From the Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada
Received for publication, August 13, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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We have previously demonstrated that
phosphatidylinositol 3-kinase (PI3-kinase) is necessary and sufficient
to account for epidermal growth factor (EGF)-induced mitogenesis in rat
primary hepatocytes. A cytosolic Gab2-containing complex accounts for >80% of the total EGF-induced PI3-kinase activity (Kong, M., Mounier, C., Wu, J., and Posner, B. I. (2000) J. Biol.
Chem. 275, 36035-36042), suggesting a key role for Gab2 in
EGF-induced mitogenesis. Here, we demonstrate that PP1, a selective
inhibitor of Src family kinases, blocks the EGF-induced Gab2 tyrosine
phosphorylation without inhibiting EGF-induced phosphorylation of the
EGF receptor, ErbB3, or Shc. We also show that Gab2 phosphorylation is
increased in Csk knockout cells in which Src family kinases are
constitutively activated. Furthermore, PP1 blocks Gab2-associated
downstream events including EGF-induced PI3-kinase activation, Akt
phosphorylation, and DNA synthesis. We demonstrate that Gab2 and Src
are constitutively associated. Since this association involves the
proline-rich sequences of Gab2, it probably involves the Src homology 3 domain of Src kinase. Mutation of the proline-rich sequences in Gab2
prevented EGF-induced Gab2 phosphorylation, PI3-kinase/Akt activation,
and DNA synthesis, demonstrating that Gab2 phosphorylation is critical for EGF-induced mitogenesis and is not complemented by ErbB3 or Shc
phosphorylation. We also found that overexpression of a Gab2 mutant
lacking SHP2 binding sites increased EGF-induced Gab2 phosphorylation and the activation of PI3-kinase but blocked activation of MAPK. In
addition, we demonstrated that the Src-induced response was down-regulated by Gab2-associated SHP2. In summary, our results have
defined the role for Src activation in EGF-induced hepatic mitogenesis
through the phosphorylation of Gab2 and the activation of the
PI3-kinase cascade.
Upon ligand binding, the activated epidermal growth factor
receptor (EGFR)1 mediates a
number of important biological responses, including the stimulation of
cell proliferation, migration, and differentiation (1-3). Src family
kinases are nonreceptor tyrosine kinases and have been described as
essential mediators of EGF signaling (4). Like most nonreceptor
tyrosine kinases, Src family kinases contain an Src homology 2 (SH2)
domain, which binds phosphotyrosine residues, and an Src homology 3 (SH3) domain, which binds proline rich sequences (reviewed in Ref. 5).
Several studies have established that Src kinases are required for
growth factor-induced mitogenesis such as that effected via receptors
for EGF (6-8), the platelet-derived growth factor (9, 10), and colony
stimulation factor-1 (6). To date, the manner in which Src family
kinases participate in effecting the mitogenic response is unclear.
Several reports have identified Gab
(Grb2-associated binder) family
proteins as key molecules for EGF-induced mitogenesis (11, 12). Gab
proteins, which include mammalian Gab1, Gab2, and Gab3, the
Drosophila homolog DOS (daughter of
sevenless), and the Caenorhabditis elegans
homolog Soc1 (Suppressor of clear), belong to a family of scaffolding proteins closely related
to insulin receptor substrates (IRS-1, IRS-2, and IRS-3), FRS2
(fibroblast growth factor substrate), LAT (linker of T cell), and Dok
(downstream of kinase) (reviewed in Refs. 13-15). They have in common
a central proline-rich domain and multiple potential binding
sites for the SH2 domains of p85, SHP2 (Src
homology 2 domain-containing protein-tyrosine phosphatase-2), phospholipase C In previous studies, we demonstrated that the activation of PI3-kinase
and not mitogen-activated protein kinase (MAPK), is necessary and
sufficient to account for EGF-induced mitogenesis (12, 20). Although
activated PI3-kinase was shown to associate with three
phosphotyrosine-phosphorylated proteins (ErbB3, Shc, and Gab2), over
80% was found in a multimeric complex consisting of Gab2-p85-SHP2-Grb2
(12). Confirming the key role of Gab2 was our finding that
overexpression of wild type Gab2 (WTGab2) augmented EGF-induced
PI3-kinase activity and DNA synthesis, whereas the Gab2 mutant
(Gab2 In the present study, we considered the possibility that Gab2 is
phosphorylated by a tyrosine kinase other than the EGFR and thus sought
to elucidate a link between Src family kinases and Gab2 in the
regulation of EGF-induced mitogenesis in primary hepatocytes. Our study
demonstrates that Src kinase(s) promote EGF-induced PI3-kinase
activation and DNA synthesis through effecting the tyrosine
phosphorylation of Gab2. In addition, we found that the proline-rich
domains of Gab2 are essential for constitutive Src association with and
tyrosine phosphorylation of Gab2. Finally, we demonstrate that these
Src-dependent responses are down-regulated by the
association of SHP2 with Gab2 and that this latter association is
critical to EGF-induced MAPK activation.
Materials--
Mouse EGF was obtained from Collaborative
Biomedical Products (Bedford, MA). Collagenase was from Worthington).
Cell culture medium and antibiotics were from Invitrogen.
Vitrogen-100 was from Collagen Corp. (Toronto, Canada).
[3H]methylthymidine, 125I- labeled goat
anti-rabbit antibody, 125I-labeled goat anti-mouse
antibody, and [ Preparation of the Gab2 Mutants--
The Adenovirus Preparation and Titration--
Large scale production
of recombinant viral particles was performed by infecting 293A cells.
