A Function for Phosphoinositide 3-Kinase 
Lipid Products
in Coupling 
to Ras Activation in Response to
Lysophosphatidic Acid*
Armelle
Yart
,
Serge
Roche§,
Reinhard
Wetzker¶,
Muriel
Laffargue,
Nicholas
Tonks
,
Patrick
Mayeux**,
Hugues
Chap, and
Patrick
Raynal
From INSERM U326, IFR 30, Hôpital Purpan,
Toulouse 31059, France, § CNRS UPR 1086, CRBM,
Montpellier 34293, France, ** INSERM U363, Hôpital
Cochin, 27 rue du Faubourg Saint-Jacques, Paris 75014, France, the
¶ Max Planck Research Unit Molecular Cell Biology, University of
Jena, Jena 07747, Germany, and the Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York 11724-2208
Received for publication, October 30, 2001, and in revised form, March 19, 2002
 |
ABSTRACT |
Although G
is thought to mediate
mitogen-activated protein kinase (MAPK) activation in response to G
protein-coupled receptor stimulation, the mechanisms involved in this
pathway have not been clearly defined. Phosphoinositide 3-kinase (PI3K)
has been proposed as an early intermediate in this process, but its
role has remained elusive. We have observed that dominant negative mutants of p110, but not of p110, inhibited MAPK stimulation in
response to lysophosphatidic acid (LPA). The role of p110 was
located upstream from Ras. To determine which of the lipid or
protein kinase activities of p110 were important for Ras activation, we produced a mutant p110 lacking the lipid but not the protein kinase activity. This protein displayed a dominant negative activity similar to a kinase-dead mutant, indicating that p110 lipid kinase activity was essentially involved in Ras activation. In agreement, overexpression of the lipid phosphatase PTEN was found to
specifically inhibit Ras stimulation induced by LPA. In addition, we
have observed that the PH domain-containing adapter protein Gab1, which
is involved in p110 activation during LPA stimulation, is also
implicated in this pathway downstream of p110. Indeed, both membrane
redistribution and phosphorylation of Gab1 were reduced in the presence
of PI3K inhibitors or dominant negative p110. Downstream of Gab1,
the tyrosine phosphatase SHP2 was found to mediate Ras activation in
response to LPA and to be recruited through PI3K and Gab1, because
transfection of Gab1 mutant deficient for SHP2 binding inhibited Ras
activation without interfering with PI3K activation. We conclude that
LPA-induced Ras activation is mediated by a p110/Gab1/SHP2 pathway.
Moreover, we present data indicating that p110 is effectively the
target of in this pathway, suggesting that the p110/Gab1/SHP2 pathway provides a novel link between and Ras by integrating two
early events of LPA signaling, i.e. G release and
tyrosine kinase receptor transactivation.
| |
INTRODUCTION |
Lysophosphatidic acid (LPA)1 is an intercellular lipid
mediator potentially involved in tissue
regeneration, brain development, tumorigenesis and atherosclerosis,
although its precise physiopathological role in vivo remains
to be explored (1-5). LPA is produced by activated cells, notably
platelets, and promotes the proliferation or survival of a large number
of cell types, similarly to a canonical growth factor. It is now well
accepted that the biological activity of LPA is mediated by at least
three different G protein-coupled receptors (GPCR), namely
LPA1, LPA2, and LPA3 (Edg 2, 4 and
7, respectively) which belong to a recently described family of
receptors to bioactive lysophospholipids. These receptors can activate
in concert the Gq, Gi and G12/13
subfamilies of G proteins, even though the coupling specificity between
each receptor and the different G proteins remains to be clearly
established (6-8).
A number of studies have started to delineate the intracellular
signaling pathways that mediate the biological activity of LPA. This
lipid was first found to activate the so-called Ras/mitogen-activated protein kinases (MAPK) pathway, which controls cell proliferation, differentiation and survival in response to numerous extracellular stimuli (see Refs. 1, 9 for recent reviews). Nevertheless, the early
mechanisms involved in MAPK activation in response to LPA are still
incompletely defined. LPA can potentially activate this pathway through
Gq-dependent Ca2+ mobilization
leading to stimulation of protein kinase C (PKC) that in turn can
activate Raf or MEK (MAP kinase/ERK kinase) (10). However, pertussis
toxin that inactivates Gi was found to inhibit MAPK
stimulation induced by LPA or other Gi-coupled receptor
agonists (9). In addition, the role of Gi was located
upstream from Ras, and the involvement of subunits has been well
documented (11, 12). In contrast, the molecular intermediates between and Ras have not been clearly identified (for recent reviews, see (9, 13)). Since Ras is classically activated downstream of tyrosine
kinases, various candidates, including Src, Pyk2 and growth factor
receptors were shown to mediate MAPK activation in response to GPCR
stimulation (14-17). However, there is little evidence for a
participation of in the activation of these tyrosine kinases,
and, in the case of Pyk2 or EGF receptor (EGFR), their recruitment has
been shown to occur through Gq-dependent pathways (14, 18, 19). Nevertheless, it has been proposed that
could participate in Src activation, leading to EGFR phosphorylation and MAPK stimulation (20). G protein-coupled receptor kinases and
-arrestins might be intermediates between and Src (21), or
even actors of the MAPK pathway (22), but these models have been
essentially described in cells overexpressing adrenergic receptors and
their relevance to LPA signaling remains to be established.
Besides tyrosine kinases, phosphoinositide 3-kinase (PI3K) has been
shown to be an early intermediate of MAPK activation in response to LPA
(9, 23). PI3K phosphorylates the 3' position of the inositol ring of
phosphoinositides to produce lipids that are now considered as crucial
spatio-temporal organizers of various signaling pathways. From a
molecular point of view, three classes of PI3Ks have been defined, and
mitogen signaling involves essentially class I enzymes that are
subdivided into two subclasses (A and B) (24, 25). Class IA
PI3Ks include the catalytic subunits p110
, , and 
associated
with a p85 regulatory subunit and activated by
phosphotyrosine-containing motifs encountered on receptors or adapter
proteins. In the case of p110, G protein subunits were shown
to dramatically increase its activation by phosphotyrosine motifs, even
though the molecular determinants of this synergism have to be further
defined (26, 27). Class IB is represented by the catalytic
subunit p110 that has the unique feature to be directly activated by
G protein subunits. p110 can be associated with a p101
regulatory subunit that has no homology with any known protein but
contributes to the sensitivity of p110 to G (28, 29). From a
mechanistic point of view, processes by which PI3Ks activate signaling
pathways have been recently unraveled. Their lipid products interact
with a number of signaling proteins, resulting in their membrane
targeting and/or modulation of their enzyme activity. For example,
phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binds to a conserved protein motif
called the pleckstrin homology (PH) domain, thereby inducing the
activation of the serine/threonine kinase Akt/protein kinase B (PKB)
and its upstream activators, the phosphoinositide-dependent
kinases (30, 31).
