The p110? isoform of phosphoinositide 3-kinase
signals downstream of G protein-coupled receptors
and is functionally redundant with p110?
Julie Guillermet-Guibert*, Katja Bjorklof*, Ashreena Salpekar*, Cristiano Gonella*, Faruk Ramadani†, Antonio Bilancio*,
Stephen Meek‡, Andrew J. H. Smith‡, Klaus Okkenhaug†, and Bart Vanhaesebroeck*§
*Center for Cell Signaling, Institute of Cancer, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom;†Laboratory
of Lymphocyte Signaling and Development, Babraham Institute, Cambridge CB2 3AT, United Kingdom; and‡Gene Targeting Laboratory, The Institute
for Stem Cell Research, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, United Kingdom
Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved April 7, 2008 (received for review August 16, 2007)
The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely
regulated by extracellular stimuli. The class IA PI3K catalytic sub-
units (p110?, p110?, and p110?) occur in complex with a Src
homology 2 (SH2) domain-containing p85 regulatory subunit,
which has been shown to link p110? and p110? to Tyr kinase
signaling pathways. The p84/p101 regulatory subunits of the
p110? class IB PI3K lack SH2 domains and instead couple p110? to
G protein-coupled receptors (GPCRs). Here, we show, using small-
molecule inhibitors with selectivity for p110? and cells derived
from a p110?-deficient mouse line, that p110? is not a major
effector of Tyr kinase signaling but couples to GPCRs. In macro-
phages, both p110? and p110? contributed to Akt activation
induced by the GPCR agonist complement 5a, but not by the Tyr
kinase ligand colony-stimulating factor-1. In fibroblasts, which
express p110? but not p110?, p110? mediated Akt activation by
the GPCR ligands stromal cell-derived factor, sphingosine-1-
phosphate, and lysophosphatidic acid but not by the Tyr kinase
of p110? in these cells reduced the contribution of p110? to GPCR
signaling. Taken together, these data show that p110? and p110?
can couple redundantly to the same GPCR agonists. p110?, which
shows a much broader tissue distribution than the leukocyte-
restricted p110?, could thus provide a conduit for GPCR-linked PI3K
signaling in the many cell types where p110? expression is low or
gene targeting ? signaling ? tyrosine kinase ? Akt ? insulin
tions such as cell growth, proliferation, differentiation, and
survival and have been implicated in cancer, inflammation, and
diabetes. Mammals have eight isoforms of PI3K, which have
been divided in three classes (1). Thus far, attention has focused
mainly on the class I PI3Ks that are acutely activated by
extracellular ligands. These heterodimers consist of a p110
catalytic subunit in complex with a regulatory subunit and have
further been subdivided into class IA and IB PI3Ks. The class IA
an Src homology 2 (SH2) domain-containing regulatory subunit
(of which there are five species, often referred to as p85s) that
binds phosphoTyr in intracellular proteins, allowing recruitment
of p85/p110 complexes to the membrane. The class IB regulatory
class IB PI3K catalytic subunit (p110?) to G protein-coupled
Over the last few years, the cellular signaling contexts and
physiological roles of p110?, p110?, and p110? have become
clearer, because of the generation of gene-targeted mice and
small-molecule inhibitors for these PI3K isoforms. In contrast,
very little is known about p110?, both at the cellular and
he lipid second messengers generated by phosphoinositide
3-kinases (PI3Ks) regulate a wide variety of cellular func-
embryonic lethality of the p110? KO mice and the fact that
proliferating cells could not be derived from these embryos (2).
