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INSERM U145 (S.R., S.T.-D., J.M., E.V.O.) 06107 Nice
Cédex 2, France
Departments of Microbiology &
Immunology and Pharmacology (M.H.-M., A.J.W.) Jefferson Cancer
Institute Philadelphia, Pennsylvania 19107
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recently, Wong and co-workers (17) cloned a new protein called Gab1 (Grb2-associated binder-1). Gab1 is found in most human tissues except lung, kidney, and liver. It has a molecular mass of 77 kDa, but migrates between 115–120 kDa on SDS-PAGE, which is thought to be due to its high level of serine/threonine phosphorylation. This protein has been identified as a substrate of the insulin and epidermal growth factor (EGF) receptors. In addition, Weidner et al. (18) have shown that Gab1 interacts directly with the c-Met tyrosine kinase.
The physiological role of Gab1 is currently unknown. However, Gab1 overexpression in epithelial cells is sufficient to generate the characteristic responses induced by c-Met tyrosine kinase receptor, such as branching morphogenesis and cell scattering. Moreover, overexpression of Gab1 in NIH3T3 cells enhances cell growth and transformation stimulated by insulin and EGF. In summary, Gab1 appears to be a key mediator in cell proliferation and transformation induced by the EGF and c-Met receptors.
Sequence analysis shows that Gab1 is homologous to IRS-1, IRS-2, and
IRS-3 especially in the Pleckstrin homology (PH) domain, which is
located at the N terminus of these proteins. In addition, Gab1
possesses 16 potential phosphotyrosine sites, some of which could serve
as binding sites for SH2 domains of the regulatory subunit of PI3-K,
Grb2, phospholipase C-
, Nck, and SHP-2. This suggests that Gab1
could serve as a docking protein, like the other IRS proteins. However,
in contrast to IRS proteins and Shc, Gab1 does not possess a
phosphotyrosine binding (PTB) domain, which is thought to be implicated
in direct binding to the IR phosphotyrosine 960 (19, 20, 21, 22).
In the present study, we used a modified version of the yeast two-hybrid system and coimmunoprecipitations in intact mammalian cells to evaluate interactions between Gab1 and PI3-K or SHP-2, and to identify the tyrosines involved in these interactions. We demonstrate that Gab1 must be tyrosine phosphorylated by IR to allow its association with PI3-K and SHP-2. Finally, we show that in intact cells, the Gab1 PH (Pleckstrin homology) domain is crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). In the presence
of methionine, expression of IR ß is repressed. Absence of methionine
allows expression of IR ß leading to phosphorylation of coexpressed
substrates in the yeast.
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). We observed that
coexpression of lexA DNA-binding domain (LDBD)-p85 and GAD-Gab1
produces a detectable level of ß-galactosidase activity only if the
IR is expressed (without methionine in the medium). In the absence of
IR ß, interaction was undetectable. These results suggest that IR ß
phosphorylates Gab1 on tyrosine residues, resulting in interaction of
Gab1 with p85. To further address this issue, lysine 1018 of the IR
ATP-binding site was mutated to obtain a kinase-deficient IR (pVJL HIR
K1018A p85). No interaction between Gab1 and p85 was detected using
mutant IR ß K1018A, suggesting that the interaction depends on
receptor tyrosine kinase activity. Similar results were obtained with
LDBD-n/c SH2 SHP-2 (data not shown). Concurrently, we verified
expression and tyrosine phosphorylation of IR. Yeast coexpressing wild
type (WT) or K1018A IR constructs were incubated in the presence or
absence of methionine, after which yeast lysates were prepared, and IR
constructs were immunoprecipitated. Phosphorylated IR was revealed by
immunoblotting with an antibody to phosphotyrosine. Expression was
measured by immunoblotting using an antibody to the hemagglutinin (HA)
epitope, which is present in the fusion protein (Fig. 2B). As shown,
expression of IR WT and IR K1018A was observed in absence of
methionine, whereas expression was repressed in the presence of
methionine (bottom panel). Moreover, as expected, IR WT is
tyrosine phosphorylated in yeast in contrast to kinase-deficient IR (IR
K1018A).
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Using this system, we identified the phosphorylated tyrosine residue(s)
of Gab1 which is (are) involved in interaction with p85.