The hypothesis that PI3K might participate in the MAPK pathway has
emerged when blockade of PI3K was found to interfere with MAPK
activation in response to LPA (9, 23). Further studies have suggested
that p110 was a strong candidate to mediate this effect (32-34),
and to date it is commonly accepted that p110 can link GPCR
stimulation to MAPK activation (13). However, the mechanism of this
connection has been incompletely characterized. Although it has been
initially reported that p110 linked G to MAPK activation
through an Shc-Grb2-Sos-Ras pathway (32), the putative tyrosine kinase
activated downstream of p110 remains to be identified. In addition,
in a particular CHO cell line displaying a deficient activation of Ras,
Takeda et al. (33) have observed that LPA can activate MAPK
at the level of MEK through a pathway involving p110 upstream of
PKC
. PKC can be stimulated directly by
PtdIns(3,4,5)P3 or by the
phosphoinositide-dependent kinases (35, 36). However this
model has been challenged by the demonstration that the protein kinase
activity of p110, but not its lipid kinase activity, was important
for MAPK activation (34). Indeed, PI3K catalytic subunits have an
intrinsic protein kinase activity that was thought to simply regulate
their lipid kinase activity (37, 38), but, at least in the case of
p110, the protein kinase activity was shown to be critical for MAPK
activation by promoting MEK phosphorylation (34). However, the protein
substrate of p110 remains to be identified. Further studies are
certainly required to complete these different models and define their
physiological relevance, but the fact that p110 seems essentially
expressed in blood cells suggests that its role between G and
MAPK might be that of a tissue-specific function.
We and others have previously reported (39-41) that LPA can activate
p110 in different non-hematopoietic cell lines. The mechanism is
thought to implicate cooperation between and a transactivation pathway involving the EGFR and the adapter protein Gab1 that provides the consensus phosphotyrosine motifs. Activation of p110 is
essential for cell cycle progression of NIH-3T3 cells stimulated with
LPA (39), which suggested that this PI3K was an important actor of the
signaling pathways involved in the mitogenic activity of LPA. We have
thus searched for a role of p110 in the MAPK pathway in a
non-transformed cell line stimulated through endogenous LPA receptors.
By using a strategy based on the transfection of dominant negative
mutants, p110 was found to contribute to MAPK activation upstream
from Ras. In addition, the lipid kinase activity of p110 was found
to be essential, pointing out a key element of Ras activation that
involves sequentially PtdIns(3,4,5)P3, the PH
domain-containing adapter Gab1, and the tyrosine phosphatase SHP2.
Moreover, we present data indicating that this pathway provides a novel
connection between and Ras.
| |
EXPERIMENTAL PROCEDURES |
Materials--
LPA, pertussis toxin, and wortmannin were from
Sigma Chemical Co. LY294002 and AG1478 were from BIOMOL. Ro-31-8220 was
from Calbiochem. Monoclonal anti-phosphoErk and anti-GST antibodies were from Sigma. Polyclonal antibodies against ERK2, Grb2, SHP2, and
EGFR and monoclonal anti-myc were from Santa Cruz Biotechnology. Polyclonal antibody against Gab1 was from Upstate Biotechnology Inc.
Polyclonal antibody against phosphoAkt/PKB (Ser-473) was from
Cell Signaling. Monoclonal anti-pan Ras was from Oncogene Research.
Monoclonal anti-His tag antibody was from Invitrogen, anti-HA tag was
from Roche Molecular Biochemicals, and anti-T7 tag was from Novagen.
Cell culture reagents were from Invitrogen.
Expression Plasmids--
A construct encoding C-terminal
Myc/His-tagged ERK1 was obtained by subcloning ERK1 (kindly provided by
Dr. E. vanObberghen, Nice, France) into pcDNA3.1-MycHis
(Invitrogen). The pRK5 plasmid encoding wild type HA-tagged p110 was
kindly provided by Drs. P. Hu and J. Schlessinger (New York University)
(44). The kinase-inactive K805R mutant of p110 was obtained by
site-directed mutagenesis (QuikChange, Stratagene) with the
following mutagenic primer: 5'-GTTGGAGTGATTTTTAGAAATGGTGATGATTTACG-3' (the changed
nucleotide is underlined). p110 "protein kinase only"
mutant was obtained by deleting amino acids 946 to 955 using the
following mutagenic primer: 5'-ATTCTTGGAAATTTCGTGCCTTTTATTCTT-3'.
The mutations were verified by sequencing, and the integrity of
the encoded proteins was controlled by immunoblotting and kinase assays
following expression in COS cells. A construct encoding full-length
p110 in antisense orientation was obtained by excising p110 from
the pRK5 construct using EcoRI and HindIII. The
insert was then subcloned through the same sites into pHM6 (Roche
Molecular Biochemicals, Meylan, France), and the construction was
verified by restriction mapping. The plasmids encoding p110, ARK,
PTEN, and Gab1 mutants have been previously described (32, 45-47). The
p85 and SHP2 constructs were kindly provided by Drs. W. Ogawa
(University of Kobe, Japan) and N. Rivard (Sherbrooke University,
Canada), respectively.
Cell Culture, Transfection, and Stimulations--
Vero cells
(ATCC CCL 81) were maintained in Dulbecco's modified Eagle medium
supplemented with 7.5% fetal bovine serum and antibiotics. For
transfection experiments, cells in 60-mm plates were incubated 3 h
with 2 ml of Dulbecco's modified Eagle medium containing 2 µg of
total DNA and 6 µl of each LipofectAMINE and Plus reagents
(Invitrogen). Cells were incubated overnight in serum-free medium
before stimulation with LPA (10 µM, 5 min). When
indicated, cells were incubated with 100 nM wortmannin or 25 µM LY294002 15 min before stimulation.
Cell Lysis, Immunoprecipitation, and Immunoblotting--
Cells
were scrapped off in lysis buffer containing 20 mM Tris, pH
7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1%
Nonidet P-40, 10 µg/ml of each aprotinin and leupeptin, 1 mM orthovanadate. After shaking for 15 min at 4 °C,
soluble material was incubated with the appropriate antibody for 2 h at 4 °C. The antigen-antibody complexes were incubated with
protein A- or protein G-Sepharose (Sigma) for 1 h, then collected
by centrifugation and washed with lysis buffer containing 0.1% Nonidet
P-40, 1 µg/ml of each aprotinin and leupeptin, 0.1 mM
orthovanadate. Pellets were then processed for in vitro
kinase assays or resuspended in electrophoresis sample buffer and
analyzed by immunoblotting. Blots were developed using chemiluminescence (Amersham Biosciences, Inc.) and semi-quantified using a Bio-Rad gel analysis device and the software IMAGE (National Institutes of Health). For immunoblotting analysis of cell lysates, cells were directly scrapped off in electrophoresis sample buffer, then
boiled and processed for immunoblotting.
