From INSERM U326, IFR 30, Hôpital Purpan,
Toulouse 31059, § INSERM U363, Hôpital Cochin, 27 rue
du Faubourg Saint-Jacques, Paris 75014,
CNRS UPR 1086, 1919 route de Mende, Montpellier 34293, France and the ¶ Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York 11724-2208
Received for publication, August 2, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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Although the mechanisms involved in the
activation of mitogen-activated protein kinases (MAPK) by receptor
tyrosine kinases do not display an obvious role for phosphoinositide
3-kinases (PI3Ks), we have observed in the nontransformed cell line
Vero stimulated with epidermal growth factor (EGF) that wortmannin and
LY294002 nearly abolished MAPK activation. The effect was observed
under strong stimulation and was independent of EGF concentration. In
addition, three mutants of class Ia PI3Ks were found to inhibit MAPK
activation to an extent similar to their effect on Akt/protein kinase B
activation. To determine the importance of PI3K lipid kinase activity
in MAPK activation, we have used the phosphatase PTEN and the
pleckstrin homology domain of Tec kinase. Overexpression of these
proteins, but not control mutants, was found to inhibit MAPK
activation, suggesting that the lipid products of class Ia PI3K are
necessary for MAPK signaling. We next investigated the location of PI3K
in the MAPK cascade. Pharmacological inhibitors and dominant negative
forms of PI3K were found to block the activation of Ras induced by EGF.
Upstream from Ras, although association of Grb2 with its conventional
effectors was independent of PI3K, we have observed that the
recruitment of the tyrosine phosphatase SHP2 required PI3K. Because
SHP2 was also essential for Ras activation, this suggested the
existence of a PI3K/SHP2 pathway leading to the activation of Ras. In
addition, we have observed that the docking protein Gab1, which is
involved in PI3K activation during EGF stimulation, is also implicated
in this pathway downstream of PI3K. Indeed, the association of Gab1
with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant
deficient for binding to SHP2 was found to inhibit Ras stimulation
without interfering with PI3K activation. These results show that, in
addition to Shc and Grb2, a PI3K-dependent pathway
involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.
The mitogen-activated protein kinases
(MAPK)1 extracellular
signal-regulated kinases (ERK) 1 and 2 transduce proliferative signals
to the nucleus (1). The mechanisms leading to their activation by
ligands of receptor tyrosine kinases appear well understood and the
GTPase Ras plays a central role (2). For example, epidermal growth
factor (EGF) activates its receptor tyrosine kinase, which
autophosphorylates, creating binding sites for SH2-domain containing
proteins, including the adapter proteins Shc and Grb2. In addition to
its SH2 domain, Grb2 binds through its SH3 domains to the guanine
nucleotide exchange factor Sos. Thus, the binding of Grb2 to
phosphorylated EGF receptor (EGFR) results in the recruitment of Sos to
the plasma membrane and has been proposed as a model for activation of
membrane-bound Ras (3). In addition, EGF-induced activation of Ras may
be transduced via Shc, which binds to activated EGFR and becomes
phosphorylated, creating an additional binding site for Grb2 (4). Once
Ras has been activated by Sos, GTP-bound Ras stimulates the
serine/threonine kinase Raf. Activated Raf stimulates the downstream
kinase MAPK/ERK kinase (MEK), which in turn phosphorylates ERK. In
addition, activation of ERK under EGF stimulation can be mediated by
Ras-independent pathways, through protein kinase C (PKC) and
calcium-mediated mechanisms (5).
The phosphoinositide 3-kinases (PI3Ks) also transduce proliferative
signals. PI3Ks phosphorylate phosphoinositides at the 3'-position of
the inositol ring, and their major lipid product is
phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is
produced during cell stimulation by various mitogens (6, 7). Three classes of PI3Ks have been defined, and class I enzymes are involved in
mitogen signaling. The members of the subclass Ia include the catalytic
subunits p110 Although the model for growth factor-induced MAPK activation described
above does not show an obvious role for PI3K, many reports have
documented inhibition of MAPK activation by pharmacological inhibitors
of PI3K (14-16). However, recent data have suggested that the role of
PI3K depends on signal strength and is in fact limited to weak
activations. In Swiss 3T3 cells, PI3K was found to be required during
stimulation induced by low, but not high, doses of platelet-derived
growth factor (17). In insulin-treated Chinese hamster ovary cells, the
requirement for PI3K was reported to depend on the number of insulin
receptors expressed on the cell surface (17). Similarly, in COS cells,
pharmacological inhibitors of PI3K were found to inhibit MAPK
activation induced only by low doses of EGF (18). On a molecular point
of view, two major mechanisms have been proposed to illustrate a
possible involvement of PI3K in MAPK activation. One involves the
ability of PIP3 to activate members of the PKC family,
directly or via the phosphoinositide-dependent kinases (19,
20). Activated PKC can then stimulate Raf (21). The second involves the
ability of p21-activated kinase, a downstream target of PI3K via Rac, to promote stimulation of Raf and MEK (22, 23).
In the nontransformed cell line Vero, we have observed that various
PI3K inhibitors block MAPK activation induced by EGF, independently of
signal strength. This led us to show that a PI3K-dependent pathway involving Gab1 and SHP2 is required, in addition to Shc and
Grb2, for the activation of Ras by EGF.
Materials--
Human recombinant EGF was from Calbiochem.
Polyclonal antibodies against Grb2, SHP2, and EGFR and monoclonal
anti-Myc were from Santa Cruz Biotechnology. Polyclonal antibodies
against Gab1, Sos, Shc, p85, and monoclonal anti-phosphotyrosine (4G10)
were from Upstate Biotechnology Inc. Anti-phospho-ERK antibody was from
Promega. 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 Life Technologies, Inc.