Phosphotyrosines in YXXM motifs are potential binding sites for p85 SH2
domains. Sequence analysis has shown that Gab1 contains at least 16
potential tyrosine phosphorylation sites, including 3 in YXXM motifs,
Y447, Y472, and Y589. We replaced tyrosine residues 447, 472, and 589
in GAD-Gab1 individually or in combination, and analyzed the ability of
the different constructs to interact with LDBD-p85 in the presence of
IR ß (Fig. 3A). As shown in Fig. 3A, mutation of
tyrosines 447 and 589 (Y447F and Y589F constructs) did not
significantly alter interaction with Gab1. Mutation of tyrosine 472, or
mutation at both tyrosines 447 and 589, decreased by approximately 50%
the interaction of Gab1 with p85. However, replacement of tyrosines 447
and 472 (GAD-Gab1 Y447F/Y472F) and tyrosines 472 and 589 (GAD-Gab1
Y472F/Y589F) completely abolished interaction between Gab1 and p85.
Lack of interaction was also obtained with a construct containing the
three mutated tyrosines (GAD-Gab1 Y447F/Y472F/Y589F). To confirm the
yeast two-hybrid results, the interaction between Gab1 and the p85 of
PI3-K was analyzed by measurement of PI3-K activity associated with
Gab1 (WT and mutants) after insulin stimulation in intact cells (Fig. 3B). After transfection, the cells were stimulated with insulin, and
Gab1 was immunoprecipitated from the cell lysates. As a control of
expression, one-tenth of the cell lysates was analyzed by SDS-PAGE and
immunoblotted with antibodies to Gab1. In all experiments, the
expression levels of Gab1 WT and mutant proteins were comparable (data
not shown). The PI3-K activity associated with Gab1 was measured as
described in Materials and Methods. In 293 EBNA cells
transfected with Gab1 WT, insulin induced a 3-fold increase in PI3-K,
which was chosen to represent 100% (Fig. 3B). In cells transfected
with Gab1 Y447F/Y589F, the insulin-induced PI3-K activity associated to
GAB was approximately increased 50% of that observed for the Gab1 WT.
In cells transfected with the other Gab1 mutants (Y447F/Y472F,
Y472F/Y589F, and Y447F/Y472F/Y589F), the insulin-stimulated PI3-K
activity associated with Gab1 was completely abolished compared with
that found with the Gab1 WT. The absence of PI3-K activity associated
with Gab1 Y447F/Y472F, Y472F/Y589F, and Y447F/Y472F/Y589F is not due to
a lack of tyrosine phosphorylation, since immunoblotting using
phosphotyrosine antibodies revealed that all Gab1 mutants are
phosphorylated on tyrosine residues (data not shown). Taken together
these results indicate that tyrosines 447, 472, and 589 of Gab1 are
important for binding p85.
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). Mutation of tyrosine 183 did not affect the
interaction of Gab1 with SHP-2 SH2 domains. In contrast, mutation of
tyrosine 627 completely abolished the interaction. Next, we examined
whether the Gab1 tyrosine 627 is involved in the interaction between
Gab1 and SHP-2 in intact cells, by mutation of this residue to
phenylalanine. We immunoprecipitated Gab1 from the transfected 293 EBNA
cells and looked for the association with SHP-2 by immunoblotting with
antibodies to SHP-2 (Fig. 4B). As a control of expression, anti-Gab1
immunoprecipitates were also analyzed by immunoblotting with antibodies
to Gab1. Gab1 was not detected in anti-Gab1 immunoprecipitates from
vector-only transfected cells (MOCK condition), and Gab1
Y627F-transfected cells expressed slightly more protein than Gab1
WT-transfected cells. In cells transfected with Gab1 WT, association of
Gab1 WT with SHP-2 was detected in unstimulated cells, and incubation
with insulin increased this association. In contrast to the findings
with Gab1 WT, SHP-2 was not bound to Gab1 Y627F. Therefore, we conclude
that tyrosine 627 of Gab1 is the major interaction site of Gab1 with
SH2 domains SHP-2 demonstrated using both two-hybrid system and intact
cells.