The titer of viral particles was determined using the Tissue Culture
Infectious Dose 50 (TCID50) method as described in the
protocol of the Ad-easy vector system (Qbiogene, Carlsbad, CA).
Cell Culture and Viral Infection--
Primary hepatocytes were
prepared as previously described (12). Before infection, cells were
bathed for 24 h in Dulbecco's modified Eagle's medium/F-12
medium containing 10% fetal bovine serum, 10 mM HEPES, 20 mM NaHCO3, 500 IU/ml penicillin, and 500 µg/ml streptomycin. Wild type and Csk Immunoprecipitation and Immunoblotting--
Cell lysates,
prepared as previously described (12), from EGF-treated or nontreated
cells were precleared with nonimmune rabbit IgG (Sigma) and protein
A-Sepharose for 1 h at 4 °C. After centrifugation, the
resulting supernatants were incubated for 2 h at 4 °C with the
antibody indicated in the figure legends. Protein A-Sepharose (50 µl
of a 50% slurry) was added to each sample, and the incubation was
continued for an additional 1 h. Immune complexes were isolated by
centrifugation, washed three times in PBS, and boiled in Laemmli sample
buffer. Immunoprecipitates or intact samples were subjected to
SDS-PAGE, transferred to Immobilon-P membranes (Millipore Ltd.,
Mississauga, Canada), and immunoblotted with the indicated first
antibody for 90 min followed by a 1-h incubation with horseradish
peroxidase-, or 125I-labeled goat anti-rabbit antibody or
goat anti-mouse antibody IgG. Immunoreactive proteins were detected by
autoradiography or by ECL system (Amersham Biosciences). Densitometric
quantification of the signals was performed using a Bio-Rad
densitometer, model GS-700.
[3H]Thymidine Incorporation Assay--
After viral
exposure, infected cells were serum-starved for 20 h in serum-free
medium, and then 100 ng/ml EGF and [3H]methylthymidine (5 µCi/ml) were added to the medium. After an 18-h incubation, cells
were rinsed three times with 3 ml of cold PBS, incubated for 15 min at
4 °C in 10% trichloroacetic acid, solubilized at room temperature
in 1 ml of 1 N NaOH, neutralized with 1 ml of 1 N HCl, and then transferred to scintillation vials and
counted for 3H.
PI3-kinase Activity Assay--
Lysates (500 µg of protein)
from EGF-treated (100 ng/ml EGF for 1 min) or nontreated cells were
immunoprecipitated in the presence of protein A-Sepharose, using
different antibodies as indicated in the figure legends.
Immunoprecipitates were extensively washed, and the Protein A-Sepharose
pellets were resuspended in 50 µl of kinase assay buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM EGTA) containing 0.5 mg/ml
L- A Key Role for Src Family Kinases in EGF-induced Gab2
Phosphorylation--
In the liver, tyrosine-phosphorylated Gab2 is
largely localized in the cytosol, could not be demonstrated to
associate with the EGFR, and is phosphorylated in a pleckstrin homology
domain-independent manner.2 EGF has been shown to activate
tyrosine kinases of the Src family (4), and studies have linked their
activation to cell proliferation (6-10). We tested the hypothesis that
Gab2 is a Src substrate by examining the effect of PP1, a selective
inhibitor of Src family kinases (23), on EGF-induced Gab2 tyrosine
phosphorylation in rat primary hepatocytes. As shown in Fig.
1A (top panels),
PP1 reduced EGF-induced tyrosine phosphorylation of Gab2 in a
dose-dependent manner. Compared with cells incubated with
vehicle only, 1 µM PP1 decreased Gab2 phosphorylation by
50% and 20 µM PP1 by more than 90%. In contrast, the
same doses of PP1 had no effect on EGF-induced tyrosine phosphorylation
of EGFR (Fig. 1A, middle panels), ErbB3, or Shc
(Fig. 1A, bottom panels). Nor did these doses of
PP1 affect insulin-induced tyrosine phosphorylation of IRS-1 or IRS-2
(Fig. 1B). PP2, another selective inhibitor of Src family
kinases, also inhibited the stimulatory effect of EGF on Gab2 tyrosine
phosphorylation (data not shown). The specificity of inhibition by PP1
and PP2 of EGF-induced tyrosine phosphorylation of Gab2 suggests that,
in rat hepatocytes, this molecule, but not other
tyrosine-phosphorylated docking proteins, is a substrate for Src family
kinase(s).
All Src family tyrosine kinases are negatively regulated by
phosphorylation at a carboxyl-terminal tyrosine (Tyr-527 in Src kinase)
carried out by another nonreceptor tyrosine kinase, Csk (C-terminal Src kinase) (24). Cells
derived from Csk-deficient embryos exhibit an order of magnitude
increase in activity of Src and the related Fyn kinase (22, 25). To
further confirm the role of Src family kinases on Gab2 phosphorylation,
we infected Csk-deficient and wild type mouse embryo fibroblasts with
recombinant Gab2 adenovirus and starved the cells for 48 h.
Western blot analysis, using a specific phospho-Src antibody (p-Src416)
(26), demonstrated that Src kinase is constitutively activated in
Csk Gab2 Tyrosine Phosphorylation by Src Family Kinases Is Essential
for EGF-dependent Downstream Signaling Events--
In
previous work, we showed that over 80% of PI3-kinase, activated
following EGF treatment, was associated with tyrosine-phosphorylated Gab2 (12) and that overexpression of Gab2 was sufficient to augment
EGF-induced DNA synthesis.2 In this study, we pretreated
hepatocytes with either vehicle (Me2SO) or 20 µM PP1 for 30 min followed by a stimulation with 100 ng/ml EGF. As shown in Fig.