Cell Culture, Transfection, and Stimulations--
Vero cells (a
monkey kidney cell line, ATCC CCL 81) were maintained in Dulbecco's
modified Eagle's 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's medium
containing 2 µg of total DNA, 6 µl of LipofectAMINE, and 6 µ of
Plus reagent (Life Technologies, Inc.). Before stimulation,
cells were blocked overnight by serum starvation. Unless otherwise
indicated, cells were stimulated for 5 min with 10 ng/ml EGF. Before
stimulation, cells were incubated for 15 min with 100 nM
wortmannin (Sigma Chemical Co.) or 25 µM LY294002 (BioMol) where indicated.
Cell Lysis, Immunoprecipitation, and Immunoblotting--
Cells
were scrapped off in lysis buffer containing 20 mM Tris, pH
7.4, 150 mM NaCl, 10 mM EDTA, 10% glycerol,
1% Nonidet P-40, 10 µg/ml of each aprotinin and leupeptin, and 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 collected with
protein A- or protein G-Sepharose (Sigma) for 1 h and washed three
times with lysis buffer. Blots were developed using chemiluminescence
(Amersham Pharmacia Biotech).
Preparation of Expression Plasmids--
A plasmid encoding wild
type HA-tagged p110 In Vitro Kinase Assays--
To measure activation of Akt/PKB,
cells were cotransfected with 0.5 and 1.5 µg of DNA encoding
HA-tagged Akt/PKB and the indicated effector, respectively. After
stimulation, cells were scrapped off in lysis buffer, then subjected to
HA immunoprecipitation using 12CA5 antibody (Roche Diagnostics).
Immunoprecipitates were washed with lysis buffer, then with kinase
buffer containing 50 mM Tris, pH 7.5, 10 mM
MgCl2, 1 mM dithiothreitol and incubated with
Histone 2B (Roche Diagnostics) and [ Activated Ras Affinity Precipitation Assay--
The assay was
performed essentially as described previously (28). The recombinant
Ras-binding domain (RBD) of Raf1 (kindly provided by Dr. F. R. McKenzie, Nice, France) was expressed as GST fusion protein in
Escherichia coli and extracted using glutathione-Sepharose beads. To measure Ras activation, 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% sodium
desoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM
phenylmethylsulfonyl fluoride, and 1 µg/ml each of 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 and then boiled, and proteins were resolved
by SDS-PAGE. Immunoblotting was performed with anti-pan Ras antibodies.
To study the activation of Ras in transfected cells, cells were
cotransfected with 1 µg of each plasmid encoding HA-tagged wild type
Ras (kindly provided by Dr. B. M. Burgering, Utrecht, The
Netherlands) and the indicated effector. 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 as
described (29). Briefly, cells were scrapped off in hypotonic lysis
buffer then Dounce-homogenized. The homogenate was centrifuged at
100,000 × g for 1 h. The pellet was dissolved in
1% Triton X-100 lysis buffer, and the insoluble material was spun out.
This was taken as the solubilized membrane fraction.
PI3K Is Required for EGF-induced MAPK Activation, Independently of
Signal Strength--
By using anti-phospho-ERK immunoblotting, we have
observed in Vero cells that the phosphorylation of ERK2 induced by 10 ng/ml EGF is abolished when cells are preincubated with wortmannin or LY2940002 (Fig. 1A). Because
the role of PI3K in ERK activation is thought to be limited to weak
stimulations, we have measured the activation of transfected ERK1-His
using an in vitro kinase assay. As shown in Fig.
1B, treatment with 10 ng/ml EGF induced a >10-fold increase
in ERK1-His activity, in agreement with the strong phosphorylation of
endogenous ERK2 observed using anti-phospho-ERK immunoblotting.
Preincubation of the cells with PI3K inhibitors reduced by over 80%
the activation of purified ERK1-His. In addition, the requirement for
PI3K appeared independent of EGF concentration, because wortmannin
treatment also inhibited the phosphorylation of endogenous ERK2 induced
by 30 or 50 ng/ml EGF (Fig. 1C),
To confirm these data obtained with pharmacological inhibitors, we have
studied the ability of three PI3K mutants to inhibit ERK1-His
activation in cotransfection experiments. In parallel, we have
determined their efficiency to inhibit PI3K signaling by measuring
their ability to interfere with the activation of Akt/PKB. This was
achieved in cotransfection experiments with HA-Akt/PKB, followed by an
in vitro kinase assay. First, cells were transfected with a
form of p85 PIP3 Is Essential for MAPK Activation--
One important question
regarding the involvement of PI3Ks in MAPK activation is the respective
role of their lipid kinase and protein kinase activities, because
PIP3 has been shown not to be necessary for
p110 PI3K Is Required Upstream of Ras and Sos--
We next investigated
the location of PI3K function in the MAPK pathway. Considering that
PI3K-dependent mechanisms have been proposed at the level
of Raf and MEK (19-23), and that MAPK activation by EGF can occur via
Ras-independent pathways (5), we have first examined the role of Ras in
Vero cells. Expression of dominant negative RasN17 was found to abolish
ERK1-His activation induced by EGF (data not shown), indicating that
MAPK activation is strictly dependent on Ras in Vero cells. Because
MAPK activation also requires PI3K, we have determined whether PI3K was
involved in the activation of Ras. This was achieved using a
precipitation assay for activated Ras. Following cell stimulation and
lysis, endogenous activated Ras was extracted using a GST fusion
protein containing the Ras-binding domain of Raf (RBD), which interacts
specifically with GTP-bound Ras (28). The amount of activated Ras in
the GST-RBD pull-down assays was determined by anti-Ras immunoblotting.
