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J Biol Chem, Vol. 274, Issue 46, 32835-32841, November 12, 1999
,From INSERM U326, IFR 30, Hopital Purpan, 31059 Toulouse, France, § Max Planck Research Unit, Molecular Cell Biology, University of Jena, 07747 Jena, Germany, and ¶ CNRS UPR 1086, CRBM, 34293 Montpellier, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Phosphoinositide 3-kinase (PI3K) has been shown
to play an essential role in G protein-induced signaling even in
non-myeloid cells where few agonists of G protein-coupled receptors are
known to activate PI3K. We have identified adherent cell lines where lysophosphatidic acid (LPA) strongly and rapidly activates the accumulation of PI3K lipid products. The process is not modified by
expression of a kinase-dead mutant of the G One of the major discovery of the 1990s in proliferative signaling
has been the emergence of the Ras/mitogen-activated protein kinase
(MAPK)1 cascade
as the main pathway used by growth factors for non-hematopoietic cells.
However, the early mechanisms of activation of this pathway by agonists
of G protein-coupled receptors (GPCR) remain elusive (1). Intensive
researches have recently focused on the identification of proteins that
could make the link between G proteins and the Ras/MAPK cascade,
leading to the identification of various protein tyrosine kinases
involved in this process, such as Pyk2 (2), Src (3), receptor tyrosine
kinases (RTKs) (4, 5), and even Syk in myeloid-derived cells (6).
However, the mechanisms of activation of these kinases by GPCR have not
been elucidated, except for Pyk2 that is recruited by a
Gq-Ca2+-dependent pathway (2). On
the other hand, phosphoinositide 3-kinase (PI3K) has been recently
shown to play a major role in the early mechanisms of GPCR-mediated
activation of the Ras/MAPK pathway (7-11). However, the fact that PI3K
inhibitors interfere with GPCR-induced signaling even in
non-hematopoietic cell lines is difficult to interpret since GPCRs
agonists are not known to induce the synthesis of PI3K lipid products
in these cells. In contrast, neutrophils for example produce large
amounts of phosphatidylinositol 3,4-bisphosphate (PI3,4P2)
and phosphatidylinositol 3,4,5-trisphosphate (PIP3) upon
stimulation with GPCR agonists (12). Similarly, stimulation of cells by
RTK agonists such as platelet-derived growth factor (PDGF) generate
PI3K lipid products through well known pathways, and PI3K inhibitors
interfere with RTK-mediated activation of the Ras/MAPK pathway,
although the mechanisms have to be clarified (13). Thus, one important
question still remains concerning the regulation of PI3K in
GPCR-induced signaling in non-myeloid cells.
Recently, two groups have identified a novel isoform of PI3K, p110 Therefore, this led us to study the activation of PI3K by GPCR in
adherent cell lines. We have measured PI3K lipid products upon
stimulation with lysophosphatidic acid (LPA), and we have identified
cell lines producing large amounts of PI3,4P2 and
PIP3 upon treatment with a GPCR agonist. The mechanism does
not seem to involve the p110 Materials--
Rabbit anti-active Erk1/2 antibody was from
Promega (catalog number V6671). Rabbit anti-phospho-specific Akt
(Ser-473) antibody was from New England Biolabs (catalog number 9271S).
Sheep anti-human EGFR (catalog number 06-129), rabbit anti-rat p85
(catalog number 06-195), rabbit anti-human Gab1 (catalog number
06-579), and monoclonal 4G10 anti-phosphotyrosine (catalog number
05-321) antibodies were purchased from Upstate Biotechnology, Inc.
Rabbit anti-human Cell Culture and Transfection--
Cos, Vero, Rat1, and IMR90
cells were maintained in DMEM supplemented with 10% fetal bovine serum
and antibiotics. For B82 L cells, 10% dialyzed newborn calf serum was
used. For transfection, cells were incubated 4 h with 10 µl of
LipofectAMINE (Life Technologies, Inc.) and 2 µg of plasmid DNA per
ml of Opti-MEM (Life Technologies, Inc.). The transfection mixture was
then replaced by DMEM supplemented with 10% serum for 24 h.
Before stimulation, cells were serum-starved for 24 h.
Plasmid Constructs--
Full-length wt-p110 Analysis of PI Polyphosphate--
Cells grown in 10-cm plates
were serum-starved for 24 h upon reaching 80-90% confluence and
then labeled for 5 h with 0.2 mCi of
[32P]H3PO4 (Amersham Pharmacia
Biotech) per ml in phosphate-free DMEM. Cells were then stimulated for
the indicated time and washed once with ice-cold phosphate-buffered
saline before addition of 3.75 ml of 2.4 M HCl solution.
Then lipid extraction was performed as described previously (27).
Briefly, lipids were solubilized by addition of 3 ml of chloroform and
4.5 ml of methanol followed by vortexing. After centrifugation, the
lower phase containing the lipids was collected, and the upper phase
was washed with 4.5 ml of chloroform. The lower phases were then
combined and evaporated under nitrogen, and lipid extracts were
solubilized in 250 µl of chloroform/methanol (1/1, v/v) and first
resolved by thin layer chromatography (TLC) using
chloroform/acetone/methanol/acetic acid/water (80/30/26/24/14,v/v). The
spots corresponding to PI4,5P2/PI3,4P2, and
PIP3 were then scraped off, deacylated by 20% methylamine, and analyzed by HPLC on a Whatman Partisphere 5 SAX column. For measurements of PI polyphosphate after transfection of Cos cells with
dominant negative mutants, the results represent the mean ± S.E.