Measurements of ERK Phosphorylation in Transfected
Cells--
Cells were transfected with 1 µg of each plasmid
encoding ERK1-His and the indicated effector protein. After
stimulation, cells were harvested in lysis buffer supplemented
with 300 mM NaCl. Soluble material was incubated with 30 µl of ProBond resin (Invitrogen) during 2 h at 4 °C then
washed three times with lysis buffer supplemented with 5 mM
imidazole. The pellets were then resuspended in electrophoresis
sample buffer and processed for anti-phosphoERK and anti-His immunoblotting.
In Vitro Akt/PKB Kinase Assay--
Vero cells were cotransfected
with 0.5 µg of DNA encoding HA-tagged Akt/PKB and 1.5 µg of plasmid
encoding the indicated dominant negative protein. In the case of
p85, only 1 µg was used to avoid nonspecific effects, supplemented
with 0.5 µg of empty vector. After stimulation, cells were scrapped
off in lysis buffer then subjected to anti-HA immunoprecipitation.
Immunoprecipitates were washed twice with lysis buffer, then twice with
kinase buffer containing 50 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol. The
reaction was performed in 25 µl of kinase buffer containing 10 µg
of histone 2B (Roche Molecular Biochemicals), 50 µM ATP, and 3 µCi of [-32P]ATP. The reaction was incubated
during 30 min at 25 °C, then stopped by addition of electrophoresis
sample buffer and analyzed by SDS-PAGE. Phosphorylation of histones was
quantified using a PhosphorImager and the software ImageQuant
(Molecular Dynamics).
In Vitro Lipid and Protein Kinase Assays--
COS cells were
transfected with the constructs encoding HA-tagged p110 mutants.
Cells were then subjected to anti-HA immunoprecipitation, followed by
lipid or protein kinase assays. To perform the lipid kinase assay,
pellets were then washed twice in the following lipid kinase buffer: 50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM
MgCl2, 1.5 mM dithiothreitol, 0.5 mM EDTA. Pellets were then resuspended in 100 µl of lipid
kinase buffer supplemented with 150 µM
phosphatidylserine, 75 µM phosphatidylinositol, 20 µM ATP and 5 µCi of [-32P]ATP. The
reaction was performed at 37 °C for 30 min, then stopped by adding
100 µl of HCl (1.5 N), followed by lipid extraction (40).
Lipids were then separated by thin layer chromatography and revealed
using a PhosphorImager. To perform the protein kinase assay,
immunoprecipitation pellets were washed twice in the following protein
kinase buffer: 50 mM Tris, pH 7.4, 100 mM NaCl,
1 mM MnCl2. Pellets were then resuspended in 50 µl of protein kinase buffer supplemented with 40 µM ATP
and 3 µCi of [-32P]ATP. The reaction was incubated
at 37 °C for 30 min, then stopped by adding electrophoresis sample
buffer and analyzed by SDS-PAGE. Autophosphorylated p110 was
revealed using a PhosphorImager.
Activated Ras Affinity Precipitation Assay--
The assay was
performed essentially as described previously (43). The recombinant
Ras-binding domain (RBD) of Raf1 (kindly provided by Dr. F. R. McKenzie, Nice, France) was expressed as a GST fusion protein in
Escherichia coli and extracted using glutathione-Sepharose beads. To measure Ras activation in stimulated cells, Vero cells were
scrapped off in 1 ml of lysis buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 10 mM MgCl2,
0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml of each aprotinin and
leupeptin. Cleared lysates were incubated at 4 °C for 30 min with 30 µg of GST-RBD bound to glutathione-Sepharose beads. Beads were washed
three times in lysis buffer then boiled, and proteins were resolved by
SDS-PAGE. Immunoblotting was performed with anti-pan Ras antibodies. To
study Ras activation in transfected cells, the cells were cotransfected
with 0.5 µg of plasmid encoding HA-tagged wild type Ras (kindly
provided by Dr. B. M. Burgering, Utrecht, The Netherlands) and 1.5 µg of the indicated effector, unless otherwise indicated. The GST-RBD
pull-down assay was performed as above, except that immunoblots were
revealed with anti-HA antibody.
Membrane Fractions--
Membrane fractions were prepared
essentially as described (48). Cells were scrapped off in lysis buffer
containing 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 mM EGTA, 2 mM
MgCl2, 10 µg/ml of each aprotinin and leupeptin, 1 mM orthovanadate, then Dounce-homogenized. The homogenate
was centrifuged at 100,000 × g for 1 h. The
pellet was washed once in lysis buffer then dissolved in lysis buffer
supplemented with 1% Triton X-100. The insoluble material was spun
out, and the supernatant was taken as the solubilized membrane fraction.
RT-PCR Analysis--
This study was performed essentially as
described (42). Total RNA were extracted from cultured cells by
the TRIzol method (Invitrogen) followed by purification on NucleoSpin
RNA II columns (Macherey-Nagel, Germany). The presence of contaminating
genomic DNA was monitored by PCR amplification of -actin. Pure RNA
preparations were then reverse-transcribed with SuperScript II
(Invitrogen) using oligo-dT or random hexamer primers as indicated. PCR
reactions were carried out in 50 µl containing 5 µl of cDNA,
200 µM dNTP, 500 µM primers, PCR buffer
(Invitrogen) supplemented with 1.5 mM MgCl2 and
2.5 units of Taq DNA polymerase (Invitrogen) unless otherwise indicated. PCR conditions were: 95 °C for 90 s
followed by 35 cycles (95 °C for 30 s, 55 °C for 45 s,
72 °C for 30 s) and final extension (72 °C for 7 min). PCR
products were separated on 2% agarose and stained with ethidium
bromide. The primers for -actin were: 5'-CTGGAACGGTGAAGGTGACA-3'
(1275-1294) and 5'-GGTCTCAAGTCAGTGTACAGG-3' (1676-1696);
LPA1: 5'-CGGAGACGACTGACTGTCAGCAC-3' (286-305) and 5'-GGTCCAGAACTATGCCGAGA-3' (664-683); LPA2:
5'-CCCAACCAACAGGACTGACT-3' (1132-1147) and 5'-GAGCCCTTATCTCTCCCCAC-3'
(1401-1420); LPA3: 5'-GGACACCCATGAAGCTAATG-3' (695-714)
and 5'-TCTGGGTTCTCCTGAGAGAA-3' (931-950).
| |
RESULTS |
MAPK Activation by LPA Requires p110 Rather Than
p110--
To examine the role of PI3K in MAPK activation, we have
used a non-transformed monkey kidney cell line (Vero) that displays a
strong increase of phosphorylated ERK during LPA stimulation (Fig.