3A, EGF induced a 20-fold
increase in Gab2-associated PI3-kinase activity in control cells.
Pretreatment with PP1 abolished this activation, that of Akt, as
reflected in Akt-Ser-473 phosphorylation (Fig. 3B), and DNA
synthesis (Fig. 3C). Similar results were obtained by
treatment of the cells with PP2 (data not shown). Our results
demonstrate that EGF-induced tyrosine phosphorylation of Gab2, mediated
by Src family kinases, is necessary for EGF-induced DNA synthesis in
primary rat hepatocytes and that the tyrosine phosphorylation of ErbB3
and Shc, which is unaffected by PP1 (Fig. 1), cannot supplant the
critical requirement for tyrosine-phosphorylated Gab2 for this
response.
Association of Src with Gab2: Significance for Gab2 Tyrosine
Phosphorylation and Downstream Signaling--
In our previous
experiments, demonstrating that Gab2 is a substrate for Src family
kinases, we observed a 60-kDa band in anti-Gab2 immunoprecipitates
(data not shown). We therefore examined the possibility that Src kinase
was associated with its substrate, Gab2. As shown in Fig.
4A (top panel), Src
family kinases are constitutively associated with endogenous Gab2, and
EGF treatment did not appear to augment this association. This
suggested that the association may result from the binding of the SH3
domain of Src with proline-rich sequences in Gab2. To test this
hypothesis, we generated a recombinant adenovirus containing a Gab2
mutant in which key proline-rich sequences were mutated (
We sought to determine the extent to which the loss of constitutive Src
binding influenced Gab2 phosphorylation by Src family kinases. Primary
hepatocytes were infected with recombinant WTGab2 or
Since overexpressing
We then examined the effect of WTGab2 and SHP2 Is a Negative Effector for PI3-kinase/Akt
Activation but Positive for MAPK Activation--
Upon EGF treatment,
Src activation leads to substantial tyrosine phosphorylation of Gab2
and activation of DNA synthesis. We previously demonstrated that
phosphorylated Gab2 associates with the SH2-containing protein-tyrosine
phosphatase, SHP2 (12). We considered that this binding might
effect dephosphorylation of Gab2 and therefore generated a Gab2 mutant
lacking the SHP2 binding sites (
Of interest is the finding that overexpression of
In contrast to these findings, whereas overexpression of WTGab2
increased the sensitivity of ERK1/2 activation to EGF (Figs. 6D and 8D, lanes 4-6),
overexpression of
The above observations identify the mechanism by which Src regulates
EGF-induced mitogenesis in hepatocytes. Upon EGF binding, Gab2-associated Src is activated, leading to activation of the PI3-kinase cascade and eventually DNA synthesis. This signal appears to
be attenuated by SHP2 through dephosphorylation of Gab2.
In response to the binding of EGF to its receptor, Gab proteins
become tyrosine-phosphorylated and bind SH2 domain-containing proteins,
including p85, the PI3-kinase subunit, and SHP2 (12, 27-30). The
mechanism by which EGF induces the phosphorylation of Gab docking
proteins is key to understanding EGF-induced signaling. Several reports
indicate that EGFR is the kinase that phosphorylates Gab1 (31-33).
Furthermore, studies have demonstrated that the pleckstrin homology
domain of Gab1 is required for its localization to membranes, thus
facilitating its phosphorylation by the EGFR kinase (31, 34, 35).
However, the process involved in phosphorylating Gab2 appears to be
different. Thus, as we have previously found, Gab2 is phosphorylated in
a pleckstrin homology domain-independent manner; nor was it observed to
associate with the EGFR. Moreover, the major multimeric Gab2 complex
was found exclusively in the cytosol of rat liver.2 For
these reasons, we examined the possibility that Gab2 is
tyrosine-phosphorylated by a cytosolic kinase and not the EGFR.
Although previous reports have established that Src family kinases are
required for EGF-induced mitogenesis (6-8), the mechanism by which Src
family kinases act has remained unclear. In the present study, we
demonstrate that EGF treatment of rat hepatocytes activates Src,
leading to Gab2 tyrosine phosphorylation, after which Gab2 recruits
SHP2, Grb2, and p85, thus forming a multimeric cytosolic complex.2 The evidence that Src family kinases are involved
in Gab2 tyrosine phosphorylation is based on the inhibitory effects of
PP1 and PP2, two widely used specific inhibitors of Src family
kinases (36, 37). Thus, in the present study, we observed that the inhibition of Gab2 tyrosine phosphorylation was
dose-dependent and occurred in the absence of inhibition of
tyrosine phosphorylation of EGFR, ErbB3, or the IRS proteins (Fig. 1).
Furthermore, using CSK To elucidate the mechanism by which Gab2 is phosphorylated by Src
family kinases, we analyzed the association between these two proteins.
In most cases, substrate recognition is dictated primarily by
interactions with noncatalytic regions of the Src family kinases such
as SH2 and SH3 domains (reviewed in Ref. 5). A constitutive association
of Src family kinases with Gab2 was observed, and this association
disappeared when the two key proline-rich regions on Gab2 were mutated
(Fig. 4), indicating that the SH3 domain of Src is probably involved in
this interaction. This is consistent with two previous reports
demonstrating that the SH3 domain of Src is required for EGF mitogenic
signaling (9, 10) and that this domain can bind to Gab2 in an in
vitro binding assay (43). The SH3 domains of Src family kinases
recognize proline-rich sequences found in a large number of substrates
of Src family kinases (44-46). The SH2 domains of Src family kinase
selectively recognize the sequence pYEEI, with a hydrophobic residue at
position +3 being an important determinant of binding (47).