of three independent experiments. For each experiment, the inhibitory
effect of the mutant has been normalized for the percentage of
transfected cells. This was determined concurrently using
Immunoblotting, Immunoprecipitation, and GST Pull-down
Experiments--
Stimulations were carried out at 37 °C in
serum-free DMEM containing 20 mM Hepes. Cells were washed
once with ice-cold phosphate-buffered saline before lysis. For
immunoblotting of crude lysates, cells were scraped off in SDS-PAGE
sample buffer and boiled 5 min, then resolved by SDS-PAGE, and analyzed
by immunoblotting using an enzyme-linked chemiluminescence system (ECL,
Amersham Pharmacia Biotech). Results obtained with anti-phospho-Akt,
and Erk antibodies were quantified by densitometry. We have verified
that results obtained with the anti-phospho-Erk antibody did not differ
from in vitro kinase assays of immunoprecipitated HA-Erk1
(construct kindly provided by Dr. J. Pouyssegur) using myelin basic
protein as a substrate. For immunoprecipitations, cells were scraped
off in lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1% Brij (Sigma catalog number P9641),
1 mM Na3VO4, and 10 µg/ml aprotinin and leupeptin. After gentle shaking during 20 min at 4 °C
and centrifugation (13,000 rpm for 10 min), the supernatants were
incubated 1 h with antibodies followed by addition of 10% (w/v)
protein A-Sepharose CL4B (Sigma) for 1 h. The immunecomplexes were
washed twice with 1 ml of lysis buffer containing 0.1% Brij, 100 µM Na3VO4, and 1 µg/ml
aprotinin and leupeptin and finally boiled in SDS-PAGE sample buffer.
For GST pull-down experiments, cells were processed similarly to the
immunoprecipitation protocol, except that cells were incubated 1 h
with 2 µg of GST fusion protein immobilized on glutathione-Sepharose
4B beads (Amersham Pharmacia Biotech).
LPA Rapidly Activates PI3K Independently of p110 Activation of PI3K by LPA Occurs through Recruitment of the EGF
Receptor--
To identify the tyrosine kinase(s) involved in this
process, we have searched for major tyrosine-phosphorylated proteins in crude lysates from cells treated 2 min with LPA. The major
phosphotyrosine signal induced by LPA in Vero or Cos cells was found in
the 180-kDa region (Fig. 3A),
suggesting an involvement of the two major RTKs expressed in
fibroblasts, i.e. the receptors for PDGF and EGF. First, we
have determined that the PDGFR-specific inhibitor tyrphostin AG1296 had
no effect on LPA-induced tyrosine phosphorylation of the 180-kDa
protein, whereas the EGFR inhibitor AG1478 nearly abolished the signal
in both Vero and Cos cells (Fig. 3A). In addition, anti-EGFR
immunoblotting of cell lysates confirmed that the 180-kDa protein
colocalized with EGFR, whereas an antibody against
We have then determined whether this event was important for PI3K
recruitment by LPA. In both Cos and Vero cells, the accumulation of
PI3,4P2 and PIP3 induced by LPA was
dramatically inhibited by the EGFR inhibitor AG1478 (Fig.
3D). This compound also abolished the activation of Akt by
LPA but did not interfere with Akt stimulation by insulin in Vero cells
(not shown). In addition, AG1478 did not alter PI3K activity itself in
Rat1 cells stimulated with PDGF (counts of PI3,4P2, control
<200; PDGF 9255 ± 475; PDGF +AG1478 100 nM,
8728 ± 836). Furthermore, Cos cells were transfected with an EGFR
mutant truncated at amino acid 688 (EGFRc688) which has a strong
ability to dimerize upon
activation.2 This mutant
inhibited by over 70% the LPA-induced accumulation of
PI3,4P2 and PIP3 after normalizing the results
for the percentage of transfection (Fig. 3E). Although these
results demonstrate that the EGFR plays a crucial role in LPA-induced
PI3K activation, EGF is not a typical agonist of PI3K, and
PIP3 levels are insensitive to EGF in various cell types.
Nevertheless, we have observed that 10 ng/ml EGF induced an important
accumulation of PI3,4P2 and PIP3 in Cos cells
(Fig. 3F). In addition, the increase in PI3K lipid products
occurred earlier upon EGF stimulation (Fig. 3F) than in the
presence of LPA (Fig. 1B), which is compatible with a
recruitment of PI3K by LPA occurring downstream the EGFR. Finally, expression of The EGFR-dependent Activation of PI3K by LPA Mobilizes
Gab1--
Since the activation of PI3K by EGF seems to differ from one
cell type to another, we have studied various pathways possibly involved in the EGFR-dependent activation of PI3K by LPA.
Using pull-down experiments with GST-p85 fusion protein, the EGFR and p85 were found to coprecipitate upon stimulation with LPA (Fig. 4A), thereby corroborating
that p85 is recruited by LPA through an EGFR-dependent
pathway. Although one of the major mechanisms of EGFR-mediated
recruitment of p85 is the heterodimerization of the EGFR with ErbB3
(28), we did not find any LPA- or EGF-induced association of ErbB3 in
p85 immunoprecipitates or GST-p85 pull-downs (not shown). To identify
other candidates possibly involved in recruitment of p85, we have
looked for tyrosine-phosphorylated proteins in GST-p85 pull-downs and
p85 immunoprecipitates from LPA- or EGF-treated cells. The major
phosphotyrosine signal appearing upon stimulation was located close to
the 115-kDa marker (Fig. 4B). This molecular mass led us to
consider the adaptor protein Gab1 as a candidate. By performing both
p85 immunoprecipitates and GST-p85 pull-downs, Gab1 was found to
associate with p85 following cell stimulation with LPA or EGF (Fig.