1A). The involvement of PI3K
was first approached using the conventional inhibitors wortmannin and
LY294002. As shown in Fig. 1A, each drug inhibited by
approximately 60% ERK phosphorylation in response to LPA. In contrast,
each compound abolished Akt/PKB phosphorylation, indicating that they
fully blocked PI3K activation in our experimental conditions. These
results suggested that MAPK activation in Vero cells stimulated with
LPA was partially dependent on PI3K. Because the function of PI3K in
the MAPK pathway is poorly understood, we have studied the involvement
of different PI3K isoforms using a dominant negative strategy. Cells
were thus transiently cotransfected with distinct PI3K mutants and
His-tagged ERK1. Following cell stimulation, ERK1-His was extracted and
its phosphorylation was analyzed by immunoblotting. We have first
examined the role of p110, because this enzyme was shown to
participate in MAPK stimulation mediated by subunits (32). This
was achieved by cotransfecting ERK1-His with the dominant negative
p110-K832R mutant. Fig. 1B shows that expression of this
mutant did not reduce ERK1-His phosphorylation induced by LPA. As a
control, we have verified that p110-K832R acted as a dominant
negative molecule by blocking ERK activation induced by
overexpression (Fig. 1C), in agreement with previous reports
(32, 40). This suggested that p110 did not significantly participate
in MAPK activation in response to LPA. In contrast, ERK1-His
phosphorylation was strongly inhibited in cells transfected with a form
of p85 lacking the p110 binding site (p85) (Fig. 1B).
This protein is a widely used dominant negative mutant for class
IA PI3K, which suggested the involvement of p110 because
other class IA enzymes are not activated by GPCRs. The role
of p110 was further studied using a kinase-inactive mutant of the
catalytic subunit (p110-K805R). This protein significantly reduced
ERK1-His phosphorylation induced by LPA, although the effect was less
pronounced than that of p85. To understand this difference, we have
compared the capacity of the two mutants to interfere with PI3K
activation in response to LPA. For convenience, the activation of
Akt/PKB was used as readout of PI3K stimulation, by cotransfecting
HA-tagged Akt/PKB with p85 or p110-K805R, followed by an in
vitro kinase assay. As shown in Fig. 1D, whereas p85
abolished the stimulation of HA-Akt/PKB induced by LPA, p110-K805R
produced only a partial inhibition, consistent with the respective
capacity of the two mutants to interfere with MAPK activation. In
addition, p110-K805R did not significantly interfere with HA-Akt/PKB
stimulation induced by LPA, further suggesting that in Vero cells
p110 does not significantly participate in LPA signaling.
Altogether, these data strongly suggested that p110 was involved in
MAPK activation in response to LPA.
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Fig. 1.
Comparative role of p110
and p110 in MAPK activation during LPA
stimulation. A, serum-starved Vero cells were incubated
for 15 min with 100 nM wortmannin (+W) or 25 µM LY294002 (+LY) when indicated before a
5-min stimulation with 10 µM LPA. Cells were then lysed
in electrophoresis sample buffer and analyzed by anti-phospho-ERK
(upper panel), anti-phospho-Akt (middle panel),
and anti-ERK2 (lower panel) immunoblotting (IB).
The graphs represent immunoblot quantification by densitometry using
the software IMAGE (National Institutes of Health), unless otherwise
indicated. B, cells were cotransfected with constructs
encoding ERK1-His and one of the following proteins: empty vector
(V); GST-tagged kinase-dead p110-K832R
(KR); HA-tagged dominant negative p85
(p85); HA-tagged kinase-dead p110-K805R
(KR). After stimulation and lysis, cells were subjected
to ERK1-His extraction, followed by anti-phosphoERK (upper
panel) and anti-His (lower panel) immunoblotting
(IB). C, the dominant negative activity of the
p110 mutant was verified on ERK1-His phosphorylation induced by
overexpression, as previously described (32). D,
cells were cotransfected with HA-tagged Akt/PKB and the indicated
constructs, following the conditions described under "Experimental
Procedures." After cell stimulation, HA-Akt/PKB was
immunoprecipitated, then subjected to in vitro kinase assay
using histone 2B (H2B) and [-32P]ATP as
substrates. Radiolabeled histones were revealed using a PhosphorImager
(upper panel) and quantified using the software ImageQuant
(graph). The amount of immunoprecipitated HA-Akt/PKB was
controlled by anti-HA immunoblotting (lower panel).
E, PI3K mutant expression was verified in lysates from
transfected cells using the appropriate antibody. Mean ± S.E. are
from at least three independent experiments. *, different from
stimulated control or empty vector; ns, not significant;
p < 0.05, paired t test.
|
|
Role of p110 Upstream from Ras--
To study p110 function
in the MAPK pathway, we have first determined at which level of the
cascade p110 was involved. Because Ras is located in a central
position, we have determined whether p110 was necessary for Ras
activation. This was achieved by using the precipitation assay for
activated Ras that takes advantage of a GST fusion protein containing
the Ras-binding domain of Raf (RBD) to extract selectively activated
GTP-bound Ras (43). The amount of activated Ras in the pull-down assays
was determined by anti-Ras immunoblotting. As shown in Fig.
2A, LPA strongly stimulated
endogenous Ras in Vero cells. Pretreatment of the cells with wortmannin
or LY294002 significantly reduced this activation, suggesting an
involvement of p110 upstream of Ras. To test this hypothesis, the
activation of HA-tagged wild type Ras was studied in cotransfection
experiments with PI3K mutants. As shown in Fig. 2B, the
p85 and p110-K805R mutants significantly inhibited HA-Ras activation induced by LPA, with a relative efficiency corresponding to
their ability to block Akt/PKB activation (Fig. 1D). In
addition, dominant negative p110 mutant did not prevent Ras
activation induced by LPA (Fig. 2B), further excluding a
role for this PI3K in this pathway. Finally, we have used an antisense
strategy to confirm the involvement of p110 in Ras activation. We
have constructed a plasmid encoding full-length p110 in antisense
orientation. Transfection of this plasmid in Vero cells produced a
partial reduction of endogenous p110, which might be due to the low
percentage of transfected cells (Fig. 2C). In these
conditions, we have tested whether this construct prevented Ras
activation in cotransfection experiments to circumvent the low
percentage of transfection. As shown in Fig. 2C,
transfection of antisense p110 led to a partial but significant
inhibition of Ras activation. Taken together, these data indicated that
p110 contributed to Ras stimulation in response to LPA.
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Fig. 2.
Involvement of p110
in LPA-induced Ras activation. A, control
(ctrl) or LPA-treated cells, preincubated or not with
wortmannin (+W) or LY294002 (+LY), were subjected
to the precipitation assay of activated Ras using a GST fusion protein
containing the Ras-binding domain of Raf (GST-RBD). The amount of
activated endogenous Ras associated to the GST-RBD beads was determined
by anti-Ras immunoblotting (upper panel). Cell lysates were
also directly subjected to anti-Ras immunoblotting to verify that equal
amounts of Ras were present in each sample (lower panel).