Interestingly, the Gab2 sequence does not contain a pYEEI motif,
suggesting that Src does not bind to Gab2 through its SH2 domain.
Although Src family kinases are constitutively associated with Gab2,
the activation of Src kinase is only observed after EGF treatment (Fig.
4C). The mechanism by which Src kinase activation is
effected by the binding of EGF to the EGFR remains to be elucidated. In
hepatocytes, Src, Fyn, and Yes are the three Src family members
expressed (48). However, in the present analysis, we could not identify
which isoform(s) phosphorylate Gab2. Indeed, the dose of PP1 we used inhibits all three Src isoforms. Furthermore, the Src antibody we used
for Western blots recognizes Fyn and Yes as well as Src.
In this paper, we show that in rat hepatocytes, inhibition of Src
family kinases prevents EGF-induced phosphorylation of Gab2 and
activation of PI3-kinase/Akt as well as DNA synthesis (Figs. 1 and 3).
Several recent studies have shown that activation of Src family kinases
is involved in signal transduction by regulating PI3-kinase activity.
Thus, erythropoietin appears to effect erythroid differentiation by
effecting Src-dependent tyrosine phosphorylation of and the
recruitment plus activation of PI3-kinase to the erythropoietin receptor (49). Also, in MCF-7 cells, estradiol was shown to promote
cell cycle progression via a Src-dependent activation of
PI3-kinase (50). Finally, in T47D cells, EGF activation of Akt was
blocked in parallel with inhibition of Src activation (36). In rat
hepatocytes, EGF-dependent Gab2 phosphorylation leads to
downstream PI3-kinase/Akt activation and DNA synthesis and is effected
by the activation of Src constitutively associated with Gab2 (Figs. 4
and 6).
SHP2 binds with Gab family proteins through a consensus
binding motif (YXX(V/I/L)) located in the C-terminal ends of Gab
proteins (15). The function of the Gab-SHP2 interaction has been
extensively studied using mutants of Gab family proteins lacking SHP2
binding sites. These studies reveal a general positive role of SHP2 in Gab-mediated signal transduction. For example, SHP2 association with
Gab1 is required for Met-dependent morphogenesis (51) as well as EGF and lysophosphatidic acid-induced MAPK activation (27, 30).
It was also found that SHP2 association with Gab2 is essential for
macrophage colony-stimulating factor-induced macrophage
differentiation (52). In the present study, we identified a positive
role for SHP2 in Gab2-potentiated EGF-induced MAPK activation (Fig.
8D). Recent reports have also identified a negative effect
of SHP2 binding on Gab protein tyrosine phosphorylation. Thus, both
Gab1 and Gab2 have been identified as substrates for SHP2 using an
in vitro phosphatase assay (18). More recently, two studies
have shown that inactive forms of SHP2 markedly increased EGF-stimulated Gab1 tyrosine phosphorylation and PI3-kinase activity (28, 53). The negative regulation of EGF-dependent
PI3-kinase activation by SHP2 appears to be through the
dephosphorylation of Gab1 p85 binding sites (53). In agreement with
this study, we have found that, in rat hepatocytes, a relatively minor
increase of In summary, we have found that, in rat hepatocytes, Src family kinases
regulate EGF-induced mitogenesis through association to and
phosphorylation of the major cytosolic docking protein, Gab2.
Furthermore, we demonstrate that the association of SHP2 with
tyrosine-phosphorylated Gab2 leads to dephosphorylation of the latter
and a corresponding decrease in EGF-induced DNA synthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, or Crk. Gab2 is
tyrosine-phosphorylated upon stimulation of hepatocytes by EGF (12) and
T cells by cytokines (16) and following activation of T- and B-cell
antigen receptors (17, 18). Phosphorylated Gab2 has been shown to bind
PI3-kinase via its 85-kDa (p85) regulatory subunit (19) as well as Grb2 (growth factor receptor-bound 2)
and SHP2.
p85) lacking p85 binding sites (pYXXM motifs as
reviewed in Ref. 21, where pY represents phosphotyrosine) effected no
such augmentation. Furthermore, we showed that following EGF treatment,
the phosphorylated multimeric Gab2 complex was exclusively cytosolic
and did not associate with membranes. Nor did overexpression of the
pleckstrin homology domain of Gab2 interfere with EGF-induced Gab2
phosphorylation or
mitogenesis.2
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP were purchased from
PerkinElmer Life Sciences. Protein A-Sepharose was from Amersham
Biosciences. PP1 was purchased from Calbiochem. The
anti-phosphotyrosine antibody (PY99) and antibodies to SHP2, Src,
IRS-2, and hemagglutinin were from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). The anti-p85, IRS-1, Shc, ErbB3, Myc, and Gab2 (for
immunoblotting) antibodies were from Upstate Biotechnology, Inc. (Lake
Placid, NY). Anti-phospho-Akt473, Akt, phospho-Src416, phospho-Erk1/2,
and Erk1/2 antibodies were from New England Biolabs (Beverly, MA).
Anti-Gab2 antibody (for immunoprecipitation) was raised by immunizing
rabbits with a glutathione S-transferase-Gab2 fusion protein
containing amino acids 376-552 of the rat Gab2 sequence. All other
reagents were obtained from Sigma and were of the highest grade available.