As shown in Fig. 4A, treatment
of Vero cells with 10 ng/ml EGF induced the coprecipitation of Ras with
GST-RBD, and preincubation of the cells with wortmannin or LY294002
strongly inhibited this association, suggesting that PI3K is involved
in Ras activation. To confirm this hypothesis, the activation of
HA-tagged Ras was studied in cells cotransfected with dominant negative
PI3Ks. As shown in Fig. 4B, expression of
Upstream from Ras, membrane translocation of Sos is thought to be the
limiting event for Ras activation. To determine whether PI3K was
involved in Sos redistribution, we have prepared membrane fractions
from EGF-treated cells preincubated or not with PI3K inhibitors. As
shown in Fig. 4C, the membrane enrichment of Sos induced by
EGF was nearly abolished by PI3K inhibitors. In addition, overexpression of Sos was found to partially overcome the need for PI3K
in ERK activation without increasing the basal activation of ERK1-His
(Fig. 4D). This suggested that PI3K was involved at the
level or upstream of Sos. However, expression of constitutively activated PI3K (p110 SHP2 and Ras Are Activated Downstream of PI3K and Gab1--
To
identify a PI3K-dependent event upstream from Sos, we have
analyzed by immunoblotting the proteins coimmunoprecipitated with Grb2.
As expected, Fig. 5A shows
that the EGFR and Shc readily precipitated with Grb2 upon EGF
stimulation and this was not modified by PI3K inhibitors. In addition,
PI3K inhibitors did not influence the constitutive association of Grb2
with Sos (data not shown). In contrast, the coimmunoprecipitation of
the tyrosine phosphatase SHP2 with Grb2 was found to depend on PI3K
(Fig. 5A). In agreement with this, the recruitment of SHP2
in anti-phosphotyrosine immunoprecipitates upon EGF stimulation was
also reduced by PI3K inhibitors (Fig. 5B), suggesting that
SHP2 is a downstream effector of PI3K in EGF signaling. To determine
whether SHP2 was important for Ras activation in Vero cells, we have
transfected a catalytically inactive mutant of SHP2 (SHP2-C/S)
and analyzed the activation of HA-tagged Ras in cotransfection
experiments. As shown in Fig. 5C, expression of SHP2- C/S
completely inhibited the precipitation of HA-Ras with GST-RBD, whereas
wild type SHP2 had no effect. This indicated that the catalytic
activity of SHP2 is involved in the activation of Ras and suggested the
existence of a PI3K/SHP2 pathway leading to Ras stimulation because
SHP2 is recruited downstream of PI3K. We next attempted to identify a
substrate of SHP2 involved in the activation of Ras using the SHP2-C/S
mutant in a "substrate trapping" experiment. Cells transfected with
SHP2-C/S, or wild type SHP2 as a control, were stimulated with EGF,
subjected to SHP2 immunoprecipitation, then anti-phosphotyrosine
immunoblotting. As shown in Fig. 5D, both wild type SHP2 and
SHP2-C/S coimmunoprecipitated with three phosphoproteins of apparent
molecular masses of around 180, 115, and 100 kDa, whereas only
the SHP2-C/S mutant associated with a protein of approximately 135 kDa.
Upon reblotting, the 180-kDa protein comigrated with the EGFR and the
115-kDa hyperphosphorylated protein comigrated with the docking protein
Gab1 (data not shown). We attempted to identify the ~135-kDa protein
using antibodies directed against Ras effectors, including
Ras-GTPase-activating protein and Sos2, but we failed to label this
protein (data not shown).
These results also suggested that the docking protein Gab1 could
mediate the PI3K-dependent recruitment of SHP2 and the
subsequent activation of Ras. Gab1 sequence displays three binding
sites for p85 and is thought to mediate the activation of PI3K during EGF stimulation (34). In addition, Gab1 contains one binding site for
SHP2 and a PIP3-specific PH domain (35, 36). However, the
role of this PH domain in EGF signaling is not clear (37), and it is
not known if the interaction of Gab1 with SHP2 requires PI3K. As shown
in Fig. 6 (A and
B), EGF induced the coimmunoprecipitation of Gab1 with SHP2,
and this interaction was reduced by PI3K inhibitors. In addition, PI3K
inhibitors nearly abolished the tyrosine phosphorylation of Gab1
induced by EGF (Fig. 6B). These data strongly suggested that
Gab1 mediates the PI3K-dependent recruitment of SHP2.
However, it is not known if Gab1 is important for Ras activation. To
examine this question, we have produced Gab1 mutants deficient for
binding to p85 or SHP2 and tested their effect on the activation of
HA-Ras in cotransfection experiments. As shown in Fig. 6C,
expression of Gab1-YF3 lacking the three p85 binding sites strongly
inhibited the binding of HA-Ras to GST-RBD, and transfection of
Gab1-Y627F deficient for SHP2 binding also blocked this association.