B, cells were cotransfected with 0.5 µg of plasmid
encoding HA-tagged Ras and one of the construct encoding the following
PI3K mutants: empty vector (V, 1.5 µg) dominant negative
p85 (p85, 1 µg + 0.5 µg of empty vector);
catalytically inactive p110 (K805R, 1.5 µg);
catalytically inactive p110 (K832R, 1.5 µg).
Following stimulation, cells were processed as in A. C, HA-tagged Ras activation was measured as in A
in cells transfected with empty vector (V) or a construct
encoding full-length p110 in antisense orientation (AS
p110). Lower panel: cell lysates were immunoblotted
with anti-p110 antibody to verify inhibition of expression. The
graphs represent quantifications (mean ± S.E.) from three
independent experiments. *, less than stimulated control;
ns, not significant; p < 0.05, paired
t test.
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|
Role of the Lipid and Protein Kinase Activities of
p110--
Because each of the lipid or protein kinase activities of
PI3K can be involved in the MAPK pathway, we have determined for p110 the respective participation of each activity in Ras
activation. We have thus prepared a protein kinase only (PKO) mutant by
deleting the region that putatively interacts with the phosphoinositide polar head, according to the sequence alignment between p110 and
p110 (34). The effect of this deletion on p110 was verified using
immune complex kinase assays following expression in COS cells. The
lipid kinase assay was performed using phosphatidylinositol as a
substrate, and autophosphorylation was used as a protein kinase assay.
As controls, the assays were also performed with wild type
(wt) and kinase-inactive p110 (K805R). Fig.
3A shows that, as expected,
the PKO protein has lost the ability to phosphorylate phosphatidylinositol. In contrast, its protein kinase activity was
conserved and was even higher than that of wild type p110 (Fig.
3B), which supported the notion that the two catalytic
activities of PI3K are antagonistic (37). Therefore, the above deletion did convert p110 into a protein kinase only mutant. To determine whether this mutant could exhibit dominant negative effects, we have
tested its ability to interfere with HA-Akt/PKB activation in
cotransfection experiments. As shown in Fig. 3C, the PKO
mutant inhibited HA-Akt/PKB stimulation as efficiently as the kinase dead K805R mutant, demonstrating that the loss of p110 lipid kinase
activity had turned the PKO protein into a dominant negative mutant for
D3-phosphoinositide-dependent processes. We have
thus tested this mutant in cotransfection experiments with HA-Ras to determine whether p110 lipid kinase activity was dispensable for Ras
activation. However, Fig. 3D shows that the PKO protein inhibited HA-Ras stimulation as efficiently as the kinase-dead mutant,
which suggested that p110 lipid kinase activity was essential to Ras
activation. To strengthen this result, Ras activation was measured in
cells overexpressing PTEN, a phosphatase that degrades PI3K lipid
products (46). As shown in Fig. 3E, overexpression of wild
type PTEN significantly inhibited the stimulation of HA-Ras induced by
LPA. Because PTEN has also a protein phosphatase activity that might
interfere with Ras activation, we have used as a control the "protein
phosphatase only" mutant of PTEN (G129E) that does not interfere with
PI3K signaling (46). Unlike wild type PTEN, this mutant did not modify
HA-Ras activation induced by LPA, which indicated that the lipid
phosphatase activity of PTEN was essentially responsible for Ras
inhibition. Taken together, these results showed that the lipid
products of p110 were critical for Ras stimulation in response to
LPA.
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Fig. 3.
Role of p110 lipid
and protein kinase activities in Ras activation.
A-B, characterization of p110-PKO mutant. COS
cells were transiently transfected with constructs encoding the
following HA-tagged p110 mutants: empty vector (V); wild
type (wt); kinase dead K805R (K805R);
protein kinase only (PKO). After 24-h expression, the
three HA-p110 variants were immunoprecipitated and assayed for their
lipid and protein kinase activities. A, lipid kinase assay:
immunoprecipitates were incubated with phosphatidylinositol and
[-32P] ATP. Lipids were then extracted and separated
using TLC. Radiolabeled lipids were revealed with a PhosphorImager.
B, protein kinase assay: immunoprecipitates were incubated
with [-32P]ATP. Autophosphorylated p110 was
resolved by SDS-PAGE and revealed using a PhosphorImager. C,
cells were cotransfected with HA-tagged Akt/PKB and the indicated
p110 constructs. Following cell stimulation, HA-Akt/PKB was
immunoprecipitated and subjected to in vitro kinase assay.
Histone phosphorylation (H2B) was revealed with a
PhosphorImager (upper panel), and HA-Akt/PKB amounts were
controlled by immunoblotting (IB HA, lower
panel). D, cells were cotransfected with HA-tagged Ras
and the indicated constructs. Following stimulation, cells were
processed for the GST-RBD assay. The amount of activated HA-Ras
associated with the beads was determined by anti-HA immunoblotting and
quantified using IMAGE. E, cells were cotransfected with
HA-tagged Ras and the indicated construct: empty vector (V);
PTEN wild type (wt); PTEN protein phosphatase only mutant
(G129E). HA-Ras activation in response to LPA was determined
as in D. Data shown represent the mean ± S.E. from
three independent experiments. *, less than stimulated control;
ns, not significant; p < 0.05, paired
t test. D and E, lower
panels: anti-HA immunoblotting of cell lysates to determine the
expression level of transfected proteins.
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Gab1 and SHP2 Link p110 to Ras--
To define p110 function
upstream from Ras, we have studied the proteins that associate with
Grb2 in a PI3K-dependent manner. This was achieved by
immunoprecipitating Grb2 from LPA-treated cells, preincubated or not
with wortmannin. Immunoprecipitates were then analyzed by
antiphosphotyrosine immunoblotting. As shown in Fig.
4A, LPA increased the
phosphorylation of six different proteins, five of which were
insensitive to wortmannin: The 180-kDa protein comigrated with the
epidermal growth factor receptor (EGFR), the 66-, 52-, and 46-kDa
proteins comigrated with Shc, and we failed to identify the 95-kDa
protein (data not shown). In contrast, wortmannin inhibited the
phosphorylation of the 115-kDa protein (Fig. 4A). This
protein comigrated with Gab1 upon reblotting the samples with anti-Gab1
antibody (data not shown). Although Gab1 is an essential activator of
p110 by providing p85-binding motifs (40), this result, and the fact
that Gab1 contains a PH domain (49), suggested that Gab1 might also be
the downstream effector of p110 lipid products. To assess this
hypothesis, we have first studied the effect of PI3K inhibitors on Gab1
phosphorylation. This was achieved by immunoprecipitating Gab1 from
control or LPA-treated cells, followed by antiphosphotyrosine
immunoblotting. As shown in Fig. 4B, Gab1 phosphorylation in
response to LPA was strongly reduced when cells were incubated with
PI3K inhibitors. In addition, Gab1 recruitment was examined by studying
its association with membrane fractions. Fig. 4C shows that
Gab1 was strongly enriched in membrane fractions prepared from
LPA-treated cells. This redistribution was almost abolished when cells
were preincubated with wortmannin or LY294002. These data showed that
PI3K was essential for Gab1 recruitment in LPA signaling, suggesting
that Gab1 was the downstream effector of p110 lipid products. To
further establish the respective role of p110 and p110 in this
pathway, we have studied the effect of their dominant negative mutants
on Gab1-myc phosphorylation. As shown in Fig. 4D, the
p110, but not p110, mutant inhibited LPA-induced Gab1
phosphorylation, further suggesting that Gab1 was the downstream target
of p110 lipid products.