SHP2Gab2 was
generated by replacing Tyr-603 and Tyr-632 with phenylalanine using the
Chameleon double-stranded site-directed mutagenesis kit (Stratagene, La
Jolla, CA) according to the manufacturer's instructions. The mutant
was verified by DNA sequencing. A plasmid containing
ProGab2 with a
deletion of 348-355 amino acids and mutations of P500A and R504A was
kindly provided by Dr. Morag Park (McGill University, Montréal,
Canada). In order to produce the adenoviruses, Gab2 mutants as well as
the full-length cDNA (to generate the WTGab2 construct) were
subcloned into pShuttle between the NheI and NotI
sites using 5'-oligonucleotides containing an NheI site and
the sequences encoding different tags (hemagglutinin for WTGab2, FLAG
for
ProGab2, and Myc for
SHP2Gab2) and 3'-oligonucleotides containing a NotI site. The recombinants of adenoviral DNA
were further generated using the Adeno-X Expression System
(Clontech, Palo Alto, CA) according to the kit's
user manual.
/
cells, kindly
provided by Dr. Philippe Soriano (Fred Hutchinson Cancer Research
Center, Seattle, WA) (22) were maintained in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum. All cells were then
infected with 10 MOI of different recombinant adenoviruses for 3 h
at 37 °C. The infected cells were then serum-starved for 48 h
before harvesting.
-phosphatidylinositol (Avanti Polar Lipids, Inc.,
Alabaster, AL) and assayed for PI3-kinase activity as previously
described (20).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Src kinase inhibitor PP1 specifically
inhibits Gab2 phosphorylation. Serum-deprived hepatocytes were
preincubated with either vehicle (Me2SO (DMSO))
or PP1 (1 or 20 µM) for 30 min followed by treatment with
(+) or without ( ) 100 ng/ml EGF (A) or 100 nM
insulin (B) for 1 min. Cell lysates were incubated with
different antibodies, and the immunoprecipitated proteins were
subjected to SDS-PAGE (7.5% gel) followed by immunoblotting with the
indicated antibodies as described under "Experimental
Procedures."
/
cells (Fig.
2A, top panel).
Gab2 tyrosine phosphorylation was then investigated in the absence of
EGF stimulation. Immunoprecipitation with
Gab2 and Western blotting
with anti-phosphotyrosine demonstrated that Gab2 phosphorylation (Fig.
2A, middle panel) is increased 1.8-fold in
Csk
/
compared with wild type fibroblasts
(Fig. 2B). This study supports the view that Gab2 is a
substrate for Src family kinases in vivo.
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Fig. 2.
Increased tyrosine phosphorylation of Gab2 in
Csk /
cells. A, wild type (WT) or Csk knockout
(Csk
/
) cells were infected with 10 MOI of recombinant
Gab2 adenovirus for 3 h and then incubated for 48 h in
serum-free medium. Cell lysates were prepared, and aliquots (50 µg of
protein) were subjected to 7.5% SDS-PAGE followed by immunoblot
analysis with anti-phospho-Src416 antibody (top panel).
Other aliquots were incubated with anti-Gab2 antibody, and the
immunoprecipitates were subjected to 7.5% SDS-PAGE followed by
immunoblot analysis with anti-phosphotyrosine antibody (middle
panel) or anti-Gab2 antibody (bottom panel).
B, the level of Gab2 tyrosine phosphorylation, quantified as
described under "Experimental Procedures," is expressed in
Csk
/
cells (solid bar) as -fold
over wild type cells (hatched bar) (mean ± S.E.; n = 3; *, p < 0.01).
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Fig. 3.
Src family kinases are required for
Gab2-mediated downstream events. A, serum-deprived
hepatocytes were preincubated with 20 µM PP1 or vehicle
(Me2SO (DMSO)) for 30 min followed by treatment
with (+) or without ( ) EGF (100 ng/ml) for 1 min. Cell lysates were
incubated with anti-Gab2 antibody, and immunoprecipitated proteins were
either assayed for PI3-kinase activity (top panel) or
subjected to SDS-PAGE (7.5% gel) followed by immunoblotting with
anti-Gab2 antibody (bottom panel). B,
serum-deprived hepatocytes were preincubated with 20 µM
PP1 or vehicle (Me2SO) for 30 min and then treated with (+)
or without (
) EGF (100 ng/ml) for 5 min. Cell lysates were subjected
to SDS-PAGE (10% gel) followed by immunoblotting with either
anti-phospho-Akt473 (top panel) or Akt antibody
(bottom panel). C, hepatocytes were incubated in
serum-free medium for 24 h followed by incubation with either
vehicle (Me2SO) (open and hatched
bars) or PP1 (20 µM) (solid
bar). After 30 min, 5 µCi of
[3H]methylthymidine was added, and the incubation was
continued with (hatched and solid
bars) or without (open bar) EGF (100 ng/ml) for 18 h in serum-free medium as described under
"Experimental Procedures." Results are expressed as -fold over
control (non-EGF-treated cells). The inhibitory effect of PP1 on EGF
stimulation of DNA synthesis was assessed in three separate experiments
(mean ± S.E.; *, p < 0.01).
ProGab2)
(see "Experimental Procedures"). Hepatocytes were infected with
either WTGab2 or
ProGab2 recombinant adenovirus and starved for
48 h before EGF treatment. In contrast to WTGab2, overexpressed
ProGab2 manifested no association with Src kinase (Fig.