4B). To confirm this observation, we have performed
anti-Gab1 immunoprecipitates, and p85 was found to coprecipitate with
Gab1 upon stimulation with LPA or EGF (Fig. 4C). Finally,
the EGFR inhibitor AG1478 was found to abolish the association of Gab1
with p85 (Fig. 4D). Altogether, these results demonstrate
that activation of PI3K by LPA occurs through an EGFR/Gab1 pathway.
The EGFR/Gab1 Pathway Is Essential to PI3K Activation by
LPA--
To determine whether LPA could activate PI3K using other
mechanisms, we have first studied IMR90 human fibroblasts where
activation of MAPK by LPA is independent of RTK activities (Fig.
5A). Although LPA and PDGF
stimulated Erk to similar levels in these cells, LPA produced only a
minor accumulation of PI3K lipid products, whereas they readily
accumulated upon treatment with PDGF (Fig. 5A), suggesting
that specific RTK transactivation is required for activation of PI3K by
LPA. To determine if RTKs other than the EGFR could participate in the
process, we have studied mouse B82 L fibroblasts that do not express
the EGFR and where transactivation of the PDGFR is required for Erk
activation by LPA (Fig. 5B) (29). In these cells, LPA
produced only a faint accumulation of PI3K lipid products that readily
accumulated upon PDGF, whereas both growth factors activated Erk to
comparable levels (Fig. 5B).
Finally, in Rat1 cells where transactivation of the EGFR is required
for Erk activation by LPA similarly to Vero cells (4, 29), we have
observed that PI3,4P2 and PIP3 were hardly
detectable in LPA-treated cells (Fig.
6A). As a control, PDGF
induced a massive accumulation of PI3K lipid products, and both LPA and
PDGF activated Erk to comparable levels. To gain insight about the
missing mechanism in Rat1 cells, we have also measured PI3K activation
upon stimulation with EGF. Interestingly, levels of PI3,4P2
and PIP3 were not modified by EGF, although Erk activation
by EGF was comparable to the PDGF response (Fig. 6A). This
suggested that the EGFR-dependent pathway of PI3K
activation present in Vero and Cos cells was deficient in Rat1 cells.
By using GST-p85 pull-downs assays, we have observed that Gab1 did not
associate with p85 in Rat1 cells stimulated with LPA or EGF (Fig.
6B). Therefore, we have compared the recruitment of Gab1 in
Cos and Rat1 cells. By immunoblotting lysates of Cos cells stimulated
with EGF, Gab1 was found to undergo a shift in its apparent molecular
weight that is typical of this adaptor (30) (Fig. 6C,
top). In contrast, Gab1 migration was hardly modified in
Rat1 cells stimulated with EGF. Similarly, following immunoprecipitation, we have found that Gab1 was not
tyrosine-phosphorylated, and its migration was unchanged in LPA- or
EGF-treated Rat1 cells (Fig. 6C, bottom).
The recent discovery of p110 In contrast, we have found that adherent cells stimulated with LPA
recruit phosphotyrosine-dependent PI3Ks, in agreement with our recent report showing a major role for p110 In addition, we did not find a significant activation of PI3K in cells
where LPA induces transactivation of the PDGFR, one of the best
activators of PI3K when activated by its ligand. These results
demonstrate that important differences exist between stimulation of
RTKs by their ligands and GPCR-induced transactivation, although both
pathways lead to similar levels of activation of Erk1/2. The
differences are most likely due to the strength of early signaling events since, for example, tyrosine phosphorylation of RTKs mediated by
GPCRs is much weaker than phosphorylation induced by RTK
ligands3 (29, 35). Thus, the LPA-induced recruitment of
PDGFR might be sufficient to fully activate Erk1/2 in B82 L cells but
not the PI3K. The difference of sensitivity between activation of these
kinases could be due to the fact that Erk1 and Erk2 are stimulated
through an amplification cascade, whereas PI3K is activated directly by
the receptor.
Various mechanisms have been proposed regarding the activation of PI3K
by EGFR which lacks the YXXM p85-binding motif found in the
PDGFR sequence for example. One of the best documented mechanisms is
the EGF-induced dimerization of EGFR with a related protein, ErbB3,
that contains several YXXM motifs (28). However, we did not
find any association of ErbB3 with p85 in LPA or EGF-treated cells, in
conditions where the EGFR and p85 were found to form a complex. A
similar mechanism of RTK heterodimerization involving association of
EGFR with Although the mechanism of GPCR-induced transactivation of RTKs remains
completely unknown, one possibility could be the secretion of EGF
induced by LPA, in light of the dependence of transactivation processes
on calcium and protein kinase C (39, 40) that are crucial factors for
secretion. Although EGF has not been found in conditioned medium of
cells treated with GPCR agonists (40, 41), one cannot exclude that
secreted EGF would remain cell-associated and work in an autocrine
fashion, as described for the fibroblast growth factor (42). However,
our study shows that activation of PI3K can be measured as early as
30 s after adding LPA, which seems hardly compatible with a
process involving the whole cellular machinery for secretion. In
contrast, a scavenger of reactive oxygen species partly inhibited PI3K
activation by LPA, due to an inhibitory effect on tyrosine
phosphorylation of the EGF,3 as described recently in HeLa
cells (43). This suggests that in unstimulated cells, protein tyrosine
phosphatases involved in maintaining RTK in an inactivated state could
play a role in the mechanism of transactivation.