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Fig. 4.
Role of PI3K in Gab1 recruitment.
A, anti-Grb2 immunoprecipitates were obtained from control
(ctrl) or LPA-treated cells, preincubated or not with
wortmannin (+W), as indicated. Samples were analyzed by
immunoblotting with antiphosphotyrosine (IB P-Tyr) and
anti-Grb2 (IB Grb2) antibodies. As a control, the
immunoprecipitation was performed without adding Grb2 antibody
( Ab). B, before stimulation with LPA, cells
were incubated with PI3K inhibitors when indicated (+W,
+LY). Cells were then subjected to Gab1 immunoprecipitation,
followed by anti-phosphotyrosine (IP P-Tyr) and anti-Gab1
(IB Gab1) immunoblotting. C, membrane fractions
from cells treated as in A were prepared by
ultracentrifugation. Membrane fractions were then analyzed by anti-Gab1
immunoblotting (upper panel), followed by anti-EGFR
immunoblotting to control gel loading (lower panel).
D, cells were cotransfected with Gab1-myc and the indicated
constructs: empty vector (V); kinase-dead p110
(KR); kinase-dead p110 (KR). After
stimulation, cells were subjected to Gab1-myc immunoprecipitation
followed by anti-phosphotyrosine (upper panel) and anti-myc
(lower panel) immunoblotting. The results displayed are
representative of at least two independent experiments performed in
duplicate.
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Gab1 is a multifunctional adapter protein that contains binding sites
for numerous signaling proteins, including Grb2, p85, and the tyrosine
phosphatase SHP2 that can be involved in Ras stimulation (47, 50). This
suggested that SHP2 might be a downstream effector of p110 and Gab1
required for Ras activation in response to LPA. To get further insight
on the role of Gab1 and its partners in this pathway, we have studied
the activation of both Ras and Akt/PKB in cells expressing Gab1 mutated
on its SHP2 binding site (Gab1-Y627F) or on its three p85 binding sites (Gab1-YF3). These mutants were previously characterized using coimmunoprecipitation experiment as unable to associate with SHP2 or
p85, respectively (47). As expected, Gab1-YF3 that prevents PI3K
activation, significantly reduced HA-Ras stimulation induced by LPA
(Fig. 5A). Interestingly,
Gab1-Y627F strongly inhibited HA-Ras activation, suggesting that the
association between Gab1 and SHP2 was important for Ras stimulation. To
determine whether this interaction was required upstream or downstream
from PI3K we have studied the ability of Gab1-Y627F to interfere with
PI3K activation using Akt/PKB as readout. As shown in Fig.
5B, Gab1-Y627F slightly increased Akt/PKB stimulation
induced by LPA compared with Gab1-wt, whereas, as a control, Gab1-YF3
nearly abolished this activation. These results suggested that the
association between Gab1 and SHP2 was important for activation of Ras,
but not of PI3K, implying that SHP2 was a downstream effector of PI3K and Gab1 in this pathway. To assess this hypothesis, we have studied the role of PI3K in the recruitment of SHP2 in Ras activation complex.
This was achieved by examining its association with Grb2 in
coimmunoprecipitation experiments. As shown in Fig. 5C, LPA induced the association of SHP2 with Grb2, and this interaction was
strongly inhibited by wortmannin. This showed that SHP2 was recruited
downstream of PI3K, and supported the idea that the phosphatase was an
intermediate between p110 and Ras. To further explore this pathway,
we have determined whether SHP2 was required for Ras activation in
response to LPA. This was achieved by studying HA-Ras activation in
cotransfection experiments with a catalytically inactive mutant of SHP2
(C/S), or wild type SHP2 as control. Fig. 5D shows that
SHP2-C/S, but not wt-SHP2, inhibited HA-Ras stimulation, which
indicated that SHP2 catalytic activity was necessary for Ras activation
induced by LPA. Taken together, these data showed that Gab1 and SHP2
mediated the participation of p110 to Ras stimulation in response to
LPA.
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Fig. 5.
Role of Gab1 and SHP2 in Ras and Akt
activation. A, cells were cotransfected with HA-tagged
Ras and the indicated construct: empty vector (V); wild type
Gab1-myc (wt); Gab1-myc mutated on its SHP2 binding site
(Y627F); Gab1-myc mutated on its three p85 binding sites
(YF3). Following stimulation, cells were lysed and processed
for GST-RBD assay. The amount of precipitated activated HA-Ras was
determined by anti-HA immunoblotting (upper panel). Lysates
were also directly subjected to anti-HA (middle panel) and
anti-myc immunoblotting (lower panel) to control expression
of HA-Ras and Gab1-myc constructs. The graph represents a
quantification (mean ± S.E.) from three independent experiments.
*, less than empty vector; ns, not significant;
p < 0.05, paired t test. B,
cells were cotransfected with HA-tagged Akt/PKB and the indicated Gab1
constructs, then processed for HA-Akt/PKB immunoprecipitation followed
by in vitro kinase assay. Phosphorylation of histones
(H2B) was quantified with a PhosphorImager (upper
panel + graph, mean ± S.E. from three experiments) and
HA-Akt/PKB was revealed by immunoblotting (lower panel).
C, anti-Grb2 immunoprecipitates were performed from cells
pretreated or not with wortmannin and stimulated with LPA as indicated.
Immunoprecipitates were then analyzed by anti-SHP2 (upper
panel) and anti-Grb2 (lower panel) immunoblotting.
D, cells were cotransfected with HA-tagged Ras and empty
vector (V), catalytically inactive SHP2 (C/S) or
wild type SHP2 (wt) as indicated. Cells were then stimulated
and subjected to the GST-RBD pull-down assay as in A.