4B, top panel, lanes 1 and
2 versus lanes 3 and
4), indicating that the proline-rich sequences are essential
for mediating the binding of Src family kinases to Gab2. Src kinase
phosphorylation was analyzed in anti-Gab2 immunoprecipitates. A
tyrosine-phosphorylated band migrating at the same position as Src
kinase (60 kDa) was detected in EGF-treated cells infected with WTGab2
but not in EGF-treated cells infected with
ProGab2 (Fig.
4C, top panel). These results suggest that Src
kinase is activated upon EGF treatment, although it is constitutively associated with Gab2.
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Fig. 4.
Constitutive association of Src with
Gab2. A, serum-deprived hepatocytes were treated with
(+) or without ( ) 100 ng/ml EGF for 1 min. Cell lysates were
incubated with anti-Gab2 antibody, and the immunoprecipitated proteins
were subjected to SDS-PAGE (7.5% gel) followed by immunoblotting with
anti-Src antibody (top panel) or anti-Gab2 antibody
(bottom panel). B and C, hepatocytes
were infected with 10 MOI of either recombinant WTGab2 or
ProGab2
adenovirus for 3 h followed by incubation in serum-free medium for
48 h. The hepatocytes were then treated with (+) or without (
)
EGF (100 ng/ml) for 1 min, and cell lysates were prepared and incubated
with anti-Gab2 antibody. The immunoprecipitated proteins were subjected
to SDS-PAGE (7.5% gel) followed by immunoblotting with anti-Src
antibody (B, top panel), anti-phosphotyrosine
antibody (C, top panel), or anti-Gab2 antibody
(B and C, bottom panel).
ProGab2
adenoviruses. As shown in Fig. 5
(top panel), EGF treatment resulted in substantial tyrosine
phosphorylation of WTGab2, whereas the phosphorylation of
ProGab2 is
reduced by more than 70% (Fig. 5, bottom panel). These
results demonstrate that, in hepatocytes, the proline-rich sequences of
Gab2 are important for Src kinase-mediated Gab2 phosphorylation.
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Fig. 5.
The proline-rich sequences of Gab2 are
required for EGF-induced Gab2 phosphorylation. Hepatocytes were
infected with 10 MOI of either recombinant WTGab2 or ProGab2
adenovirus for 3 h and then incubated in serum-deprived medium for
48 h. Cell lysates were incubated with anti-Gab2 antibody, and the
immunoprecipitates were subjected to 7.5% SDS-PAGE followed by
immunoblotting with either anti-phosphotyrosine antibody or anti-Gab2
antibody (top panel). The bar graph
(bottom panel) represents quantification of Gab2 tyrosine
phosphorylation in EGF-treated cells. The extent of Gab2
phosphorylation in
ProGab2-infected cells is expressed as a fraction
of that in control (WTGab2-infected) hepatocytes (mean ± S.E.;
n = 3; *, p < 0.005).
ProGab2 dramatically reduced EGF-induced Gab2
tyrosine phosphorylation, we examined whether this resulted in a
decrease of Gab2-dependent downstream signaling. Cells were infected with either control recombinant adenovirus (LacZ) or Gab2
constructs (WTGab2 or
ProGab2). As previously observed, overexpression of WTGab2 potentiated EGF-induced PI3-kinase activation by 25-fold (Fig. 6A, top
panel, lane 2 versus
lane 4), whereas overexpression of
ProGab2
reduced EGF-induced PI3-kinase activity by more than 70% (Fig.
6A, top panel, lane 4 versus lane 6). This parallels the
association of Gab2 with p85 (Fig. 6A, middle
panel). Interestingly,
ProGab2 did not exert a dominant
negative effect in that it did not reduce PI3 kinase activity to less
than control levels despite being expressed at 20-fold the level of
endogenous Gab2. Corresponding to the PI3-kinase activity, we found
that EGF treatment augmented Akt serine 473 phosphorylation in cells overexpressing WTGab2 but not in those overexpressing
ProGab2 (WTGab2- versus
ProGab2-infected cells, expressed as a
percentage of LacZ-infected cells was 146 ± 4.7 versus
110 ± 4.1 (n = 3, mean ± S.E.,
p < 0.01)) (Fig. 6B). Parallel results were
obtained when we measured EGF-induced DNA synthesis (Fig.
6C). The observations in Fig. 6 are consistent with a
greater sensitivity to EGF of PI3-kinase activation and p85 recruitment
than to Akt phosphorylation and DNA synthesis. This could
explain the closer correlation between Akt activation and the extent of
stimulation of DNA synthesis. These findings indicate that the
proline-rich sequences in Gab2, which mediate Src binding, are
important for EGF-induced PI3-kinase activation and DNA synthesis.
View larger version (42K):
[in a new window]
Fig. 6.
Influence of Gab2 proline-rich sequences on
EGF-induced activation of PI3-kinase/Akt, MAPK, and DNA synthesis.