This indicated that interaction of Gab1 with p85 and SHP2 is required for Ras activation. To determine whether Gab1 mutants blocked the
pathway upstream or downstream of PI3K, we have studied their effect on
the activation of HA-Akt/PKB. As shown in Fig. 6D, this activation was not impaired in cells transfected with Gab1-Y627F in
comparison with cells expressing wild type Gab1. In contrast, activation of HA-Akt/PKB was strongly inhibited when Gab1-YF3 was
expressed. In addition, Gab1-Y627F did not bind less p85 than wild type
Gab1 in coimmunoprecipitation experiments (Fig. 6E). This
demonstrated that Gab1-YF3 blocked the activation of Ras upstream of
PI3K, whereas Gab1-Y627F interfered with Ras activation without
preventing PI3K activation. Altogether, these results show that, in
addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 participates to the activation of Ras under EGF
stimulation.
These data also suggested that the major function of PIP3
in the activation of Ras is to promote the recruitment of Gab1 in EGF
signaling, leading to the recruitment of SHP2. However, Gab1 can
associate to the activated EGFR through Grb2 (34) and directly through
the Met-binding domain (MBD), which constitutes a novel phosphotyrosine-binding motif (36, 38). We have thus further examined
the role of PIP3 in Gab1 recruitment by preparing a mutant deleted of the PH domain (Gab1-
As a first approach to answer this question, we have studied the
ability of Gab1- Although the mechanisms involved in growth factor-induced
activation of MAPK do not display an obvious role for PI3K,
pharmacological inhibitors of PI3K were found to strongly interfere
with MAPK activation in Vero cells stimulated with EGF. In agreement
with this, expression of mutants of class Ia PI3K inhibited the
activation of MAPK to an extent similar to their effect on the
activation of Akt/PKB, a major effector of PI3K. Moreover, the
requirement for PI3K was observed under strong activation and
independently of EGF concentration, which indicated that this enzyme
can play an important function in the mechanisms leading to MAPK activation.
We have first studied the role of PI3K lipid products in this pathway,
because it has been reported that only the protein kinase activity of
p110 Based on biochemical approaches, multiple reports have suggested an
involvement of PI3K in MAPK activation downstream of Ras, considering
that the activation of Raf or MEK can be mediated by targets of PI3K
signaling (19-23). In contrast, we have observed using inhibitors and
dominant negative mutants that PI3K has a function upstream of Ras
during EGF stimulation. PI3K was also found to be necessary for the
redistribution of Sos, but expression of constitutively activated PI3K
was not sufficient to activate MAPK. This suggested that
PIP3 cannot directly induce the membrane translocation of
Sos, although Sos contains a PH domain that has some affinity for this
lipid (33). Nevertheless, we cannot exclude that PIP3
participates in Sos redistribution by stabilizing its interaction with
the plasma membrane. In addition, although PI3K does not seem to
contribute to the formation of the complex between Grb2 and the EGFR,
Shc, or Sos, we have observed that the recruitment of SHP2 in this
complex was dependent on PI3K. Because the activation of Ras in
response to EGF was also strongly dependent on SHP2, this suggested the
existence of a PI3K/SHP2 pathway that is important, in addition to Shc
and Grb2, for the activation of Ras.
The docking protein Gab1 was found to participate in the process at two
different levels (Fig. 8). First,
we have verified that Gab1 is effectively involved in PI3K activation
during EGF stimulation, because expression of Gab1 deficient for p85
binding sites abolished the activation of Akt/PKB. Second, Gab1
mediates the recruitment of SHP2 downstream of PI3K and the subsequent activation of Ras. Indeed, the phosphorylation of Gab1 and its association with SHP2 induced by EGF were blocked by PI3K inhibitors, and disruption of its SHP2 binding site suppressed the stimulation of
Ras without interfering with PI3K activation. In addition, we have
observed that the PH domain is important for efficient phosphorylation
of Gab1 and binding to SHP2. Therefore, these data support the notion
that PIP3 is essential for the recruitment of Gab1 in EGF
signaling, as suggested by the fact that Gab1 contains a PH domain that
binds specifically PIP3 (35, 36). Yet, one may wonder what
precise role PIP3 plays, because Gab1 can associate to the
phosphorylated EGFR through the MBD and indirectly through Grb2 (34,
36). PIP3 could simply stabilize the association of Gab1
with the EGFR, leading to an enhanced phosphorylation of Gab1. However,
the results obtained with Gab1-
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ABSTRACT
INTRODUCTION
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, p110
, and p110
associated with a regulatory subunit p85 and activated through protein tyrosine kinases, and subclass Ib is represented by p110
, which is activated by
heterotrimeric G proteins. On a functional point of view, subclass Ia
PI3Ks are required for growth factor-induced mitogenesis (8, 9), and it
was recently reported that embryos of p110
knockout mice die at
early age due to a profound proliferative defect (10). The mechanisms
by which PI3Ks activate signaling pathways have been recently
unraveled. PIP3 has binding affinity for a conserved peptidic sequence called the pleckstrin homology (PH) domain, thereby
inducing the localization of PH-domain-containing proteins to
membrane-associated signaling complexes (6). Several targets for PI3K
lipid products have been proposed, including the proto-oncogene product
Akt/protein kinase B (PKB) and its upstream activators, the
phosphoinositide-dependent kinases. These kinases activate various enzymes that are important for cell growth, including the
p70-S6 kinase and the glycogen synthase kinase 3 (6, 7). In addition,
the catalytic subunits of PI3Ks possess an intrinsic protein kinase
activity, which is involved in the down-regulation of their lipid
kinase activity (11, 12). Interestingly, at least in the case of
p110
, this protein kinase activity has been reported to participate
to MAPK signaling, whereas its lipid kinase activity appeared not to be
necessary (13).