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These results also suggested that the pathway described herein might
provide a novel link between and Ras, because is thought
to participate in p110 activation. On the other hand, this pathway
could be simply activated downstream of Gq, because it
requires EGFR transactivation. To gain insight on the G protein specificity of this pathway, we have first studied Ras activation in
cells treated with pertussis toxin or transfected with the C-terminal
domain of the -adrenergic receptor kinase (ARK-CT) that acts as a
-scavenger molecule (51). As shown in Fig. 6 (A and B),
pertussis toxin and ARK-CT strongly inhibited Ras activation in
response to LPA, which indicated that Gi-derived
played an essential role in this pathway. Although p110 might be the
downstream effector of , it has also been proposed that
could participate in EGFR recruitment (20). To distinguish between
these two possibilities, we have compared the effect of pertussis toxin
and ARK-CT on LPA-induced EGFR transactivation and p110
activation. As shown in Fig. 6 (C and D),
pertussis toxin and ARK-CT significantly inhibited Akt/PKB
activation that was used as marker of p110 activation. In contrast,
pertussis toxin did not modify EGFR phosphorylation induced by LPA
(Fig. 6E). We have also studied EGFR transactivation in
cells transfected with ARK-CT. To circumvent the low efficiency of
transfection, we have attempted to cotransfect Vero cells with a
His-tagged EGFR and analyze its phosphorylation in response to LPA, but
this produced a constitutive activation of this receptor (data not shown). Vero cells were thus transfected with a construct encoding myc-tagged Grb2, and its association with EGFR and Shc was used as a
readout of EGFR transactivation. As shown in Fig. 6F, LPA induced the coimmunoprecipitation of Grb2-myc with the EGFR and Shc,
and these associations were not modified in cells expressing ARK-CT.
Altogether, these data indicated that Gi and were involved in p110 activation but not in EGFR transactivation, suggesting that p110 and its downstream effectors provide a novel connection between and Ras.
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Fig. 6.
Role of Gi,
, and PKC in activation of Akt/PKB,
Ras, and EGFR. A, before stimulation cells were
incubated with pertussis toxin (+PTX, 100 nM,
16 h) when indicated, followed by the precipitation assay of
activated Ras. Throughout these experiments, the EGFR inhibitor AG1478
(+AG, 250 nM, 30 min) was used as a control,
whereas the PKC inhibitor Ro-31-8220 was used to investigate the role
of PKC in this pathway. The amount of activated endogenous Ras in the
pull-down assays was determined by immunoblotting (upper
panel). Lower panel: immunoblotting of cell lysates to
verify the amount of Ras in each sample. B, cells were
cotransfected with HA-tagged Ras and empty vector (V) or the
C-terminal domain of ARK carrying a T7 epitope tag
(T7-ARK). Cells were then stimulated and
subjected to GST-RBD precipitation. The amount of activated HA-Ras in
the pull-down assays was determined by anti-HA immunoblotting
(upper panel). Lysates were subjected to anti-HA
(middle panel) and anti-T7 immunoblotting (lower
panel) to verify expression of HA-Ras and T7-ARK. C,
the effect of pertussis toxin on the activation of endogenous Akt was
studied using a phosphospecific anti-Akt antibody (upper
panel). D, cells were cotransfected with HA-Akt and
either empty vector (V) or ARK-CT (T7-ARK).
Following stimulation, HA-Akt was immunoprecipitated and processed for
in vitro kinase assay. Radiolabeled histones
(H2B) were visualized using a PhosphorImager (upper
panel), and the amount of immunoprecipitated HA-Akt was verified
by anti-HA immunoblotting (middle panel). Lower
panel: expression of T7-ARK in the lysates. E, the
phosphorylation of EGFR in response to LPA was monitored in cell
lysates using a phosphospecific anti-EGFR antibody (upper
panel). F, cells were cotransfected with myc-tagged
Grb2 and either empty vector (V) or ARK
(T7-ARK). Following stimulation, Grb2-myc was
immunoprecipitated and samples were analyzed by anti-EGFR and anti-Shc
immunoblotting as indicated (two upper panels). Two
lower panels: Expression of Grb2-myc and T7-ARK in lysates.
G, cells were incubated with the following inhibitors, alone
or in combination as indicated: Ro-31-8220 (+Ro 10 µM, 30 min), wortmannin (+W, 100 nM, 30 min), pertussis toxin (+PTX, 100 nM, 16 h). Following stimulation with LPA, cells were
lysed in electrophoresis sample buffer and analyzed by anti-phospho-ERK
(upper panel) and anti-ERK2 (lower panel)
immunoblotting. The results displayed are representative of at least
two independent experiments performed in duplicate.
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As shown in Fig. 1, the PI3K-dependent pathway described
herein seems to account for about half of ERK activation induced by
LPA. As a preliminary study of the other half of this activation, we
have used Ro-31-8220, an inhibitor of all PKC family members, alone or
in combination with wortmannin or pertussis toxin. As shown in Fig.
6G, Ro-31-8220 partially reduced ERK phosphorylation induced
by LPA. In addition, when cells were treated with both Ro-31-8220 and
wortmannin, ERK phosphorylation was nearly abolished, suggesting that
PKC and PI3K acted on distinct pathways leading to MAPK activation. In
agreement with this, Ro-31-8220 displayed also an additive effect with
pertussis toxin that was shown to block the PI3K pathway at the level
of Gi (see Fig. 6C). Moreover, Ro-31-8220 did
not interfere with LPA-induced activation of both Akt/PKB and Ras (Fig.
6, A and C), further suggesting that PKC and the
Gi/p110/Ras pathway provide two distinct routes leading to ERK activation in response to LPA.
The ability of LPA to stimulate signaling pathways is thought to be
mediated through at least three different GPCR, namely LPA1
(Edg2), LPA2 (Edg4), and LPA3 (Edg7). As a
first approach to define their role in the pathway described herein, we
have studied their expression using RT-PCR analysis. As shown in Fig. 7, both LPA1 and
LPA3 could be amplified from Vero cell cDNA. However,
LPA1 was produced more readily, suggesting that it was expressed to a higher level. Because LPA1 and
LPA3 preferentially activate Gi and
Gq, respectively (6-8), these results suggested that
LPA1 might be involved in the Gi/p110/Ras
pathway whereas LPA3 could be responsible for the
PKC-dependent pathway leading to MAPK activation.
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Fig. 7.
RT-PCR analysis of LPA receptors in Vero
cells. RNA from Vero cells was extracted, purified, and
reverse-transcribed using random hexamers (hex) or oligo-dT
(dT) primers. cDNAs were then amplified by PCR using
specific primers for LPA1 (A), LPA2
(B), or LPA3 (C) and -actin as
control. Amplification of -actin and LPA1 was performed
with 2.5 units of Taq DNA polymerase, whereas
LPA2 and LPA3 were amplified with 5 units.
cDNAs from the human T-cell leukemia cell line Jurkat
(LPA1 and LPA2) and human fibroblast cell line
IMR-90 (LPA3) were used as positive controls.
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DISCUSSION |
Although PI3Ks are important effectors of LPA signaling,
their role in the MAPK pathway has remained somewhat elusive. p110 appeared to be the best candidate to participate in this pathway, but
the mechanisms connecting p110 to MAPK activation have been incompletely characterized. We report here a novel mechanism involving p110, a widely expressed PI3K, upstream of a complex signaling pathway leading to Ras activation in response to LPA.