Hepatocytes were infected with 10 MOI of either recombinant LacZ,
WTGab2, or ProGab2 adenovirus for 3 h followed by incubation in
serum-free medium for 48 h. A, serum-deprived
hepatocytes were treated with (+) or without (
) EGF (100 ng/ml) for 1 min, and cell lysates were prepared, equally divided, and incubated
with anti-Gab2 antibody. Immunoprecipitated proteins were assayed for
PI3-kinase activity (top panel) or subjected to SDS-PAGE
(7.5% gel) followed by immunoblotting with anti-p85 antibody
(middle panel). The membrane was stripped and reblotted with
an anti-Gab2 antibody (bottom panel). B,
serum-deprived hepatocytes were treated with (+) or without (
) EGF
(100 ng/ml) for 5 min. Cell lysates were subjected to SDS-PAGE (10%
gel) followed by immunoblotting with an anti-phospho-Akt473 (top
panel), anti-Akt (middle panel), or anti-Gab2
(bottom panel) antibodies. C, virus-infected
hepatocytes were incubated in serum-free medium for 24 h followed
by the addition of 5 µCi of [3H]methylthymidine without
(hatched bars) or with (solid bars) EGF (100 ng/ml) for 18 h. Incorporation of
[3H]methylthymidine into DNA was determined as described
under "Experimental Procedures." Results are expressed as -fold
over control (LacZ adenovirus-infected cells without EGF) cells. The
difference between EGF-treated hepatocytes infected with WTGab2
versus
ProGab2 was determined in three separate
experiments (mean ± S.E.; *, p < 0.005).
D, hepatocytes were treated with the indicated doses of EGF
for 5 min, and cell lysates were prepared and subjected to SDS-PAGE
(10% gel) followed by immunoblotting with anti-phospho-Erk1/2
(top panel), anti-Erk1/2 (middle panel), or
anti-Gab2 (bottom panel) antibodies.
ProGab2 on ERK
phosphorylation. As shown in Fig. 6D, ERK phosphorylation
was barely observed with 0.2 ng/ml EGF in LacZ-infected cells, whereas,
at this dose, there was clear phosphorylation of ERK in cells infected with WTGab2. We also demonstrated that ERK1/2 phosphorylation at higher
doses of EGF (10 or 100 ng/ml) is much greater than that at 0.2 and 1.0 ng/ml. At these concentrations, we did not observe any effect of
overexpressed Gab2 (data not shown). This suggests that overexpressed
Gab2 increases the sensitivity of ERK to EGF, which correlated with
previous studies showing that at a low dose of EGF (0.25 ng/ml),
overexpression of Gab1 potentiated EGF-induced ERK activation in HEK293
cells (27). These results may indicate that, at higher doses of EGF,
the influences of Gab2 could be obscured by input(s) from
Gab2-independent signaling pathways. Alternatively, a high sensitivity
of ERK phosphorylation to EGF may result in its saturation at the 10 ng/ml dose of EGF. As shown in Fig. 6D,
ProGab2
comparably potentiated EGF-induced ERK1/2 activation as WTGab2 at the
low dose of EGF (i.e. following EGF (0.2 ng/ml) treatment,
ERK activation in WTGab2 and
ProGab2 was 218 ± 4.2 and
236 ± 12, respectively, when expressed as a percentage of values
in LacZ-infected cells (n = 3, mean ± S.E., p < 0.05 LacZ versus either construct)).
Thus, the reduced level of tyrosine phosphorylation of
ProGab2 would
appear to be sufficient to activate ERK1/2 but not PI3-kinase.
SHP2Gab2; see "Experimental
Procedures"). Overexpression of
SHP2Gab2 in hepatocytes totally
abolished SHP2 binding to Gab2 (Fig.
7A, top panel,
lane 2 versus lane
4) and was tyrosine-phosphorylated (Fig. 7B,
lane 4 versus lane
6) to an extent 25% greater than WTGab2 (Fig.
7C).
View larger version (26K):
[in a new window]
Fig. 7.
SHP2 binding negatively regulates EGF-induced
Gab2 phosphorylation. A, hepatocytes were infected with
10 MOI of either recombinant WTGab2 or SHP2Gab2 adenovirus for
3 h, followed by incubation in serum-free medium for 48 h.
Cells were then treated with (+) or without (
) EGF (100 ng/ml) for 1 min, and cell lysates were incubated with either anti-hemagglutinin or
anti-Myc antibody as described under "Experimental Procedures."
Immunoprecipitated proteins were subjected to 7.5% SDS-PAGE followed
by immunoblotting with anti-SHP2 (top panel), or anti-Gab2
(bottom panel) antibody. B, hepatocytes were
infected with 10 MOI of either recombinant WTGab2 or
SHP2Gab2
adenovirus for 3 h followed by incubation for 48 h in
serum-free medium. The cells were treated with (+) or without (
) EGF
(100 ng/ml) for 1 min. Cell lysates were incubated with anti-Gab2
antibody, and immunoprecipitated proteins were subjected to SDS-PAGE
(7.5% gel) followed by immunoblotting with anti-phosphotyrosine
antibody (top panel). The membrane was stripped and
reblotted with anti-Gab2 antibody (bottom panel). The
bar graph represents determination of Gab2
tyrosine phosphorylation in EGF-treated cells infected with
SHP2Gab2
versus that in cells infected with WTGab2 (mean ± S.E.; n = 3; *, p < 0.005).
SHP2Gab2
potentiated EGF-induced PI3-kinase activation 2.6-fold compared with
that observed in cells overexpressing comparable levels of WTGab2 (Fig.
8A, top panel,
lane 4 versus lane
6). As expected, this correlated with augmented p85-Gab2
association (Fig. 8A, middle panel,
lane 4 versus lane
6). In parallel, we found that following EGF treatment, Akt
serine 473 phosphorylation, expressed as a percentage of LacZ-infected
cells, was 146 ± 4.7 versus 201 ± 25, in WTGab2
versus
SHP2Gab2 (n = 3, mean ± S.E., p < 0.01) (Fig. 8B, top
panel, lane 4 versus
lane 6). Similar results were observed when we
looked at the EGF-induced DNA synthesis by -fold (Fig. 8C).