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PH to interact with p85 in the presence or absence of IR
was tested using the modified yeast two-hybrid system. In the absence
of IR, we did not see binding of Gab1 to p85 (Fig. 5A). In contrast, in the presence of IR, interaction
was detectable with GAD-Gab1 as well as with GAD-Gab1 PH. No
significant difference was observed between GAD-Gab1 PH and GAD-Gab1
WT. These results suggest that, in this system, the Gab1 PH domain is
not involved in Gab1 phosphorylation by IR.
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PH HA-Gab1 were immunoprecipitated with antibodies to
HA from lysates of 293 EBNA cells overexpressing the respective
constructs. Purified receptors activated by addition of insulin were
added to pellets containing Gab1 WT or Gab1 PH. Phosphorylation was
initiated by the addition of 30 µM ATP, 8 mM
MgCl2, and 4 mM MnCl2, and the
reaction was stopped after 30 min. Phosphorylated Gab1 was revealed by
Western blotting using an antibody to phosphotyrosine. Figure 5B
shows
that Gab1 WT and Gab1 PH are phosphorylated in vitro by
activated IRs. No tyrosine phosphorylation was detected in the absence
of receptor or in the absence of insulin. These results indicate that
in vitro, Gab1 is a direct substrate of the purified IR, and
that deletion of the Gab1 PH domain does not prevent Gab1 tyrosine
phosphorylation.
Finally, we studied in 293 EBNA cells insulin-induced
phosphorylation of WT Gab1 and Gab1 PH and their hormone-induced
association with SHP-2. Transfected cells expressing WT or PH
HA-Gab1 constructs were incubated with insulin or buffer, after which
cell lysates were prepared and HA-Gab1 was immunoprecipitated with an
antibody to HA. Tyrosine- phosphorylated Gab1 was revealed by Western
blotting with an antibody to phosphotyrosine, and its association with
SHP-2 was monitored by immunoblotting with antibodies to SHP-2. As a
control of expression and immunoprecipitation, one third of the HA-Gab1
immunoprecipitates was analyzed by immunoblotting with antibodies to
HA. In all experiments, the expression levels of WT and PH Gab1 were
similar (data not shown). As shown in Fig. 6, basal
phosphorylation of Gab1 WT and association with SHP-2 were detected in
unstimulated cells. Insulin stimulation enhanced tyrosine
phosphorylation of Gab1 WT by approximately 5-fold and concomitantly
increased Gab1 association to SHP-2. In contrast, we did not observe
phosphorylation of Gab1 PH and association with SHP-2 either in
stimulated or in unstimulated cells. These data indicate that, in
intact cells, activated IRs induce Gab1 tyrosine phosphorylation and
its association with SHP-2. In addition, the Gab1 PH domain appears to
be crucial for the occurrence of these insulin actions. Taking our
results as a whole, we conclude that Gab1 is a direct substrate of the
IR, and that in intact cells Gab1 PH domain is necessary to allow Gab1
tyrosine phosphorylation and association with SHP-2 after insulin
stimulation. However, the PH domain does not appear to be required in a
cell-free system or in the yeast two-hybrid system.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present work, using different approaches, we characterized interaction between 1) Gab1 and IR, and 2) Gab1 and SH2 domain-containing proteins such as p85-regulatory subunit of PI3-K and phosphotyrosine phosphatase, SHP-2.
With the classic two-hybrid system, we failed to detect interaction between Gab1 and IR, or IGF-I R. Similarly, we did not find interaction of Gab1 with p85 or with SHP-2.
Therefore, we engineered a modified two-hybrid vector in which the IR ß cDNA was subcloned under control of the methionine-repressible promoter MET25. We found that the presence of a functional IR ß leads to interaction between Gab1 and p85, or between Gab1 and SHP-2. Therefore, we conclude that phosphorylation of Gab1 by IR is a prerequisite for the induction in the yeast two-hybrid system of an efficient interaction with the SH2 domains of p85 and of SHP-2.
Previous studies performed in our laboratory have shown that IRS-1 interacts with p85 in the classic yeast two-hybrid system, i.e. without expression of IR. However, compared with the interaction seen in the absence of IR, a 4-fold increase in interaction of IRS-1 with p85 is seen when IR is expressed (data not shown). These data suggest that IRS-1 tyrosine phosphorylation by tyrosine kinases present in yeast is insufficient to generate the full-blown interaction between IRS-1 and p85.