The involvement of p110 in this pathway is based on the dominant
negative effects of three different PI3K mutants. First, p85, the
standard inhibitor of class IA PI3Ks, inhibited MAPK activation induced by LPA, which strongly suggested the involvement of
p110, because other class IA enzymes are not known to be
activated by GPCR stimulation. In agreement with this, a
kinase-inactive mutant of p110 catalytic subunit was found to
interfere with the stimulation of both Ras and MAPK, although less
efficiently than p85. This difference seems to be due to the
differential ability of p85 and p110 mutants to interfere with PI3K
stimulation, as shown by studying Akt/PKB activation. An explanation
for this latter observation could be that p110 mutants must displace a pre-existing complex between endogenous p85 and p110 to produce dominant negative effects, whereas p85 can act on its own. Moreover, through a different approach using antisense cDNA of p110, we have also observed that this protein was important for Ras activation in response to LPA. Finally, the fact that the PKO mutant also produced
a dominant negative effect on Ras activation further demonstrated the
involvement of p110.
The PKO mutant was produced to distinguish which of the protein kinase
or lipid kinase activities of p110 were involved in this pathway,
considering that each activity can potentially participate in MAPK
activation. However this mutant displayed a dominant negative activity
on Ras activation, suggesting that the lipid kinase activity of p110
was essentially involved. In addition, overexpression of the
phosphatase PTEN that degrades PI3K lipid products was found to
specifically interfere with Ras activation, further supporting a major
role for p110 lipid products in this pathway. Therefore, the
functional link between PI3K protein kinase activity and the MAPK
pathway remains thus far restricted to p110, although its protein
substrate remains to be identified.
Moreover, it seems that the notion that p110 is a major intermediate
between GPCR stimulation and the MAPK pathway must be somewhat
moderated. Although it has been reported that dominant negative p110
interfered with MAPK activation in COS or CHO cells stimulated with
LPA, we failed to reproduce this result in Vero cells. However, the
study in COS cells was performed with a very low dose of LPA (40 nM), suggesting that the role of p110 is limited to weak
stimulations of the MAPK pathway (34). A common theme may also be
evoked to explain the results in CHO cells, because the involvement of
p110 was essentially demonstrated in a particular cell line where
Ras activation had been compromised (33). This suggests that p110
provides in these cell lines a limited contribution to MAPK activation
that has been masked under our experimental conditions. Indeed, we have
studied the response of normal cells stimulated with a mitogenic dose
of LPA (10 µM) that engages at least two pathways,
i.e. PKC and Ras, leading to MAPK activation. In addition,
although p110 is abundant in neutrophils or platelets, its level of
expression is much lower in Vero and other adherent cell lines (COS,
Rat1, and NIH-3T3) (data not shown). We therefore assume that the
capacity of p110 to contribute significantly to the MAPK pathway,
and other cellular responses, might be restricted to hematopoietic
cells. In agreement with this, the phenotypes described to date in
p110 knockout mice are associated with blood cell functions
(52-55), taking into account that the role of p110 in colorectal
cancer has been recently challenged (56). Nevertheless, additional
studies are necessary to determine whether p110 is physiologically
important for MAPK activation in hematopoietic cells.
Several biochemical mechanisms have suggested a role for PI3K in the
MAPK pathway at the level of Raf or MEK, because these enzymes are
potential targets of kinases activated through
PI3K-dependent processes (10, 36, 57-59). However, we have
observed that PI3K can play an important role upstream from Ras in
cells stimulated with LPA. Indeed, the lipid products of p110 were
found to facilitate the recruitment of the docking protein Gab1 that
participates in Ras activation. Gab1 was thought to provide the
phosphotyrosine-binding motifs required for p110 activation, but we
have observed that it is also a downstream target of p110. We thus
propose the following model connecting LPA to Ras where Gab1 is
involved at two different steps (Fig. 8).
Following LPA stimulation, Gab1 is recruited to phosphorylated EGFR
through Grb2 or direct binding to the EGFR (49, 60). This induces the
phosphorylation of Gab1 on p85 and SHP2 binding sites, leading to
p110 activation in synergy with subunits released during
stimulation of LPA receptors. Activation of p110 produces
PtdIns(3,4,5)P3 that will recruit additional Gab1 molecules
in the vicinity of the EGFR through binding to its PH domain. We assume
that this mechanism provides a way to amplify the recruitment of p85
and SHP2, because the physical association of Gab1 with the EGFR is
limited by the low number of EGFR molecules that are phosphorylated in
response to LPA (data not shown). Downstream of Gab1, we have observed
that SHP2 could provide a link with Ras, although the nature of this connection remains obscure. It has been proposed that SHP2 can function
as an adapter protein in platelet-derived growth factor signaling,
because it can bind to both the receptor and Grb2, and therefore
contributes to the recruitment of Grb2-Sos (61). However, we have
observed that the catalytic activity of SHP2 was important for Ras
activation in response to LPA, in agreement with a recent report
showing that this phosphatase was necessary for MAPK activation in
HEK293 cells stimulated through LPA1 (62). This suggests
that SHP2 activates by dephosphorylation a protein involved in Ras
activation, or down-regulates an inhibitor of Ras. One possible
candidate could be the GTPase-activating proteins that promote Ras
deactivation. Interestingly, in U937 cells, PI3K was found to play a
permissive role in basal activation of Ras through inhibition of
GTPase-activating proteins (63). However, the mechanism of this
regulation is not known, and additional studies are required to define
whether Ras GTPase-activating proteins are a downstream target of
SHP2.
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Fig. 8.
Model outlining the role of
p110 between
and Ras. Following LPA stimulation, the EGFR is
transactivated and Gab1 is recruited through Grb2 or direct binding to
the EGFR. This induces Gab1 phosphorylation on p85 and SHP2 binding
sites, leading to p110 activation in synergy with subunits
released by stimulation of LPA receptors. Activation of p110
produces PtdIns(3,4,5)P3 that will recruit additional Gab1
molecules in the EGFR vicinity through binding to its PH domain. This
mechanism allows the amplification of the recruitment of p85 and SHP2,
because the physical association of Gab1 with the EGFR is limited by
the low number of EGFR molecules that are phosphorylated in response to
LPA. Downstream of Gab1, SHP2 dephosphorylates an unidentified
substrate that facilitates the activation of Ras.
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One important aspect of the pathway described herein is that it
provides a substantial connection between and the MAPK pathway.
Although it is well established that is important for Ras
activation, the effectors of this pathway have not been clearly
identified (9, 13). Interesting models have suggested that might
be involved in EGFR recruitment through -arrestin and Src (20, 21),
but we have observed that was required at the level of p110<
TOP
ABSTRACT
INTRODUCTION