Thus, SHP2 binding to tyrosine-phosphorylated Gab2 acts as a negative
regulator of EGF-induced PI3-kinase activation and DNA synthesis.
View larger version (41K):
[in a new window]
Fig. 8.
SHP2 binding negatively affects EGF-induced
PI3-kinase/Akt activation and DNA synthesis but positively affects MAPK
activation. Hepatocytes were infected with 10 MOI of either
recombinant LacZ, WTGab2, or SHP2Gab2 adenovirus for 3 h
followed by incubation in serum-free medium for 48 h.
A, serum-deprived hepatocytes were treated with (+) or
without (
) EGF (100 ng/ml) for 1 min, and cell lysates were equally
divided and incubated with anti-Gab2 antibody. The immunoprecipitated
proteins were either assayed for PI3-kinase activity (top
panel) or subjected to SDS-PAGE (7.5% gel) followed by
immunoblotting with anti-p85 antibody (middle panel). The
membrane was then stripped and reblotted with anti-Gab2 antibody
(bottom panel). B, serum-deprived hepatocytes
were treated with (+) or without (
) EGF (100 ng/ml) for 5 min, and
cell lysates were subjected to SDS-PAGE (10% gel) followed by
immunoblotting with anti-phospho-Akt473 (upper panel),
anti-Akt (middle panel), or anti-Gab2 (bottom
panel) antibodies. C, virus-infected hepatocytes were
incubated in serum-free medium for 24 h followed by the addition
of 5 µCi of [3H]methylthymidine without (hatched
bars) or with (solid bars) EGF (100 ng/ml) for 18 h. Incorporation of [3H]methylthymidine into DNA was
determined as described under "Experimental Procedures." Results
are expressed as -fold over control (LacZ adenovirus-infected cells
without EGF). The difference between EGF-treated hepatocytes infected
with WTGab2 versus
SHP2Gab2 was determined in three
separate experiments (mean ± S.E.; *, p < 0.005). D, serum-deprived hepatocytes were treated with the
indicated dose of EGF for 5 min, and cell lysates were subjected to
SDS-PAGE (10% gel) followed by immunoblotting with anti-phospho-Erk1/2
(top panel), anti-Erk1/2 (middle panel), or
anti-Gab2 (bottom panel) antibodies.
SHP2Gab2 had no capacity to effect EGF-induced
activation of ERK1/2 (Fig. 8D, lanes
7-9) (i.e. following EGF treatment (0.2 ng/ml),
ERK activity in WTGab2- versus
SHP2Gab2-infected cells,
expressed as percentage of LacZ-infected cells, was 218 ± 4.2 versus 130 ± 9 (n = 3, mean ± S.E., p < 0.05)) (Fig. 8D). These data
indicate that SHP2 binding to tyrosine-phosphorylated Gab2 influences
EGF downstream signaling by negatively affecting EGF-dependent PI3-kinase activation and positively
affecting activation of the MAPK pathway.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mouse embryonic cells, in which
Src family kinases are constitutively activated, we confirmed a key
role for these kinase(s) in the phosphorylation of Gab2. Consistent
with our results are previous studies demonstrating that the target
involved in Src-dependent mitogenic activity is a cytosolic
molecule accessible to unmyristoylated Src. Thus, Src deletion mutants,
lacking the amino-terminal one-third of the molecule, including the
membrane binding domain, were shown to still induce cell proliferation
(38). It has been known for some time that EGF signaling involves c-Src
substrates with molecular sizes of 120-130, 100, and 75 kDa (39).
Further studies have identified p75 as cortactin (40) and p120-130 as
comprising several proteins, including p125FAK (41) and p130CAS (42). However, the identity of p100 has hitherto remained unknown. Our results appear to establish the p100 Src kinase substrate as Gab2.
SHP2Gab2 phosphorylation compared with WTGab2 (Fig.
7B) was accompanied by a more noticeable augmentation of
PI3-kinase activity and DNA synthesis, suggesting that SHP2 is involved
in the dephosphorylation of specific p85 binding sites on Gab2 (Fig. 8,
A-C). Moreover, the fact that the
SHP2Gab2 mutant blocks
EGF-induced MAPK activation and augments PI3-kinase and DNA synthesis
further confirms, in primary hepatocytes, the key role of PI3-kinase
and not MAPK in EGF-induced DNA synthesis.
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FOOTNOTES |
---|
* This work was supported by the Medical Research Council and the National Cancer Institute of Canada as well as the Cleghorn Fund at McGill University, and the Maurice Pollack Foundation of Montreal.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: Polypeptide Hormone
Laboratory, Strathcona Anatomy Bldg., 3640 University St., Montreal,
Quebec, Canada H3A 2B2. Tel.: 514-398-4101; Fax: 514-398-3923; E-mail:
barry.posner@staff.mcgill.ca.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M208286200
2 M. Kong, C. Mounier, A. Balbis, G. Baquiran, and B. I. Posner, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
EGFR, epidermal
growth factor receptor;
EGF, epidermal growth factor;
PI3-kinase, phosphatidylinositol 3-kinase;
MAPK, mitogen-activated protein kinase;
MOI, multiplicity of infection;
SH2 and -3, Src homology 2 and 3, respectively;
IRS, insulin receptor substrate;
WTGab2, wild type Gab2;
IP, immunoprecipitation;
WB, Western blot;
ERK, extracellular
signal-regulated kinase;
PY, anti-phosphotyrosine.
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