-responsive PI3K p110. In contrast, it is inhibited by genistein or expression of a
dominant negative mutant of p85 and potentiated by overexpressing wild-type p110
or - but not -. By using a specific chemical inhibitor of the epidermal growth factor receptor (EGFR) and expression of a dominant negative mutant, we have observed that recruitment of
p85/p110 PI3Ks occurs through transactivation of the EGFR by LPA and
downstream mobilization of the docking protein Gab1 that associates
with p85 upon LPA stimulation. Finally, we show that LPA cannot
activate PI3K in cell lines lacking the EGFR/Gab1 pathway, including
cells that transactivate the PDGF receptor. Altogether, these results
demonstrate that activation of PI3K by LPA is conditioned by the
ability of LPA to transactivate an EGFR/Gab1 signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
which contains a pleckstrin homology domain in its N terminus region.
Interestingly, p110 can be directly activated by G protein subunits
(14, 15), due both to a constitutive association with a p101
-sensitive protein (15) and to a direct interaction with the
complex (16). Although this isoform of PI3K plays a role in the
activation of the Ras/MAPK pathway by G proteins (17-19), it is not
clear yet whether this enzyme is involved in any GPCR-induced
PIP3 production in non-hematopoietic cell lines (15, 17,
18). On the other hand, p110 was recently found to play a role in
GPCR-induced signaling and mitogenesis (20, 21).
isoform of PI3K but recruits the
p85/p110 isoforms through LPA-induced mobilization of the EGF receptor (EGFR) and subsequent engagement of the docking protein Gab1. Finally,
we show that LPA cannot activate PI3K in various cell types lacking the
EGFR/Gab1 pathway, thereby demonstrating the pivotal role of this
transactivation pathway for PI3K activation by LPA in
non-myeloid-derived cell lines.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PDGFR antibody was from PharMingen (catalog number
15746E). Rabbit anti-human ErbB3 antibody was from Santa Cruz
Biotechnology (catalog number sc-285-G). Monoclonal 12CA5 anti-HA and
anti-Myc antibodies were from Roche Molecular Biochemicals and
Invitrogen, respectively. Rabbit anti-GST antibody was from Sigma
(catalog number G-7781). Horseradish peroxidase-conjugated anti-mouse,
-rabbit, and -sheep IgG antibodies were from Sigma, New England
Biolabs, and Rockland, respectively. LPA was from Sigma. Human
recombinant EGF and PDGF BB were from Calbiochem and Upstate
Biotechnology, Inc., respectively. Genistein and tyrphostin AG1478 were
from Biomol. Tyrphostin AG1296 was from Calbiochem.
was subcloned
into the NheI/KpnI sites of pcDNA3.1/Myc-His
(Invitrogen) using polymerase chain reaction with appropriate primers,
Pfu DNA polymerase (Stratagene), and pBS-p110wt as a
template (14). The construct was verified by sequencing. The plasmid
for bacterial expression of GST-p85wt and other mammalian expression
constructs has already been described: pGEX-p85wt (22), SR-
p85
(23), pSG5- p110wt (24), pRK5-p110wt (25), pCMV-p110K832R
(17). The EGFRc688 mutant (26) was subcloned into pRcCMV (Invitrogen)
in Dr. G. Gill's laboratory.
-galactosidase as reporter and following a standard procedure. The
efficiency of transfection was routinely around 40%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
but Recruits
p85/p110 Isoforms--
To gain insight into the regulation of PI3K by
LPA, we have measured the amount of PI3K lipid products in
serum-starved cells stimulated with LPA. 1 µM LPA induced
an important accumulation of PI3,4P2 and PIP3
in both Vero and Cos cells, at a level up to 10-fold higher than
control, with a maximum after 2 min stimulation (Fig.
1, A and B).
Dose-response assays indicated that the effect was detectable with as
low as 0.1 µM LPA and increased up to 10 µM
LPA (Fig. 1C). In contrast, another PI3K product,
phosphatidylinositol 3-monophosphate, was found in relative abundance
in resting cells and poorly accumulated upon LPA stimulation (counts in
resting, 5600 ± 1980; stimulated 2 min, 8850 ± 1280). To
elucidate the mechanism of PI3K activation by LPA, we have first
studied p110 which can be directly activated by G protein subunits.
The accumulation of PI3,4P2 and PIP3 upon LPA
was measured in Cos cells transiently transfected with a kinase-dead
mutant of p110 (K832R) that inhibits the G-induced activation
of the Ras/MAPK pathway (Fig.
2B). Expression of this mutant
somewhat reduced the number of cells retrieved 2 days after
transfection, leading to a small decrease of extracted lipids including
PI4,5P2 (Fig. 2A). However, upon stimulation by
LPA, PIP3 levels were only moderately lowered by expression
of kinase-dead p110, even after normalizing the results for the
percentage of transfection. Thus, the inhibition on PIP3 production appeared similar to the inhibitory effect on
PI4,5P2. Moreover, PI3,4P2 production was not
modified in Cos cells expressing the K832R p110 mutant. In contrast,
the accumulation of PI3,4P2 and PIP3 induced by
10 µM LPA was nearly abolished in Cos cells treated with
genistein (Fig. 2C), thereby suggesting the implication of
p85/p110 PI3Ks. To evaluate this hypothesis, we have measured PI3K
activation by LPA in Cos cells transfected with a dominant negative
form of p85 lacking the p110-binding site (p85) (23). After
normalization of the results for the percentage of transfection, we
have observed that expression of p85 nearly abolished the accumulation of PI3,4P2 and PIP3 induced by 10 µM LPA (Fig. 2D). In addition, we have
determined whether overexpression of wild-type p110, -, or -
had any potentiating effect on PI3K activation triggered by a
submaximal dose of LPA (1 µM). Analysis of PI3K stimulation limited for convenience to measurements of
PI3,4P2 showed that p110 and p110 to a greater extent
potentiated PI3K activation by LPA, whereas overexpression of p110
had no effect (Fig. 2E). Altogether, these results
demonstrate that the activation of PI3K by LPA is mediated by the
tyrosine kinase-dependent p85/p110 isoforms of PI3K.