Holgado-Madruga et al. (17) have shown that Gab1 isolated from insulin-induced A431 cells coimmunoprecipitated with the p85 regulatory subunit of PI3-K and with the phosphotyrosine phosphatase SHP-2. Using the modified two-hybrid system and coimmunoprecipitation in intact cells, we studied the phosphorylated Gab1 tyrosines possibly involved in interaction between Gab1 and p85, or between Gab1 and SHP-2. In relation to association with p85, tyrosines 447, 472, and 589 appear to be crucial for the interaction of Gab1 with PI3-K. Previous studies have shown that tyrosyl-phosphorylated IRS-1 peptides containing a single YXXM motif activate PI3-K in vitro. Furthermore, mutation of either SH2 domain significantly reduced phosphopeptide binding to p85 and decreased PI3-K activation by IRS-1 by 50% (25). Taking these findings together, it is tempting to imagine a two-step process in which first phosphotyrosine (e.g. tyrosine 472) interacts with one of the two SH2 domains (amino- or carboxy-terminal), and subsequently the other tyrosine (447 or 589) binds to the second SH2 domain. This bivalent binding would stabilize the interaction between Gab1 and p85 and would lead to full-blown PI3-K activation by Gab1.
Tyrosines 183 and 627 are present in a potential binding site for the SH2 domains of SHP-2 (YXXL or YXXI). We found that substitution of tyrosine 627 on Gab1 abolishes interaction between Gab1 and SHP-2 in yeast and in intact mammalian cells. In contrast, mutation of tyrosine 183 does not modify interaction of Gab1 with SHP-2. We have previously demonstrated that IRS-1 phosphotyrosines 1172 and 1222, present in YXXL and YXXI motifs, respectively, are the interaction sites of IRS-1 with SHP-2 in intact cells (5). Contrary to IRS-1, only one site is important for interaction of Gab1 with the SH2 domains of SHP-2. It remains to be determined whether SHP-2 is capable of dephosphorylating Gab1 and whether this dephosphorylation is dependent on its association with Gab1.
Finally, we investigated the role of Gab1 PH domain in Gab1 interaction with IR and its phosphorylation and association with SHP-2. It has been shown previously in hematopoietic 32D cells, that the IRS-1 PH domain, but not its PTB domain, is essential for insulin-stimulated IRS-1 tyrosine phosphorylation and subsequent stimulation of PI3-K (24). Knowing that Gab1 contains only a PH domain and no PTB domain, we examined the role of the Gab1 PH domain in the Gab1/IR interaction. We found that in intact cells the Gab1 PH domain is essential for insulin-induced Gab1 tyrosine phosphorylation and its association with SHP-2. In contrast, when the IR is expressed at high levels or when Gab1 and IR are colocalized, for example in in vitro experiments and in yeast two-hybrid system, deletion of the PH domain has no effect on Gab1 tyrosine phosphorylation by IRs. Our observations are in agreement with recent studies in 32D hematopoietic cells showing that, in the presence of low levels of receptor, the PH domain of IRS-1 is essential for insulin-stimulated IRS-1 tyrosine phosphorylation, PI3-K activity, and p70s6k stimulation (24). In addition, other studies have shown that deletion of the PH domain of IRS-1 has no effect on in vitro phosphorylation by the purified IR (26). Crystal structure analysis of PH domains found in several unrelated proteins suggests that the end of the structure is open and may represent an interaction site with another protein(s) (27). Further, it has been demonstrated that PH domains may associate with membrane phospholipids and thereby recruit signaling proteins to the membrane (28, 29). Taking these findings together, we propose that the PH domain of Gab1 may interact with membrane phospholipids to permit recruitment of Gab1 to proximity of IR, resulting its subsequent phosphorylation and transduction of insulin responses. Since Gab1 does not have a PTB domain, it remains to be determined which region on Gab1 drives the interaction with IR leading to its phosphorylation.
In summary, using a modified version of the yeast two-hybrid system, we have demonstrated which phosphorylated tyrosine residues of Gab1 are required for interaction of 1) Gab1 and p85 or 2) Gab1 and SHP-2. Further, our data indicate that in intact mammalian cells, the Gab1 PH domain plays an important role in mediation of interaction with IR.