Recent progress has been made by the generation of small-
molecule inhibitors with selectivity for p110?, allowing scientists
to define a role for p110? in platelet function and thrombus
formation (3). Some evidence has also been presented for the
coupling of p110? to GPCRs, either by in vitro studies that
documented activation of p110? by G?? subunits (4, 5) or in
cellular experiments where p110? function was probed by mi-
croinjection of neutralizing antibodies to p110? (6, 7), RNAi
against p110? (8), or overexpression of p110? (8, 9). Here, we
have used pharmacological tools with selectivity for p110? (3),
in conjunction with cells derived from a mouse line in which
p110? has been inactivated by gene targeting, to investigate the
role of p110? in PI3K signaling downstream of Tyr kinase and
line in which exons 21 and 22 of the kinase domain of p110? are
flanked by loxP sites [supporting information (SI) Fig. S1]. This
floxed p110? allele, which is further referred to as p110?flox, is
schematically shown in Fig. 1A. Exon 22 contains the DFG motif
and the activation loop, which are critical for the activity of
kinases. The loxP sites were positioned in such a way that, after
treatment with Cre, the locus is expected to give rise to an
mRNA in which exon 20 is spliced in-frame onto exon 23,
encoding an internally truncated p110? protein with a predicted
Mrof ?112 kDa (further referred to as p110??21,22) instead of
the ?122-kDa WT protein.
From crosses with heterozygous parents, we obtained ?30% of
expected numbers of viable mice, homozygous for the p110??21,22
allele (p110??21,22/?21,22mice), indicating lethality with incomplete
embryonic fibroblasts (MEFs) and bone marrow-derived macro-
phages (BMMs) from viable p110??21,22/?21,22mice were used for
further study. RT-PCR and DNA sequencing on mRNA prepared
from p110??21,22/?21,22MEFs showed that the mutant p110? locus
gave rise to the expected truncated mRNA (Fig. S1I and data not
In control p110?flox/floxMEFs, lipid kinase activity in p110?
Author contributions: J.G.-G., K.B., A.S., C.G., F.R., A.B., S.M., A.J.H.S., K.O., and B.V.
designed research; J.G.-G., K.B., A.S., C.G., F.R., A.B., and S.M. performed research; J.G.-G.,
K.B., A.S., C.G., F.R., A.B., S.M., A.J.H.S., K.O., and B.V. analyzed data; and J.G.-G. and B.V.
wrote the paper.
Conflict of interest: B.V. is a consultant for PIramed (Slough, UK) and AstraZeneca.
This article is a PNAS Direct Submission.
§To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
June 17, 2008 ?
vol. 105 ?
immunoprecipitates, made using Abs against the C terminus of
p110?, was sensitive to the p110? inhibitor TGX-155 (Fig. 1B).
in p110? immunoprecipitates (IPs) from p110??21,22/?21,22MEFs
(Fig. 1B). PI3K activity associated with a phosphoTyr peptide
matrix (which binds all p85 species; referred to as pY peptide)
was unaffected in p110??21,22/?21,22MEFs (Fig. 1B), indicating
that p110? contributes minimally to p85-associated PI3K activity
in unstimulated fibroblasts.
Transient overexpression in HEK293 cells of the p110??21,22
cDNA cloned from p110??21,22MEFs revealed that the
p110??21,22protein could be translated from this mRNA (Fig.
S1K). As expected, the ?122-kDa WT p110? protein could no
longer be detected in p110??21,22/?21,22MEFs and BMMs upon
immunoblotting with C-terminal (Fig. 1C Right) or N-terminal
antibodies to p110? (Fig. S1J). However, no evidence for the
?112-kDa p110??21,22protein could be found (Fig. 1C and Fig.
S1J), indicating that this truncated protein is either not pro-
duced, is further truncated, or is unstable [note that the lower Mr
signal observed with the p110? Cter Abs in MEFs is a nonspe-
cific signal that is not altered in p110??21,22/?21,22cells (Fig. 1C);
this lower MW signal is not observed in BMMs]. The levels (Fig.
1C) and activity (Fig. S1L) of p110? were unaffected. The
observation that expression of the p85 regulatory subunit and
1C) argues against the degradation of the entire p110? protein,
which would most likely lead to a reduction in p85 levels (10).
Thus, in p110??21,22/?21,22cells, p110? activity is absent, yet the
p85/p110 stoichiometry is unaltered.
p110? and p110? Signal Downstream of the Same GPCR Ligands in
Cells Expressing All Class I PI3Ks.Wefirstassessedtheroleofp110?
in early PI3K signaling in BMMs that express all class I p110
isoforms, including p110? (Fig. 1C).