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Fig. 1.
LPA induces the accumulation of PI3K lipid
products in Vero and Cos cells. A, serum-starved Vero
cells were labeled 5 h with
[32P]H3PO4 and then stimulated
with 1 µM LPA. After extraction of lipids and
purification on TLC, the PI4,5P2 region was deacylated and
analyzed by HPLC. Left graph, measurements at 0 and 2 min
expressed in crude counts from the radioactivity detector. Right
graph, time course measurements expressed in percent of the
maximum value. B, same as A with Cos cells.
C, dose-response of PI3K activation by LPA in Cos
cells.
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Fig. 2.
Activation of PI3K by LPA is independent of
p110 but involves tyrosine phosphorylations
and p110/p85 isoforms. A, Cos cells were transiently
transfected with a kinase-dead mutant of p110 (K832R) or vector as a
control. After serum starvation, PI3K lipid products were measured as
described in Fig. 1 following a 2-min stimulation with 10 µM LPA. The results represent the percentage of
inhibition of PI polyphosphate production in transfected cells
normalized for the percentage of transfection, as described under
"Experimental Procedures." B, the effect of the K832R
mutant on G-induced phosphorylation of hemagglutinin
(HA)-tagged Erk1 was measured in Cos cells transfected with
the indicated constructs (V, empty vector). C,
Cos cells were labeled with
[32P]H3PO4, incubated or not
(control) with 100 µM genistein for 15 min, and then
treated for 2 min with 10 µM LPA before analysis of PI3K
lipid products. D, Cos cells were transiently transfected
with a dominant negative mutant of p85 (p85) or vector as a control.
After serum starvation, cells were stimulated 2 min with 10 µM LPA and analyzed for their content in PI3K lipid
products. E, Cos cells were transfected with constructs
expressing wild-type p110, -, and - or vector as indicated.
The cellular contents of PI4,5P2 and PI3,4P2
was determined following a 2-min stimulation with 1 µM
LPA. For each transfection, the level of PI4,5P2 and
PI3,4P2 in unstimulated cells has been subtracted.
Inset, immunoblots of cells transfected with empty vector
(
) or the various p110 (+) using their respective anti-tag
antibodies.
PDGFR gave no
signal in Vero cells and a very faint band in Cos cells (Fig.
3B). Finally, following immunoprecipitation, the EGFR was
found to be tyrosine-phosphorylated after treatment of Cos cells with
LPA (Fig. 3C). Thus, these data demonstrate that
phosphorylation of the EGFR is the major early tyrosine phosphorylation event induced by LPA in Cos and Vero cells.
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Fig. 3.
Activation of PI3K by LPA is dependent on the
EGFR. A, serum-starved Cos and Vero cells were treated
or not with 10 µM LPA for 2 min following a 15-min
incubation with tyrphostins specific for the PDGFR (AG1296, 10 µM), the EGFR (AG1478, 100 nM), or
Me2SO as control. Then cells were processed for
anti-phosphotyrosine (pY) immunoblotting (IB) of
crude lysates. B, 30 µg of protein from lysates of Cos,
Vero, and Rat1 cells were immunoblotted with anti-EGFR or anti- PDGFR
antibodies. C, the EGFR was immunoprecipitated
(IP) from Cos cells treated 2 min with 10 µM
LPA or 10 ng/ml EGF and immunoblotted with 4G10 antibody. D,
analysis of phosphatidylinositol polyphosphates was performed in Cos
and Vero cells stimulated with 10 µM LPA for 2 min
following a preincubation with AG1478 or Me2SO as control.
E, Cos cells were transiently transfected with a dominant
negative mutant of the EGFR (EGFRc688) or vector as a control. The PI3K
lipid products were measured in cells treated 2 min with 10 µM LPA. The results are normalized for the percentage of
transfected cells. F, Cos cells were stimulated with 10 ng/ml EGF for the indicated time followed by measurements of PI3K lipid
products. G, Cos cells were transfected with the p85
mutant or vector as control and stimulated for 2 min with 10 ng/ml
EGF.
p85 in Cos cells suppressed the synthesis of PI3K lipid products induced by EGF to an extent similar to that upon LPA
stimulation (Figs. 3G and 2D). This suggested
that both LPA and EGF use a same p85-dependent pathway to
stimulate PI3,4P2 and PIP3 production.
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Fig. 4.
LPA induces the association of p85 with Gab1
in an EGFR-dependent fashion. A, quiescent
Cos cells were stimulated 2 min with 10 µM LPA or 10 ng/ml EGF and then lysed and incubated with 2 µg of Sepharose-GST-p85
fusion protein for GST-p85 pull-down (PD) assays.
Precipitated proteins were analyzed by imunoblotting (IB)
using an anti-EGFR antibody. B, left, cells were
stimulated as above and then processed for GST pull-down assays,
followed by anti-phosphotyrosine (pY) and Gab1
immunoblotting. Right, anti-p85 immunoprecipitates
(IP) from control or stimulated cells were immunoblotted
with the indicated antibodies (NRS, normal rabbit serum).
C, Gab1 was immunoprecipitated from control
(Ctrl) or stimulated cells and then the precipitated
proteins were revealed with the indicated antibodies. D,
before stimulation, cells were incubated with AG1478 when indicated and
then association of Gab1 with p85 was analyzed using GST-p85 pull-down
experiments.
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Fig. 5.