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was kindly provided by Drs. P. Hu and J. Schlessinger (New York University) (24). The kinase-inactive K805R
mutant of p110
was obtained by replacing with arginine the lysine
805 present in the putative ATP binding site using site-directed
mutagenesis (QuikChange, Stratagene) with the following mutagenic
primer: 5'-GTTGGAGTGATTTTTAGAAATGGTGATGATTTACG-3' (the
changed nucleotide is underlined). The cDNA of Tec was cloned using
RT-PCR (Superscript II, Life Technologies) from poly(A)+ RNA purified
from the megakaryoblastic cell line Dami and inserted into pET21
(Novagen) to introduce a N-terminal T7 epitope tag. The cDNA of
T7-Tec was then subcloned into pCI-neo (Promega) for mammalian
expression. A construct encoding the N-terminal PH domain was obtained
by deleting the fragment between the two StuI internal sites
which encompasses the SH3, SH2 and kinase domains. The R29C mutant was
obtained using the following mutagenic primer:
5'-CGCCCTTAAACTACAAAGAGTGCCTTTTTGTACTTACAAAGTCC-3'. The
cDNA of Gab1 was kindly provided by Dr. A. Ullrich (Max Planck Institute, Germany). To generate the mutant of Gab1 defective for p85
binding (YF3), tyrosines 447, 472, and 589 were replaced with
phenylalanines using the following primers, respectively: 5'-CTGGATGAAAATTTCGTCCCAATGAATC-3';
5'-CAGGAAGCAAATTTTGTGCCAATGACTC-3'; 5'-CAGTGAAGAGAATTTTGTTCCCATGAACC-3'. The SHP2 binding site
of Gab1 was mutated by replacing tyrosine 627 with phenylalanine using
the following primer:
5'-GGAGACAAACAGGTGGAATTCTTAGATCTCGACTTAGA-3'. Gab1 deleted of the PH domain was generated by polymerase chain reaction amplification of the cDNA sequence encoding amino acids 104-694. In this mutant, translation is initiated on the second methionine at position 104. All Gab1 constructs have a C-terminal Myc
tag. In addition, HA-tagged Gab1 was obtained by subcloning the entire
coding sequence of wild type Gab1 into pcDNA-HA using polymerase
chain reaction amplification. All the mutations were verified by
sequencing. A construct encoding His/Myc-tagged ERK1 was obtained by
subcloning ERK1 (kindly provided by Dr. E. vanObberghen, Nice, France)
into pcDNA3.1-MycHis (Invitrogen). The constructs encoding
HA-tagged PTEN and its G129E mutant have been already described
(25). The vectors encoding
p85 and p110
-K802R mutants were kindly
provided by Drs. W. Ogawa (University of Kobe, Japan) and M. Wymann
(University of Fribourg, Switzerland), respectively. The plasmids
encoding SHP2 and its catalytically inactive mutant (C/S) have been
kindly provided by Dr. N. Rivard (Sherbrooke University, Canada).
-32P]ATP as
described previously (26). To measure MAPK activation, cells were
transfected with 1 µg of each DNA 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), then washed with
lysis buffer supplemented with 5 mM imidazole, followed by
washes with kinase buffer. Phosphorylation of myelin basic protein
(MBP) was performed as described (27). Reactions were stopped by
addition of Laemmli sample buffer and analyzed by SDS-PAGE.
Phosphorylation of MBP and histones was quantified using a PhosphorImager.
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ABSTRACT
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View larger version (28K):
[in a new window]
Fig. 1.
PI3K inhibitors block ERK activation under
strong stimulation and independently of EGF concentration.
A, serum-starved Vero cells were incubated 15 min with 100 nM wortmannin (W) or 20 µM
LY294002 (LY) when indicated before a 5-min stimulation with
10 ng/ml EGF. Cell lysates were analyzed by anti-phospho-Erk
(upper panel) and anti-ERK2 (lower panel)
immunoblotting (IB). B, cells transfected with
ERK1-His were incubated with PI3K inhibitors as indicated before
stimulation with EGF. Following extraction, ERK1-His was incubated with
myelin basic protein (MBP) and [ -32P]ATP.
The reaction was analyzed using a PhosphorImager (upper
panel) and anti-His immunoblotting (lower panel).
Bottom graph, mean ± S.E. of MBP phosphorylation from
three experiments. C, Vero cells were preincubated with
wortmannin when indicated (Wort) then stimulated with
increasing EGF concentrations. The phosphorylation of endogenous ERK in
the lysates was determined by immunoblotting (upper panel).
Lysates were also subjected to anti-ERK2 immunoblotting (lower
panel).
lacking the p110 binding site (
p85), which is a
widely used dominant negative mutant for class Ia PI3Ks (30). As shown
in Fig. 2A, expression of
p85 almost completely inhibited the activation of ERK1-His induced by EGF. Because
p85 contains SH2 and SH3 domains that might
interfere with MAPK activation, we have tested catalytically inactive
mutants of p110
(
K802R) and p110
(
K805R) subunits.
Expression of these mutants was found to produce a partial inhibition
of ERK1-His activation, the p110
construct being more efficient than
p110
. However, these mutants also inhibited partially the activation of HA-Akt/PKB, and the p110
construct was more efficient (Fig. 2B). Thus, the ability of the PI3K mutants to inhibit MAPK
activation was correlated to their capacity to interfere with PI3K
signaling. Taken together, these results indicate that, in Vero cells
stimulated with EGF, PI3K plays an essential role in MAPK
activation.
View larger version (19K):
[in a new window]
Fig. 2.