Phosphorylation of Akt and Erk induced by the Tyr kinase
ligand colony-stimulating factor-1 (CSF-1) was not affected by
pharmacological or genetic inactivation of p110? or p110? (Fig.
S2 A-C; p110? was inhibited by using AS604850, a small-
molecule inhibitor with selectivity for p110? (11)). These data
indicate that p110? and p110? do not play a major role in Tyr
kinase-induced PI3K activation in BMMs. This was previously
reported for p110? (11–13), but is somewhat unexpected for
p110?, given that injection of neutralizing Abs to p110? has been
shown to block CSF-1-induced chemotaxis in a macrophage cell
In contrast to Tyr kinase ligands, Akt phosphorylation in-
duced by the GPCR agonist complement 5a (C5a) was substan-
tially, but not completely, inhibited by inactivation of p110?,
either genetically (Fig. 2 A and C) or pharmacologically (Fig. 2
D and F). The residual C5a-induced Akt phosphorylation in the
absence of p110? activity could be blocked by the p110?
inhibitor AS604850 (Fig. 2 G and I), indicating that p110?
controlled this component of the signal.
p110β β∆ ∆21,22
relative to flox/flox
lower band (nonspecific)
upper band (= p110β)
16 17 18 19 2021 22 23
16 17 18 19 20 23
16 17 18 19 20 21 22 23
16 17 18 19 20 2324
PI3K activity (%)
Exon 22 encodes the DFG domain and activation loop that are critical for p110? kinase activity. Constitutive deletion of exons 21 and 22 in mice was achieved
by intercrossing p110?flox/floxmice with Cre deleter mice, creating the p110??21,22allele. The loxP sites were positioned in such a way that, after treatment with
Cre, the locus is expected to give rise to mRNA in which exon 20 is spliced in-frame onto exon 23, now encoding an internally truncated p110? protein lacking
protein and to retain reactivity with antisera raised against the extreme C terminus of p110?. (Left) Exon sequences are represented by filled black rectangles,
Exon boundaries are represented from exons 16 to 24. The positions of the primers used for PCR screening are designated by arrows together with the expected
exons 16 to 24. (B) Effect of p110? deletion on in vitro lipid kinase activity. Homogenates of the indicated MEFs were immunoprecipitated by using p110? Abs
or absorbed onto PDGF receptor phosphoTyr peptide (pY peptide) immobilized to Sepharose (which binds all class IA PI3K regulatory subunits), followed by in
vitro lipid kinase assay with or without 100 nM TGX-155. The level of p110? and p85 in the indicated cell fractions was verified by immunoblotting (data not
shown). (C) Effect of p110? deletion on PI3K isoform expression. (Left) Analysis of p110? protein expression in MEFs from p110?flox/flox, p110?flox/?21,22, and
p110? expression was not affected in each genotype. p110? and p110? were hardly detectable in fibroblasts and could not be reliably quantified. (Right) Total
cell lysates or pY-peptide pull-downs were immunoblotted by using the indicated Abs. In NIH 3T3 cells and MEFs (but not in BMMs), the anti-p110? Abs used
for Western blotting (sc-602) recognize a nonspecific protein just below the specific p110? signal, indicated as lower band (aspecific) and upper band (p110?),
respectively. It is only the upper band that disappears upon p110? deletion.
Genetic inactivation of p110? in mice. (A) Schematic representation of genomic DNA, mRNA, and protein primary structure of the p110?floxand
Guillermet-Guibert et al.
June 17, 2008 ?
vol. 105 ?
no. 24 ?
In line with previous reports (12), C5a-induced Akt phosphor-
ylation was decreased in p110? KO BMMs (Fig. 2 B and C) and
in WT BMMs upon treatment with AS604850 (Fig. 2 E and F).
However, residual C5a-stimulated Akt phosphorylation was also
observed under those conditions (see quantification of Western
by p110?, because TGX-155 treatment inhibited the residual p-Akt
signal in C5a-stimulated p110? KO BMMs (Fig. 2 H and I).