A general picture emerges in which Gab1 is a direct substrate of the IR and plays a role of adaptor for several SH2-containing proteins. In addition, this newly identified IRS protein is also a substrate for other tyrosine kinases such as EGF receptor and c-Met receptor. An urgent challenge is to elucidate the precise role of Gab1 compared with other IRS proteins in transmission of the insulin pleiotropic effects.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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cDNA was a gift from J. E.
Pessin (Iowa City, IA). Human IR cDNA was provided by A. Ullrich
(Munich, Germany). Antibodies to phosphotyrosine and to human Gab1 were
obtained from Upstate Biotechnology (Lake Placid, NY). Antibodies to
human SHP-2 were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Synthetic complete (SC) minimal yeast media lacking the
appropriate amino acids were from BIO 101 (La Jolla, CA).
Oligonucleotides were purchased from Genset (Paris, France) and
chlorophenol red-ß-D-galactopyranoside (CPRG) was from
Boehringer Mannheim (Meylan, France). Insulin was kindly provided by
Novo-Nordisk (Copenhagen, Denmark). Triton X-100 and reagents for
SDS/PAGE were from Bio-Rad (Richmond, CA). All other chemical reagents
were obtained from Sigma Chemical Co. (St. Louis, MO).
Plasmid Construction
For most constructions, we introduced convenient restriction
endonuclease sites to each end of the desired cDNA fragment by PCR to
allow the in-frame insertion into the expression vector. The
full-length human Gab-1 cDNA was subcloned in frame with the Gal4
activation domain into the two-hybrid expression vector pACT II. The
coding sequence of the IR cytoplasmic domain (amino acids 944-1343)
(34) was amplified by PCR and then inserted in the plasmid pVJL9 3H
downstream of the repressible promoter MET25 in frame with the HA
epitope and a nuclear localization signal. The full-length human p85
and the n/c SH2 SHP-2 cDNA were subcloned in pVJL9 3H in frame with the
DNA-binding domain of lexA. The plasmid encoding GAD-IRS-1 construct
(IRS-1 amino acids 5–1235) was obtained as previously described (22).
All point mutations and deletions of different proteins were generated
by site-directed mutagenesis using the Stratagene QuikChange Kit (La
Jolla, CA). Point mutations were verified by DNA sequence analysis.
Yeast Strain, Culture Media, Transformation, and Reporter Gene
Expression
The genotype of the Saccharomyces cerevisiae reporter
strain L40 is MAT a, trp1, leu2, his3,
LYS2::lexA-HIS3,
URA3::lexA-lacZ (30). L40 were grown at
30 C in YPD media containing 1% (wt/vol) yeast extract, 2% (wt/vol)
Bacto-Peptone, and 2% (wt/vol) glucose, or in SC yeast media lacking
the appropriate auxotrophic amino acids.
Yeast L40 was transformed simultaneously with the two indicated plasmids by the improved lithium acetate method of Gietz et al. (35). The transformants were grown on SC plates lacking tryptophan and leucine to select for the presence of pBTM116 and pACTII, respectively. Where indicated, medium without methionine was used to allow expression of IR ß-subunit.
After 48 h, the double transformants were patched on SC plates lacking tryptophan, leucine, and methionine for ß-galactosidase assays or on SC plates lacking tryptophan, leucine, methionine, and histidine to study histidine prototrophy. After 2 days at 30 C, the ß-galactosidase assay was performed by a color filter assay using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) as previously described (36). For quantitative studies of ß-galactosidase activity, liquid culture assays using CPRG as a substrate were carried out as described by Bartel et al. (36). Yeast extracts were incubated with 8 mM CPRG, and the increase in A574 was measured after 10 or 30 min. Results were expressed as Miller’s units: one unit of ß-galactosidase was defined as (A574 x 1000)/[A600 x volume (ml) x time (min)] (37).