Inhibition of ERK1-His and HA-Akt/PKB
activation by expression of PI3K mutants. A, cells were
cotransfected with ERK1-His and one of the indicated constructs: empty
vector (V); dominant negative p85 ( p85);
catalytically inactive p110
(
K802R);
catalytically inactive p110
(
K805R). After
cell stimulation and lysis, the activation of purified ERK1-His was
determined by in vitro kinase assay. Right
panels, expression of PI3K mutants was verified in lysates from
cells transfected with the indicated construct. B, cells
were cotransfected with HA-tagged Akt/PKB and the same constructs as in
A. Following cell stimulation, HA-Akt/PKB was
immunoprecipitated and incubated with histones (H2B) and
[
-32P]ATP. Phosphorylation of histones was revealed
using a PhosphorImager. The bottom graph represents the
mean ± S.E. of three independent experiments.
-mediated activation of MAPK (13). To define the importance of
PI3K lipid products in EGF signaling, we have taken advantage of PTEN,
a protein phosphatase that is also capable of dephosphorylating PI3Ks
lipid products (25). As shown in Fig. 3,
overexpression of PTEN inhibits ERK1-His activation induced by EGF. To
determine whether the PTEN effect is due to its protein or lipid
phosphatase activity, we have used as a control the "protein
phosphatase only" mutant of PTEN (G129E), which does not interfere
with PI3K signaling (25, 31). Transfection of PTEN-G129E did not
significantly inhibit ERK1-His activation, whereas this mutant was
somewhat more expressed than wild type PTEN (Fig. 3). To confirm the
importance of PI3K lipid products, we have used the PH domain of Tec as
a competitor for binding to PIP3. Tec belongs to a family
of tyrosine kinases containing a PIP3-sensitive PH domain
(32, 33). As shown in Fig. 3, expression of the Tec PH domain
significantly inhibited ERK1-His activation induced by EGF. As a
control, we have used the Tec PH domain containing the R29C mutation,
which decreases the affinity of Tec kinases for PIP3 (32,
33). This mutant did not significantly inhibit ERK1-His activation.
Altogether, these results suggested that PIP3 produced
during EGF stimulation is necessary for MAPK activation.
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Fig. 3.
MAPK inhibition in cells overexpressing PTEN
and Tec PH domain. Cells were cotransfected with ERK1-His and the
plasmids encoding the indicated proteins: empty vector (V);
wild type PTEN; PTEN-G129E mutant; Tec PH domain; Tec PH domain
carrying the R29C mutation. After stimulation with EGF, ERK1-His was
extracted and its activation was measured as above. Data shown
represent the mean ± S.E. from three independent experiments. *,
less than control; ns, not significant; p < 0.05, paired t test. Cell lysates were subjected to
immunoblot analysis with anti-HA or anti-T7 antibodies to verify the
expression level of the transfected proteins (bottom
panels).
p85 or
p110
-K805R inhibited the amount of HA-Ras associated with the
GST-RBD protein. Taken together, these results indicate that PI3K plays
a critical role in the activation of Ras induced by EGF.
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Fig. 4.
Role of PI3K in the activation of Ras.
A, Cells treated with wortmannin (W) or LY294002
(LY) when indicated were stimulated with EGF then lysed.
Cleared lysates were incubated with glutathione-Sepharose beads bound
to a GST fusion protein containing the Ras-binding domain of Raf
(GST-RBD). The beads were then washed and proteins resolved by
SDS-PAGE. 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). As a control, the pull-down
assay was also performed from cells transfected with constitutively
activated RasV12 (right lane). B, cells were
cotransfected with HA-tagged Ras and the indicated constructs: empty
vector (V); dominant negative p85
( p85); catalytically inactive p110
(
K805R). Following stimulation, cells were
lysed and incubated with GST-RBD beads. The amount of HA-Ras associated
with the beads was determined by anti-HA immunoblotting. The
immunoblots shown are representative of at least two independent
experiments. C, membrane fractions from control or
EGF-treated cells were prepared by ultracentrifugation and analyzed by
anti-Sos immunoblotting (upper panel), followed by anti-EGFR
immunoblotting to verify that an equal amount of fractions were loaded
on the gel (lower panel). D, cells were
cotransfected with ERK1-His and a construct encoding wild type Sos or
empty vector (V) as indicated. Following treatment or not
with wortmannin (W) and stimulation with EGF as indicated,
measurements of ERK1-His activation were performed. *, different than
empty vector; p < 0.05, paired t test.
Right panel, cell lysates were subjected to Sos
immunoblotting to verify expression of transfected Sos.
-CAAX) was not sufficient to
activate MAPK in unstimulated cells (data not shown). This indicated
that PIP3 cannot directly induce the redistribution of Sos,
suggesting the existence of a PIP3-dependent
mechanism upstream from Sos.
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Fig. 5.
SHP2 is recruited downstream of PI3K and is
essential for Ras activation. A, anti-Grb2
immunoprecipitation was performed from cells treated with PI3K
inhibitors when indicated (W, wortmannin; LY,
LY294002) and stimulated, or not (Ctrl), with EGF.
Immunoprecipitates were analyzed by immunoblotting with the indicated
antibodies. As a control, the immunoprecipitation was performed without
adding Grb2 antibody ( Ab). B,
anti-phosphotyrosine immunoprecipitations were performed from cells
treated with PI3K inhibitors when indicated and stimulated with EGF.
Immunoprecipitates were then analyzed by anti-SHP2 (upper
panel) and anti-EGFR (lower panel) immunoblotting.