Pertussis toxin (PTx) treatment blocked the C5a-induced
pAkt signal in p110? KO and p110??21,22/?21,22cells (Fig. S2 D
and E), indicating that C5a-stimulated Akt phosphorylation
mediated by p110? and p110?, respectively, is G protein-
Taken together, these data show that p110? signals effectively
downstream of a GPCR in cells and that p110? and p110? are
activated downstream of the same GPCR ligand to mediate Akt
phosphorylation. Genetic or pharmacological inactivation
of p110? or p110? did not affect CSF-1 or C5a-induced Erk
phosphorylation (Fig. 2 A, B, D, and E).
p110? Controls GPCR-Induced Early PI3K Signaling in Fibroblasts. We
next tested the role of p110? in Tyr kinase and GPCR signaling
in NIH 3T3 and MEFs, which mainly express p110? and p110?
and have undetectable or low levels of p110? and p110?,
respectively (Fig. 1C and Fig. S3A). Absolute quantification of
class IA p110 isoforms in NIH 3T3 cells showed that p110? is
expressed at much higher levels than p110? and p110? [12,000
versus 2,000 and 1,000 molecules per cell, respectively (15)].
However, given that barely detectable levels of p110? can have
a major biological impact [such as in cardiomyocytes (16)], we
first tested the potential role of p110? in these cells. The p110?
inhibitor AS604850 did not impair lysophosphatidic acid (LPA)-
induced phosphorylation of Akt in NIH 3T3 cells (Fig. S3B).
Moreover, in p110? KO MEFs, GPCR stimuli such as sphin-
gosine-1-phosphate (S1P) and LPA could still induce Akt phos-
phorylation (Fig. S3C). This Akt phosphorylation was sensitive
to TGX-155 but not to AS604850 (Fig. S3C), suggesting that
p110? is the main GPCR-coupled PI3K isoform in fibroblasts.
To further substantiate these findings, a broader range of
agonists was tested in NIH 3T3 cells. Akt phosphorylation
induced by PDGF, insulin, or insulin-like growth factor 1 (IGF1)
was sensitive to LY294002 but was not blocked by TGX-155 (Fig.
3A Top). This finding is in stark contrast to Akt phosphorylation
induced by each of the GPCR ligands tested [S1P, stromal
cell-derived factor (SDF-1?), LPA], which were equally sensitive
to TGX-155 and LY294002 in each case (Fig. 3A Middle; for
quantification of the results of three independent experiments
with all six stimuli, see Fig. 3A Bottom). Similar results were
obtained in WT MEFs upon stimulation with PDGF or GPCR
ligands such as S1P and LPA (Fig. S3D) and upon using
TGX-115 and TGX-221, two alternative inhibitors from the
same chemical series as TGX-155 (3) (data not shown).
was also inhibited by TGX-155 when tested at different time
points (Fig. S3E). TGX-155 inhibited LPA-induced T308 and
S473 phosphorylation of Akt in a dose-dependent manner, with
an IC50 of 0.1 ?M (Fig. S3F), in line with the PI3K isoform
specificity of this compound (see Materials and Methods). Oc-
casionally, an increase in LPA-induced S473 phosphorylation of
Akt was observed at very low doses of TGX-155 (0.01 ?M; data
not shown) for reasons that are unclear. PTx pretreatment de-
creased LPA-induced Akt phosphorylation in NIH 3T3 cells, but
had no effect on PDGF-induced Akt phosphorylation (Fig. S3G).
In p110??21,22/?21,22MEFs, Tyr kinase (PDGF, insulin, IGF1,
EGF)-induced Akt phosphorylation was largely unaffected, in
contrast to Akt phosphorylation induced by the GPCR ligands
LPA, SDF-1, and IL-8, which was almost completely blocked
(Fig. 3B). Similar results were obtained with MEFs derived from
mice that are homozygous for a kinase-dead allele of p110?
(p110?D931A/D931A; data not shown). Also acute genetic deletion
of p110? [by 4-hydroxytamoxifen treatment of MEFs from
p110?flox/floxmice crossed onto the ROSA-CreERT2 mice (17),
resulting in activation of Cre by its translocation from the cytosol
to the nucleus], reduced LPA-induced Akt phosphorylation,
with the residual pAkt signal being no longer sensitive to
TGX-155 (Fig. S3H).
