of PI3K is
the down-regulation of p110-mediated lipid kinase activity (42, 43),
suggesting that populations of PI3K comprising a complex of the p110
and p85 subunits or the p110 subunit alone would have different
activities. A 100-kDa PI3K activity has been detected in lysates of
bovine thymus by size-exclusion chromatography (SEC) (44) suggesting
that the p110 subunit may exist on its own and implying that the p85
regulatory subunit may also exist as a separate entity with a
biological function distinct from its role in the regulation of p110
activity. Another potential role of a pool of dimeric p85 in
vivo could be to bind and stabilize the p110 catalytic subunit as
soon as it is translated, as p110
has been shown to be prone to
inactivation and degradation at 37 °C without the bound regulatory
subunit (42).
The existence of forms of the regulatory subunit of PI3K, such as
p49
or p55
, that do not have the potential for self-association may represent a mechanism for divergent signaling. PI3K-mediated signaling through the insulin receptor and insulin receptor substrate 1 has been shown to preferentially utilize forms of the PI3K regulatory subunit that do not have the potential for self-association (8, 20). As
yet it is unclear what implication a lack of dimerization of some
regulatory subunit isoforms has for PI3K signaling. The use of
protein-protein interactions of SH3 and BH domains and proline-rich
motifs to regulate the nature and localization of PI3K activity would
not be an option for the lower molecular mass isoforms. The regulatory
subunit of PI3K may therefore represent both an adaptor for the
catalytic subunit and a mediator of other protein-protein interactions
that utilize these domains. This study has highlighted that these same
domains are also implicated in self-association of the p85 protein,
which adds a further layer to the potential complexity of the
regulation of PI3K activity in cell signaling events.
 |
ACKNOWLEDGEMENTS |
We thank Akunna Akpan and Krishna Pitrola for
assistance with Sf9 cell culture, Professor I. D. Campbell
and Dr. K. Drickamer (University of Oxford) for access to the
analytical ultracentrifuge, and Dr. R. Wallis for kind assistance in
the implementation and interpretation of the SE-AUC experiments.
 |
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.
§
Current address and to whom correspondence should be addressed:
Cell Biophysics Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Tel.: 44-171-269-3054; Fax: 44-171-269-3094; E-mail:
A.Harpur{at}icrf.icnet.uk.
¶
Supported by C. J. Martin Fellowships from the National
Health and Medical Research Council, Australia. Both authors
contributed equally to this work.
Current address: The Ludwig Institute for Cancer Research, PO
Royal Melbourne Hospital, Parkville, 3050, Australia.

Supported by a Wellcome Trust Prize Studentship. Current
address: Structural Biology Programme, EMBL, Meyerhofstrasse, 1, Postfach 10.2209, Heidelberg D-69126, Germany.
2
F. Pagès and M. D. Waterfield,
unpublished results.
3
Layton, M. J., Harpur, A. G., Panayotou, G.,
Bastiaens, P. I. H., and Waterfield, M. D. (1998) J. Biol. Chem.
273, 33379-33385.
 |
ABBREVIATIONS |
The abbreviations used are:
PI3K, phosphatidylinositol 3-kinases;
BH, BCR homology;
PRM, proline-rich
motif;
pY, phosphotyrosine;
HP-SEC, high performance-size exclusion
chromatography;
DTT, dithiothreitol;
GST, glutathione
S-transferase;
PAGE, polyacrylamide gel electrophoresis;
SE-AUC, sedimentation equilibrium-analytical ultracentrifugation.
 |
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