LPA does not induce the synthesis of PI3K
lipid products in cells that do not transactivate RTKs or cells that
transactivate the PDGF receptor. A, IMR90 human
fibroblasts were serum-starved for 3 days and then stimulated 5 min
with 10 µM LPA, 10 ng/ml EGF, or 30 ng/ml PDGF. Top
panel, activation of Erk1/2 was determined using anti-phospho-Erk
immunoblotting (IB). When indicated, cells have been
preincubated with 100 nM AG1478 or 10 µM
AG1296. Bottom graph, cells were labeled with
[32P]H3PO4 before stimulation and
then processed for measurements of PI3K lipid products. B
same as A in B82 L mouse fibroblasts. Ctrl,
control.
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Fig. 6.
LPA stimulation does not generate PI3K lipid
products in Rat1 cells that transactivate the EGF receptor but do not
recruit Gab1. A, quiescent Rat1 fibroblast cells were
labeled with [32P]H3PO4,
stimulated with 10 µM LPA, 10 ng/ml EGF, or 30 ng/ml PDGF
and then analyzed for their content in PI3K lipid products.
Inset, anti-phospho-Erk immunoblots (IB) of Rat1
cells stimulated 5 min as indicated. B, quiescent Cos and
Rat1 cells were stimulated with LPA or EGF and processed for analysis
of the p85-Gab1 association using GST-p85 pull-downs. C,
top, Gab1 was immunoblotted in crude lysate from EGF-treated
Cos and Rat1 cells. Bottom, Gab1 was immunoprecipitated
(IP) from stimulated Cos and Rat1 cells and then
immunoblotted as indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
that can be directly activated by
G protein subunits (14) led us first to evaluate the role of this
enzyme in LPA-mediated activation of PI3K. However, our results
demonstrate that the synthesis of PI3K lipid products induced by LPA
occurs independently of p110, based on the lack of inhibitory effect
of a kinase-dead mutant and supported by the non-potentiating effect of
the overexpressed wild-type enzyme. This observation was rather
surprising in light of recent well documented reports showing that
p110 is involved in G-induced signaling, such as activation of
the Ras/MAPK pathway (17, 31). However, we have investigated the
natural signaling resources of cells stimulated by endogenous LPA
receptor(s), whereas the studies by Lopez-Ilasaca and co-workers (17,
31) were based mainly on transient expression of G subunits and
cotransfection of effectors. Substantial differences might exist
between these two complementary models in terms of recruitment of
effectors, such as G protein subunits for example. In addition,
although p110 is not apparently involved in generation of PI3K lipid
products, a recent report demonstrated that the protein kinase activity of p110 is essential for MAPK activation (18), in agreement with our
observation that a catalytically dead mutant of p110 moderately
inhibited cell growth without influencing PIP3 levels. However, it is also important to consider that the expression of
p110 in fibroblasts is marginal in comparison to blood
platelets,3 a cell type where
activation of PI3K by the thrombin GPCR has been reported to engage
p110 (32, 33). Similarly, in neutrophils where p110 is readily
expressed (15), wortmannin has been shown to inhibit GPCR-induced
signaling independently of p85 (34). Therefore, the facts that p110
seems preferentially expressed in myeloid-derived cells and the
inability of most adherent cells to produce PIP3 upon GPCR
agonists (12) suggest that the role of p110 in GPCR-induced
signaling could be restricted to hematopoietic cells.
in LPA-induced mitogenesis of fibroblast cells (21). Interestingly, this isoform can
be activated synergistically by G subunits and phosphotyrosyl peptides (20), which may account for a more pronounced effect of
overexpressing p110 than p110 on PI3,4P2 production
induced by LPA. In addition, we have shown that engagement of p85/p110 PI3K by LPA occurs mainly through transactivation of an
EGFR/Gab1-signaling pathway. Although the GPCR-induced tyrosine
phosphorylation of the EGFR has been described as a docking effect for
downstream effectors (5), it has been shown using AG1478 that the
kinase activity of the EGFR is also required for LPA-induced activation of the Ras/MAPK pathway (4). Here we show that inhibition of EGFR
activity blocks PI3K activation by LPA, indicating that transactivation of the EGFR is an essential step for crucial events in LPA-induced signaling, including activation of MAPK and PI3K.
PDGFR has been recently described (36). However, this
possibility can be excluded in the case of the events described herein,
based on the very weak expression of PDGFR in Cos cells that we and
others have observed (29, 37), as well as their very weak
responsiveness to PDGF in terms of activation of PI3K and MAPK (not
shown) and the undetectable expression of PDGFR in Vero cells.
Recently, a novel docking protein associated to Grb2, Gab1, has been
shown to mediate PI3K activation by various RTKs (30), through an
interaction YXXM/SH2 domains of p85 (38). Our data
demonstrate that this adaptor is also involved in the EGFR-dependent activation of PI3K by LPA, since it is the
major tyrosine-phosphorylated protein associated with p85 in
LPA-treated cells. In addition, we have observed that Gab1 was
indispensable to the EGFR-dependent activation of PI3K by
LPA. Indeed, in Rat1 cells, neither LPA which transactivates EGFR nor
EGF itself are able to recruit p85, due to a poor recruitment of Gab1
by EGFR in this cell type. The reason for this observation remains
undetermined but could be due to a fewer number of EGFR molecules in
Rat1 than in Cos cells3 that is sufficient to activate the
Ras/MAPK pathway but not to significantly phosphorylate Gab1.