Taken together, these data show that p110? is critical for early
PI3K signaling induced by GPCRs but is not a major transducer
downstream of receptor Tyr kinases.
p110? Can Complement p110? Function in GPCR-Induced PI3K Signal-
ing in Fibroblasts. To test whether p110? could in principle signal
downstream of GPCRs in fibroblasts, we stably transfected
p110?, together with its p101 regulatory subunit, into NIH 3T3
cells (Fig. 3C Left). In control transfectants, the p110? inhibitor
AS604850 did not decrease LPA-induced Akt phosphorylation
(Fig. 3C). In contrast, NIH 3T3 cells expressing p110? became
sensitive to AS604850 and also showed reduced sensitivity to the
p110? inhibitor TGX-155 (Fig. 3C). These data further substan-
0 1 5 15 0 1 5
+ + +
p110γ γ KO
- + + +
151 5 15 0 1 5
+ + +
p110β βΔ Δ21,22/Δ Δ21,22
- + ++
15015 15 0 15
- +- + ++ +
Hp110γ γ KO
p110β βΔ Δ21,22/Δ Δ21,22
fold over basal
- + + -
Time (min)Time (min)
0 15 15 0 1 5
-+ + ++ +
Starved BMMs of the indicated genotype were stimulated with C5a, followed
by C5a stimulation and immunoblotting using the indicated Abs. (C and F)
Quantification of at least two independent experiments was performed, and
data are shown as fold over P-Akt under unstimulated conditions. (G–I)
Starved BMMs of the indicated genotype were treated for 30 min with
AS604850 (1 ?M) or TGX-155 (0.5 ?M) and stimulated for 5 min with C5a,
followed by immunoblotting using the indicated Abs. I shows the quantifica-
Role of p110? and p110? in cell signaling in macrophages. (A and B)
www.pnas.org?cgi?doi?10.1073?pnas.0707761105Guillermet-Guibert et al.
tiate the notion that p110? and p110? can signal in the same
GPCR signaling pathways.
Here, we define a key role for p110? in early PI3K signaling
downstream of GPCRs. This is not only the case in cells that do
not express p110?, the other well established GPCR-linked
PI3K, but also in leukocytes that coexpress p110? and p110?.
Our data show that in the latter cells p110? acts in concert with
p110? to provide full PI3K activity downstream of GPCRs,
contradicting previous reports that indicated that p110? inacti-
vation leads to full blockade of Akt phosphorylation by GPCR
ligands in macrophages (11–13) but also in other leukocytes such
as neutrophils (18) or mast cells (19). Further studies using a
broader range of ligands and cell types will be essential to
determine the relative importance of p110? and p110? in
signaling and biological outputs.
Somewhat unexpectedly, our data show that p110? does not
contribute substantially to Akt activation by Tyr kinase ligands.
This finding is surprising, given that the absolute amount of
p110? in the cell types studied here is three to six times higher
than that of p110? (15). However, our observations are in line
with recent reports that p110? is the major PI3K isoform in Tyr
kinase signaling by insulin and IGF1, with a minimal contribu-
tion of p110? (20, 21). Also, p110? has been found to be
recruited to the PDGF receptor, without contributing to Akt
activation and the biological functions induced by PDGF (7, 22,
23). Key differences between p110? and p110? in insulin sig-
naling include selective recruitment of p110? over p110? to
IRS-1/2 complexes (20) and the lack of p110? lipid kinase
activity in these complexes (20, 21). It is possible that p110? is
precluded from effective recruitment to membrane-bound re-
ceptors because of its association with the Rab5 small GTPase
influence the low activity of p110? in Tyr kinase complexes
include the low specific activity of p110? compared with p110?
(25) and the notion that full activation of p110? upon Tyr-
mediated recruitment may require the presence of G?? subunits
(4, 5, 8, 26). p110? could thus become activated by Tyr kinase
receptors under conditions whereby cells receive parallel stim-
ulation through GPCRs, a scenario that may very well be
operational in vivo when cells are confronted with a multitude of
stimuli. For example, this could occur upon direct activation of
Src-family Tyr kinases by GPCRs (27).