Immunoblot Analysis of Expression and Tyrosine Phosphorylation of
Hybrid IR and GAD-Gab1 in Yeast
A single colony of each strain expressing the different IR and
Gab1 hybrids was cultured in 100 ml selective medium at 30 C until the
cells reached a density of 1.2 x 107 cells/ml. The
cells were pelleted and washed with water (10 ml). The dry pellet was
frozen on dry ice and then at -20 C for 10 min, resuspended in 500
µl of lysis buffer (50 mM HEPES, 150 mM NaCl,
10 mM EDTA, 10 mM
Na4P2O7, 2 mM vanadate,
100 mM NaF, 1% (vol/vol) Triton X-100, 0.5 mM
phenylmethylsulfonylfluoride, 100 U/ml aprotinin, and 20
µM leupeptin) and vortexed with glass beads (425–600
µm). Fusion proteins (HA-IR and GAD-Gab1) were immunoprecipitated
using antibodies to HA. After separation of the samples by SDS-PAGE
under reducing conditions, the proteins were transferred to a
polyvinylidene difluoride membrane (Immobilon, Millipore Corp.,
Bedford, MA). The membrane was probed with antibodies to
phosphotyrosine (1 µg/ml) and, as expression and immunoprecipitation
control, with antibodies to HA, as previously described (5). Finally,
antibody binding was visualized using [125I] protein A
and quantified using the PhosphoImager system (Bio-Rad).
Cell Culture and Transfection of 293 EBNA Cells
293 EBNA cells are human embryo kidney cells that constitutively
express the EBNA-1 protein from the Epstein Barr Virus (Invitrogen, San
Diego, CA). These cells were grown in DMEM supplemented with 5%
(vol/vol) FCS in the presence of 500 µg/ml geneticin (G418, GIBCO,
Grand Island, NY). Cells were transfected as described by Chen and
Okayama (38). Briefly, exponentially growing cells were trypsinized,
seeded at 3 x 106 cells per 10-cm plate, and
incubated overnight in 10 ml of growth medium. Then 10 µg of
supercoiled DNA were mixed with 0.5 ml of 0.25 M
CaCl2 and 0.5 ml of 2 x BBS (buffered saline
containing 50 mM BES, 280 mM NaCl, 1.5
mM Na2HPO4, pH 6.95). The mixture
was incubated for 30 min at room temperature before being added
dropwise to the cells. After incubation for 15–18 h at 35 C under 3%
CO2, the medium was removed, and cells were incubated with
growth medium for 8 h and then starved for 14 h in DMEM
containing 0.5% (vol/vol) FCS.
Tyrosine Phosphorylation of Gab1 in Intact Cells
Transfected 293 EBNA cells in 10-cm plates were stimulated with
insulin (10-7 M) for 5 min at 37 C and
solubilized on ice in lysis buffer B [50 mM HEPES, 150
mM NaCl, 10 mM EDTA, 10 mM
Na4P2O7, 2 mM vanadate,
100 mM NaF, 1% (vol/vol) Triton X-100, 0.5 mM
phenylmethylsulfonyl fluoride, 100 IU/ml Aprotinin, and 20
µM leupeptin]. Gab-1 was immunoprecipitated during 90
min at 4 C with antibodies to HA (ascites fluid 1:100) or with
antibodies to human Gab1 (2 µg/plate) preadsorbed on protein
G-Sepharose beads. Samples were analyzed by SDS-PAGE followed by
Western blotting with antibodies to phosphotyrosine (1 µg/ml) as
previously described (5). Proteins were revealed using
[125I]protein A followed by autoradiography.
Association of Gab-1 with SHP-2 in Intact Cells
Transfected 293 EBNA cells in 10-cm plates were incubated with
10 mM vanadate for 10 min before stimulation with insulin
(10-7 M) for 5 min at 37 C. After
solubilization of the cells in ice-cold lysis buffer B and
immunoprecipitation with antibodies to HA or with antibodies to Gab1,
the proteins were separated by SDS-PAGE and immunoblotted with
antibodies to HA, to Gab1, or to SHP-2 depending on the experiment.
PI3-Kinase Assay
Transfected 293 EBNA cells in 10-cm plates were stimulated with
insulin (10-7 M) for 5 min at 37 C. The PI3-K
activity was measured after immunoprecipitation of Gab1 with antibodies
to Gab1 as previously described (39). The phospholipids were analyzed
by TLC and autoradiography [32P]phosphate. Incorporation
into phosphatidylinositol 3-phosphate was quantified using the
PhosphoImager system (Bio-Rad).