C, cells were cotransfected with HA-tagged Ras and the
indicated constructs: empty vector (V); catalytically
inactive SHP2 (C/S); wild type (wt) SHP2. Cells
were then stimulated and incubated with GST-RBD beads to measure the
activation of HA-Ras. The amount of HA-Ras associated with the beads
was determined by anti-HA immunoblotting (upper panel). As a
control, one pull-down assay was performed from a lysate of stimulated
cells incubated with GST alone (lane
RBD). Lower
panel, lysates were subjected to anti-HA immunoblotting to verify
that equal amounts of HA-Ras were present in each sample. D,
cells were transfected with wild type (wt) or C/S SHP2 as
indicated, before stimulation or not (ctrl) with EGF. The
cells were then subjected to anti-SHP2 immunoprecipitation, followed by
anti-phosphotyrosine (upper panel) and anti-SHP2
(lower panel) immunoblotting. The immunoblots shown in this
figure are representative of at least two independent experiments
performed in duplicate.
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Fig. 6.
Role of Gab1 in the recruitment of SHP2 and
Ras activation. Vero cells were preincubated with PI3K inhibitors
when indicated before stimulation with EGF. Cell lysates were then
subjected to SHP2 (A) or Gab1 (B)
immunoprecipitation. Immunoprecipitates were analyzed by anti-Gab1,
anti-SHP2, and antiphosphotyrosine immunoblotting as indicated.
C, cells were cotransfected with HA-tagged Ras and the
indicated construct: empty vector (V); wild type
(wt) Gab1-Myc; Gab1-Myc mutated on its three p85 binding
sites (YF3); Gab1-Myc mutated on its SHP2 binding site
(Y627F). Following stimulation, cells were lysed and
incubated with GST-RBD beads. The amount of HA-Ras precipitated with
the beads was determined by anti-HA immunoblotting. Lysates were also
directly subjected to anti-HA (middle panel) and anti-Myc
immunoblotting (lower panel) to verify the expression of
HA-Ras and Gab1-Myc constructs. D, cells were cotransfected
with HA-Akt and the indicated Gab1 constructs. Following cell
stimulation, activation of HA-Akt was determined using in
vitro kinase assay. E, cells expressing the indicated
Gab1-Myc constructs were treated or not with EGF and subjected to
anti-Myc immunoprecipitation followed by immunoblotting analysis using
the indicated antibodies.
PH). As expected, this mutant has
lost the ability to associate with membrane fractions in a PI3K-dependent manner (Fig.
7A). We next studied its
involvement in EGF signaling. As shown in Fig. 7B, the
phosphorylation of Gab1-
PH and its coimmunoprecipitation with SHP2
were reduced in comparison to Gab1-wt, and these events were
insensitive to wortmannin. This indicates that the PH domain is
important for the recruitment of Gab1 in EGF signaling. However, it is
not clear if the interaction between the PH domain and PIP3
simply stabilizes the association of Gab1 with the EGFR, or whether
PIP3 promotes the recruitment of additional Gab1 molecules
in the vicinity of the receptor.
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Fig. 7.
Role of the PH domain in the recruitment of
Gab1. A and B, cells were transfected with
Gab1-Myc containing (wt) or not
( PH) the PH domain before preincubation with
wortmannin (+W) and stimulation with EGF when indicated.
Following cell lysis, membrane fractions were prepared by
ultracentrifugation and analyzed by anti-Myc immunoblotting (A,
upper panel), followed by anti-EGFR immunoblotting to control gel
loading (A, lower panel). B, lysates were
subjected to anti-Myc immunoprecipitation, followed by immunoblotting
with the indicated antibodies. C, cells were cotransfected
with ERK1-His and the following constructs: empty vector
(V); wild type Gab1-Myc (wt); Gab1-Myc deleted of
the PH domain (
PH); Gab1-Myc mutated on its
SHP2 binding site (Y627F). After stimulation with EGF,
ERK1-His was extracted, and its activation was measured using an
in vitro kinase assay with MBP. Data shown represent the
mean ± S.E. from three independent experiments. D,
cells were cotransfected with 0.1 µg of DNA encoding HA-tagged wild
type Gab1 and 2 µg of the indicated Gab1-Myc constructs. After
stimulation, cells were processed for anti-HA immunoprecipitation
followed by immunoblotting with the indicated antibodies. Lysates were
also directly subjected to anti-Myc immunoblotting to verify the
expression level of the Gab1-Myc constructs (bottom
panel).
PH to interfere with EGF signaling. This was
achieved by measuring MAPK activation in cotransfection experiments. Fig. 7C shows that Gab1-
PH did not significantly modify
Erk1-His activation. As a control, transfection of the Gab1-Y627F
mutant that inhibited the activation of Ras (Fig. 6C) was
found to reduce by more than 70% MAPK activation (Fig. 7C).
Because Gab1-
PH is less phosphorylated than Gab1-wt (Fig.
7B), the fact that Gab1-
PH did not inhibit EGF signaling
suggested that this mutant, albeit overexpressed, could not prevent the
recruitment of endogenous wild type (wt) Gab1. To test this hypothesis,
we have determined whether overexpression of Gab1-
PH interferes or
not with the phosphorylation of Gab1-wt induced by EGF. This was
achieved by studying the phosphorylation of HA-tagged Gab1-wt
cotransfected with Gab1-
PH in a 1:20 ratio. As shown in Fig.