Our studies do not exclude a role for p85-mediated recruit-
ment of p110?, given that our screen of Tyr kinase ligands and
cell types, as well as kinetics and dose of stimulation, has not
been exhaustive. Indeed, other stimuli and biological responses
that activate Tyr kinases may engage p110?, including apoptotic
cell and Fc?R-mediated phagocytosis and CSF-1-stimulated
chemotaxis in macrophages (28), EGF-induced DNA synthesis
in breast cancer cells (29), Fc?RI-activated calcium influx in
mast cells (30), and insulin signaling in endothelial and hepatic
cell lines (22, 31). It is possible that p110?, while not being a
major effector in early PI3K signaling, could contribute to Tyr
kinase-driven PI3K signaling at later time points and in different
signaling contexts and/or modulate signaling through other PI3K
isoforms. Evidence for the latter has been documented in insulin
signaling, whereby ‘‘basal’’ p110? activity seems to set the
threshold for activation of p110? (21, 31). It is tempting to
speculate that p110?-mediated GPCR-PI3K signaling
through serum components (such as LPA) in the cell models
used in these studies contributes to this basal PI3K activity.
Taken together, our data suggest an analogy between p110?/
p110? and p110?/p110? in the coverage of Tyr kinase- and
GPCR-mediated PI3K signaling in distinct cell types (schemat-
ically shown in Fig. 4). Indeed, all evidence suggests that the
ubiquitously expressed p110? plays an important role in Tyr
kinase-driven PI3K signaling in all cell types (including leuko-
cytes), with p110? providing additional Tyr kinase-driven PI3K
signaling in cell types in which it is expressed at high level, such
as leukocytes. Under these conditions, the contribution of p110?
can also exceed that of p110?, for example in lymphocytes (ref.
32 and unpublished results). A similar scenario could be envis-
TG LYDD D D
p110γ + p101
p110γ + p101
/ total Akt
DASD TG AS
/ total Akt
P-Akt (T308)/total AktP-Akt (S473)/total Akt
P-Akt/ total Akt
(% of stimulated)
+ - + - + -
+ - + - + - +
Middle) Starved NIH 3T3 cells were treated for 1 h with TGX-155 (TG; 1 ?M),
min with the indicated ligands. Total cell lysates were immunoblotted with
the indicated Abs. A representative immunoblot is shown. (Bottom) Quanti-
fication of three independent experiments was performed, and data are
presented as percentage of ligand-induced P-Akt/total Akt. (B) Starved MEFs
of indicated genotype were stimulated with indicated stimuli for 10 min and
immunoblotted with the indicated Abs. A representative experiment per-
formed with MEFs isolated from different embryos is shown. (C) p110 isoform
expression in NIH 3T3 cells transfected with empty vector (NIH 3T3 control) or
p110? and its regulatory subunit HA-p101 (p110? ? p101). Starved cells were
treated for 1 h with TGX-155 (1 ?M) or AS604850 (1 ?M), followed by
stimulation for 10 min with LPA and immunoblotting with the indicated Abs.
Role of p110? and p110? in cell signaling in fibroblasts. (A) (Top and
Guillermet-Guibert et al.
June 17, 2008 ?
vol. 105 ?
no. 24 ?
aged for the ubiquitously expressed p110? that could control
GPCR-driven PI3K in all cell types, with p110? providing
additional GPCR-PI3K signaling capacity in white blood cells.