Alternatively, the EGFR-dependent phosphorylation of Gab1
might require an intermediate protein tyrosine kinase poorly expressed
in Rat1. Nevertheless, these data obtained in Rat1 cells further
confirm the pivotal role of the EGFR/Gab1 pathway for activation of
PI3K by LPA in non-myeloid-derived cell lines.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Dr. G. Gill for providing
us the EGFRc688 construct and B82 L cells; Drs. M. Kasuga and W. Ogawa
for p85 constructs; Drs. M. Waterfield, B. Vanhaesebroeck, and J. Downward for the p110 plasmid; Drs. P. Hu and J. Schlessinger
for the p110 construct; Dr. J. Pouyssegur for the HA-Erk1 plasmid;
Dr. B. Stoyanov for constructs; C. Viala for preparing GST
fusion proteins; and Dr. G. Mauco for help with HPLC.
| |
FOOTNOTES |
|---|
* This work was supported by grants from Association pour la Recherche sur le Cancer, Ligue Nationale Contre le Cancer, and Conseil Régional de Midi-Pyrénées.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.
To whom correspondence should be addressed. Fax: 33 561 77 94 01;
E-mail: raynal@cict.fr.
2 K. Tanner, J. Kyte, and G. Gill, manuscript in preparation.
3 M. Laffargue, P. Raynal, A. Yart, C. Peres, R. Wetzker, S. Roche, B. Payrastre, and H. Chap, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; EGF epidermal growth factor, EGFR epidermal growth factor receptor; GPCR, G protein-coupled receptors; LPA, lysophosphatidic acid; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI3K, phosphoinositide 3-kinase, PI3,4P2, phosphatidylinositol 3,4-bisphosphate; PI4, 5P2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; RTK, receptor tyrosine kinase; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; wt, wild type; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Gutkind, J. S.
(1998)
J. Biol. Chem.
273,
1839-1842 |
| 2. | Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737-745[CrossRef][Medline] [Order article via Infotrieve] |
| 3. |
Luttrell, L. M.,
Hawes, B. E.,
van Biesen, T.,
Luttrell, D. K.,
Lansing, T. J.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
19443-19450 |
| 4. | Daub, H., Weiss, F. U., Wallasch, C., and Ullrich, A. (1996) Nature 379, 557-560[CrossRef][Medline] [Order article via Infotrieve] |
| 5. |
Luttrell, L. M.,
Dellarocca, G. J.,
van Biesen, T.,
Luttrell, D. K.,
and Lefkowitz, R. J.
(1997)
J. Biol. Chem.
272,
4637-4644 |
| 6. | Wan, Y., Kurosaki, T., and Huang, X. Y. (1996) Nature 380, 541-544[CrossRef][Medline] [Order article via Infotrieve] |
| 7. |
Ferby, I. M.,
Waga, I.,
Sakanaka, C.,
Kume, K.,
and Shimizu, T.
(1994)
J. Biol. Chem.
269,
30485-30488 |
| 8. | Sakanaka, C., Ferby, I., Waga, I., Bito, H., and Shimizu, T. (1994) Biochem. Biophys. Res. Commun. 205, 18-23[CrossRef][Medline] [Order article via Infotrieve] |
| 9. |
Hawes, B. E.,
Luttrell, L. M.,
Vanbiesen, T.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
12133-12136 |
| 10. | Kranenburg, O., Verlaan, I., Hordijk, P. L., and Moolenaar, W. H. (1997) EMBO J. 16, 3097-3105[CrossRef][Medline] [Order article via Infotrieve] |
| 11. |
Hu, Z.-W.,
Shi, X.-Y.,
Lin, R. Z.,
and Hoffman, B. B.
(1996)
J. Biol. Chem.
271,
8977-8982 |
| 12. | Stephens, L. R., Jackson, T. R., and Hawkins, P. T. (1993) Biochim. Biophys. Acta 1179, 27-75[Medline] [Order article via Infotrieve] |
| 13. |
Duckworth, B. C.,
and Cantley, L. C.
(1997)
J. Biol. Chem.
272,
27665-27670 |
| 14. |
Stoyanov, B.,
Volinia, S.,
Hanck, T.,
Rubio, I.,
Loubtchenkov, M.,
Malek, D.,
Stoyanova, S.,
Vanhaesebroeck, B.,
Dhand, R.,
Nurnberg, B.,
Gierschik, P.,
Seedorf, K.,
Hsuan, J. J.,
Waterfield, M. D.,
and Wetzker, R.
(1995)
Science
269,
690-693 |
| 15. | Stephens, L. R., Eguinoa, A., Erdjument-Bromage, H., Lui, M., Cooke, F., Coadwell, J., Smrcka, A. S., Thelen, M., Cadwallader, K., Tempst, P., and Hawkins, P. T. (1997) Cell 89, 105-114[CrossRef][Medline] [Order article via Infotrieve] |
| 16. |
Leopoldt, D.,
Hanck, T.,
Exner, T.,
Maier, U.,
Wetzker, R.,
and Nurnberg, B.
(1998)
J. Biol. Chem.
273,
7024-7029 |
| 17. |
Lopez-Ilasaca, M.,
Crespo, P.,
Pellici, P. G.,
Gutkind, J. S.,
and Wetzker, R.
(1997)
Science
275,
394-397 |
| 18. |
Bondeva, T.,
Pirola, L.,
Bulgarelli-Leva, G.,
Rubio, I.,
Wetzker, R.,
and Wymann, M. P.
(1998)
Science
282,
293-296 |
| 19. | Takeda, H., Matozaki, T., Takada, T., Noguchi, T., Yamao, T., Tsuda, M., Ochi, F., Fukunaga, K., Inagaki, K., and Kasuga, M. (1999) EMBO J. 18, 386-395[CrossRef][Medline] [Order article via Infotrieve] |
| 20. |
Kurosu, H.,
Maehama, T.,
Okada, T.,
Yamamoto, T.,
Hoshino, S.,
Fukui, Y.,
Ui, M.,
Hazeki, O.,
and Katada, T.