7D, overexpression of Gab1-
PH did not modify the
phosphorylation of HA-Gab1-wt. As a control, overexpression of other
Gab1 constructs (Gab1-Myc-wt or Gab1-Myc-Y627F) strongly inhibited the
phosphorylation of HA-Gab1-wt. These results indicate that Gab1-
PH
cannot prevent the phosphorylation of Gab1-wt by the EGFR, suggesting
that PIP3 alone is sufficient to recruit Gab1 in the
vicinity of the receptor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is necessary for MAPK activation (13). We have shown that
overexpression of two proteins interfering with PIP3
impaired MAPK activation, suggesting that, in contrast to p110
, the
lipid kinase activity of class Ia PI3Ks is essential for MAPK
signaling. PTEN has already been shown to interfere with growth
factor-induced MAPK activation, but it has been proposed that this
effect was due to its ability to dephosphorylate proteins potentially
involved in MAPK signaling, including the Fak kinase and Shc (39, 40).
We have thus used as a control the G129E mutant of PTEN, which retains
the catalytic activity but has lost its ability to interact with
phosphoinositides and, consequently, does not interfere with PI3K
signaling (25, 31). In our model, this mutant was not active on MAPK
signaling, suggesting that the lipid phosphatase activity of PTEN is
primarily responsible for MAPK inhibition. In agreement with this,
overexpression of the PH domain of Tec also inhibited MAPK, whereas
mutation of an amino acid residue involved in PIP3 binding
produced an inactive protein.
PH suggest that PIP3
alone could promote the recruitment of Gab1 molecules in the vicinity
of the EGFR. Indeed, the observations that Gab1-
PH is phosphorylated
upon EGF stimulation, albeit less strongly than Gab1-wt, and that this
phosphorylation is independent of PI3K suggest that Gab1-
PH can
compete with Gab1-wt for binding to the receptor through Grb2- or
MBD-mediated interactions. Nevertheless, overexpression of Gab1-
PH
does not have a dominant negative effect on EGF signaling,
i.e. phosphorylation of Gab1-wt and MAPK activation. This
indicates that Gab1-wt is effectively recruited to the receptor in
cells overexpressing Gab1-
PH, through a mechanism for which Gab1-
PH cannot compete. This is most likely the interaction between the PH domain and PIP3. In any case, this question must be
further investigated, by studying, for example, the recruitment of Gab1 mutants deleted of the MBD and the Grb2-binding regions.
View larger version (27K):
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Fig. 8.
Model outlining the roles of PI3K, Gab1, and
SHP2 in the activation of Ras during EGF stimulation. The
activated receptor autophosphorylates on tyrosine residues involved in
binding to Grb2 and Gab1 MBD. This leads to the association of Gab1
with the receptor and its phosphorylation on SHP2 and p85 binding
sites, which triggers the activation of PI3K and the production of
PIP3. This lipid recruits additional Gab1 molecules in the
vicinity of the EGFR through binding to Gab1 PH domain, leading to an
increased recruitment of p85 and SHP2. Downstream of SHP2, the
relocation of Sos to membrane-bound Ras is facilitated by
dephosphorylation of an unidentified SHP2 substrate.
An important question is the physiological significance of this
PI3K-dependent pathway in regard to the canonical
Shc/Grb2/Sos pathway. Further studies are necessary to determine
whether the PI3K-dependent pathway is cell-type specific or
if it is more generally involved in MAPK activation. A recent study in
Cos cells has shown that the role of PI3K in MAPK activation is limited to weak stimulations induced by low doses of EGF (18), suggesting that
the Shc/Grb2/Sos pathway is prominent in this cell type. In contrast,
in cells derived from SHP2 or Gab1 knockout mice, MAPK are activated by
EGF at much lower levels than in control cells (41, 42). It is not
known if MAPK activation is also altered in p110 knockout mice that
have profound proliferative defects (10), but these findings strongly
support the notion that activation of Ras by EGF does not depend
entirely on the Shc/Grb2 pathway in normal cells.
The next step in the understanding of this PI3K-dependent
pathway will be brought by the identification of the SHP2 substrates involved in the activation of Ras. It has been proposed that SHP2 can
function as an adaptor in PDGF signaling, because it can bind to both
the receptor and the SH2 domains of Grb2 and therefore contributes to
the recruitment of Grb2-Sos (43). However, we have found that
expression of catalytically inactive SHP2 abolished Ras activation in
EGF-treated Vero cells, which is consistent with the fact that
catalytically inactive SHP-2 was shown to inhibit MAPK activation
induced by EGF in other cell types (37, 44). This suggests that SHP2
activates by dephosphorylation a protein promoting the translocation of
Sos or down-regulates an inhibitor of Sos redistribution. A candidate
could be the 135-kDa protein that is associated with catalytically
inactive SHP-2 in our substrate trapping experiment. Another candidate
could be an unidentified 90-kDa protein associated to Gab1 and for
which phosphorylation is up-regulated in SHP2 knockout mice (41).
Further efforts are required to identify these proteins.
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ACKNOWLEDGEMENTS |
---|
We are grateful to the following researchers for constructs used in this study: Drs. P. Hu, M. Wymann, F. R. McKenzie, El. vanObberghen, C. Susini, N. Rivard, J. Downward, W. Ogawa, P. Chardin, P. van Bergen en Henegouwen, B. M. Burgering, and A. Ullrich.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from Ministère de la Recherche et de l'Enseignement Supérieur, Association pour la Recherche sur le Cancer, and Ligue Nationale Contre le Cancer.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: Tel.: 33-561-779-412; Fax: 33-561-779-401; E-mail: raynal@purpan.inserm.fr.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M006966200
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ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular-regulated protein kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MBD, Met-binding domain; MEK, MAP kinase/ERK kinase; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PKB, protein kinase B; PKC, protein kinase C; RBD, Ras-binding domain of Raf1; wt, wild type; HA, hemagglutinin; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
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