Materials and Methods
Reagents. Small-molecule inhibitors were dissolved in DMSO, with final con-
centration of DMSO in the assays maximally 0.2%. LY294002 was from Cal-
0.03 ?M; p110?: ?20 ?M; p110?: 0.34 ?M (3, 33)] and p110? inhibitor
AS604850 [in vitro IC50for p110?: 3.4 ?M; p110?: ?20 ?M; p110?: 0.19 ?M;
p110?: ?20 ?M (11)] were a gift from Merck/Serono (Geneva). Abs to class IA
PI3Ks were generated in-house. p110? Ab used for IP was generated by using
a C-terminal peptide of p110? (KVNWMAHTVRKDYRS) as immunogen, affin-
ity-purified over this peptide, and further purified over GST-p110? and GST-
p110? to deplete cross-reactivity with p110? and p110?. p110? Abs used for
immunoblot were from Santa Cruz Biotechnology (sc-602). These recognize
the last 19 aa of p110? (SWTTKVNWMAHTVRKDYRS) and are referred to as
p110? C-ter Abs. Anti-N-terminal p110? Abs used for Western blotting were
generated in-house with p110? peptides present in exon 3, 5, 9, and 12
VSSRGGK, respectively) as immunogen and used as a mix at 1 ?g/ml each; this
mix is referred as p110? Nter Abs. Additional PI3K Abs were from Upstate
(p85pan; catalogue no. 06-195) or Alexis (p110?; clone H1). Abs to Akt,
?-tubulin, and GAPDH were from Cell Signaling, Sigma, and Abcam, respec-
S1P, LPA, CSF-1, and C5a were from Preprotech. PTx (Bordetella pertussis) and
4-hydroxytamoxifen were from Sigma.
Mice. Mice were kept in individually ventilated cages and cared for according
were intercrossed to generate p110??21,22/?21,22and p110?flox/floxmice. Em-
bryos from timed pregnant mice were used to prepare MEFs.
Fibroblast Culture and Stimulation. Cells were cultured at 37°C in a humidified
that were minced and dissociated with trypsin, and cells were allowed to
adhere on dishes. NIH 3T3 cells and MEFs were cultured in DMEM supple-
stimulation, NIH 3T3 cells and early-passage (P2–P5) MEFs were seeded in
10-cm dishes (106cells per dish), starved in DMEM without FBS and antibiotics
for 24 h, treated for 1 h with the indicated doses of TGX-155 (TG), AS604850
ng/ml), insulin (1 ?M), EGF (20 ng/ml), IGF1 (30 ng/ml), S1P (200 nM), SDF (30
ng/ml), or LPA (1 ?M) for 10 min, unless stated otherwise. PTx (100 ng/ml) was
added for 15 h in DMEM without FBS before cell stimulation.
Macrophage Culture and Stimulation. Cells were cultured at 37°C in a humid-
ified 5% CO2atmosphere. BMMs were derived from bone marrow precursor
for 5 min, unless stated otherwise.
Cell Transfection. NIH 3T3 cells were transfected with pcDNA3 without or with
the coding sequences for human p110? (untagged) and porcine p101 (HA-
tagged) by using Superfect (Qiagen), followed by selection in 800 ?g/ml
geneticin (Invitrogen) for at least 15 days. Geneticin-resistant clones from a
single well were pooled and maintained under continuous selection in 200
Immunoblotting. Cell lysis and immunoblotting of NIH 3T3 cells and MEFs was
performed as described (34). Total cell lysates of macrophages were prepared
of DNA by passing five times through a 0.45-mm needle. Samples were
resolved by 10% SDS/PAGE.
Lipid Kinase Assay. PI3K activity assays were performed as described with PIP2
as a substrate.
ACKNOWLEDGMENTS. We thank A. Candi, P. Cutillas, P. Gonzalez-Gomez, A.
of Torino, Torino, Italy), M. Wymann (University of Basel, Basel, Switzerland),
R. Wetzker (University of Jena, Jena, Germany), and C. Rommel (Merck-
U.K.) for small-molecule inhibitors; and members of the Center for Cell Sig-
naling, especially M. Graupera, for feedback and support. This work was
funded by Diabetes U.K. Grant BDA:RD 01/0002179, Biotechnology and Bio-
logical Science Research Council Grants BB/C505659/1 and BB/C50989/1, Euro-
pean Union Grant FP6-502935, and the Ludwig Institute for Cancer Research.
J.G.-G. was supported by Fondation pour la Recherche Me ´dicale Grant FRM-
SPE20051105175, European Molecular Biology Organization Grant ALTF676–
2005, and European Union Grant MEIF-CT-2006-039676). K.B. was supported
by PIramed. F.R. was supported by a Medical Research Council capacity build-
ing award. K.O. was supported by Biotechnology and Biological Science
Research Council David Phillips Fellowship JF1928.
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no. 24 ?