(1997)
J. Biol. Chem.
272,
24252-24256 |
| 21. |
Roche, S.,
Downward, J.,
Raynal, P.,
and Courtneidge, S.
(1998)
Mol. Cell. Biol.
18,
7119-7129 |
| 22. | Kotani, K., Yonezawa, K., Hara, K., Ueda, H., Kitamura, Y., Sakaue, H., Ando, A., Chavanieu, A., Calas, B., Grigorescu, F., Nishiyama, M., Waterfield, M. D., and Kasuga, M. (1994) EMBO J. 13, 2313-2321[Medline] [Order article via Infotrieve] |
| 23. |
Hara, K.,
Yonezawa, K.,
Sakaue, H.,
Ando, A.,
Kotani, K.,
Kitamura, T.,
Kitamura, Y.,
Ueda, H.,
Stephens, L.,
Jackson, T. R.,
Hawkins, P. T.,
Dhand, R.,
Clark, A. E.,
Holman, G. D.,
Waterfield, M. D.,
and Kasuga, M.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
7415-7419 |
| 24. | Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J., Waterfield, M. D., and Downward, J. (1994) Nature 370, 527-532[CrossRef][Medline] [Order article via Infotrieve] |
| 25. |
Hu, P.,
Mondino, A.,
Skolnik, E. Y.,
and Schlessinger, J.
(1993)
Mol. Cell. Biol.
13,
7677-7688 |
| 26. | Chen, W. S., Lazar, C. S., Lund, K. A., Welsh, J. B., Chang, C. P., Walton, G. M., Der, C. J., Wiley, H. S., Gill, G. N., and Rosenfeld, M. G. (1989) Cell 59, 33-43[CrossRef][Medline] [Order article via Infotrieve] |
| 27. |
Ireton, K.,
Payrastre, B.,
Chap, H.,
Ogawa, W.,
Sakaue, H.,
Kasuga, M.,
and Cossart, P.
(1996)
Science
274,
780-782 |
| 28. |
Soltoff, S. P.,
Carraway, K. L. R.,
Prigent, S. A.,
Gullick, W. G.,
and Cantley, L. C.
(1994)
Mol. Cell. Biol.
14,
3550-3558 |
| 29. |
Herrlich, A.,
Daub, H.,
Knebel, A.,
Herrlich, P.,
Ullrich, A.,
Schultz, G.,
and Gudermann, T.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8985-8990 |
| 30. | Holgado-Madruga, M., Emlet, D. R., Moscatello, D. K., Godwin, A. K., and Wong, A. J. (1996) Nature 379, 560-564[CrossRef][Medline] [Order article via Infotrieve] |
| 31. |
Lopez-Ilasaca, M.,
Gutkind, J. S.,
and Wetzker, R.
(1998)
J. Biol. Chem.
273,
2505-2508 |
| 32. |
Zhang, J.,
Benovic, J. L.,
Sugai, M.,
Wetzker, R.,
Gout, I.,
and Rittenhouse, S. E.
(1995)
J. Biol. Chem.
270,
6589-6594 |
| 33. |
Tang, X. W.,
and Downes, C. P.
(1997)
J. Biol. Chem.
272,
14193-14199 |
| 34. |
Ferby, I. M.,
Waga, I.,
Hoshino, M.,
Kume, K.,
and Shimizu, T.
(1996)
J. Biol. Chem.
271,
11684-11688 |
| 35. | Daub, H., Wallasch, C., Lankenau, A., Herrlich, A., and Ullrich, A. (1997) EMBO J. 16, 7032-7044[CrossRef][Medline] [Order article via Infotrieve] |
| 36. |
Habib, A. A.,
Hognason, T.,
Ren, J.,
Stefansson, K.,
and Ratan, R. R.
(1998)
J. Biol. Chem.
273,
6885-6891 |
| 37. | Akimoto, K., Takahashi, R., Moriya, S., Nishioka, N., Takayanagi, J., Kimura, K., Fukui, Y., Osada, S., Mizuno, K., Hirai, S., Kazlauskas, A., and Ohno, S. (1996) EMBO J. 15, 788-798[Medline] [Order article via Infotrieve] |
| 38. |
Holgado-Madruga, M.,
Moscatello, D. K.,
Emlet, D. R.,
Dieterich, R.,
and Wong, A. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12419-12424 |
| 39. |
Zwick, E.,
Daub, H.,
Aoki, N.,
Yamaguchi-Aoki, Y.,
Tinhofer, I.,
Maly, K.,
and Ullrich, A.
(1997)
J. Biol. Chem.
272,
24767-24770 |
| 40. |
Eguchi, S.,
Numaguchi, K.,
Iwasaki, H.,
Matsumoto, T.,
Yamakawa, T.,
Utsunomiya, H.,
Motley, E. D.,
Kawakatsu, H.,
Owada, K. M.,
Hirata, Y.,
Marumo, F.,
and Inagami, T.
(1998)
J. Biol. Chem.
273,
8890-8896 |
| 41. | Tsai, W., Morielli, A. D., and Peralta, E. G. (1997) EMBO J. 16, 4597-4605[CrossRef][Medline] [Order article via Infotrieve] |
| 42. |
Vlodavsky, I.,
Folkman, J.,
Sullivan, R.,
Fridman, R.,
Ishai-Michaeli, R.,
Sasse, J.,
and Klagsbrun, M.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
2292-2296 |
| 43. |
Cunnick, J. M.,
Dorsey, J. F.,
Standley, T.,
Turkson, J.,
Kraker, A. J.,
Fry, D. W.,
Jove, R.,
and Wu, J.
(1998)
J. Biol. Chem.
273,
14468-14475 |
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