In Vitro Phosphorylation of Gab1 by Wheat Germ
Agglutinin (WGA)-Purified IRs
Antibodies to HA were incubated with protein G-Sepharose for 45
min at 4 C. The pellets were washed twice with 50 mM HEPES,
150 mM NaCl, pH 7.6. Lysates from Gab1-transfected cells
were incubated with the HA antibody-containing pellets for 90 min at 4
C. The Gab1-containing pellets were washed twice with 50 mM
HEPES, 150 mM NaCl, containing 1% (vol/vol) Triton X-100.
WGA-purified IRs (300 fmol) (40) were incubated for 45 min with insulin
(10-7 M) before being added to the
Gab1-containing pellets. The phosphorylation reaction was initiated by
addition of 30 µM ATP, 8 mM
MgCl2, 4 mM MnCl2. After 30 min,
the pellets were washed three times with 50 mM HEPES, 150
mM NaCl, 10 mM EDTA, 10 mM
Na4P2O7, 2 mM vanadate,
100 mM NaF, 10% (vol/vol) glycerol, and 1% (vol/vol)
Triton X-100. Samples were resuspended into Laemmli sample buffer and
separated by SDS-PAGE followed by Western blotting with antibodies to
phosphotyrosine (1 µg/ml) as previously described (5). Proteins were
visualized using [125I]protein A followed by
autoradiography.
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ACKNOWLEDGMENTS |
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cDNA, J. Pouysségur for PECE-HA vector, and J.
Camonis for pVJL-modified vector. We also thank V. Baron and C. Sable
for critical reading of the manuscript. |
FOOTNOTES |
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This work was supported by funds from INSERM, Université de Nice-Sophia-Antipolis, Association pour la Recherche contre le Cancer (ARC Grant 6432), Ligue Nationale contre le Cancer, and Groupe LIPHA (Contract 9323). Stephane Rocchi has a student-fellowship from Ligue Nationale contre le Cancer.
Received for publication October 3, 1997. Revision received March 5, 1998. Accepted for publication April 8, 1998.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 H. Kameda, J. I. Risinger, B.-B. Han, S. J. Baek, J. C. Barrett, W. C. Glasgow, and T. E. Eling Identification of Epidermal Growth Factor Receptor- Grb2-associated Binder-1-SHP-2 Complex Formation and Its Functional Loss during Neoplastic Cell Progression Cell Growth Differ., June 1, 2001; 12(6): 307 - 318. [Abstract] [Full Text] [PDF]
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 U. Schaeper, N. H. Gehring, K. P. Fuchs, M. Sachs, B. Kempkes, and W. Birchmeier Coupling of Gab1 to C-Met, Grb2, and Shp2 Mediates Biological Responses J. Cell Biol., June 26, 2000; 149(7): 1419 - 1432. [Abstract] [Full Text] [PDF]
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J. M. Cunnick, J. F. Dorsey, T. Munoz-Antonia, L. Mei, and J. Wu Requirement of SHP2 Binding to Grb2-associated Binder-1 for Mitogen-activated Protein Kinase Activation in Response to Lysophosphatidic Acid and Epidermal Growth Factor J. Biol. Chem., April 28, 2000; 275(18): 13842 - 13848. [Abstract] [Full Text] [PDF]
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 L. Delahaye, S. Rocchi, and E. Van Obberghen Potential Involvement of FRS2 in Insulin Signaling Endocrinology, February 1, 2000; 141(2): 621 - 628. [Abstract] [Full Text] [PDF]
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J. M. Korhonen, F. A. Said, A. J. Wong, and D. R. Kaplan Gab1 Mediates Neurite Outgrowth, DNA Synthesis, and Survival in PC12 Cells J. Biol. Chem., December 24, 1999; 274(52): 37307 - 37314. [Abstract] [Full Text] [PDF]
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H. Maegawa, M. Hasegawa, S. Sugai, T. Obata, S. Ugi, K. Morino, K. Egawa, T. Fujita, T. Sakamoto, Y. Nishio, et al. Expression of a Dominant Negative SHP-2 in Transgenic Mice Induces Insulin Resistance J. Biol. Chem., October 15, 1999; 274(42): 30236 - 30243. [Abstract] [Full Text] [PDF]
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 C. Lecoq-Lafon, F. Verdier, S. Fichelson, S. Chretien, S. Gisselbrecht, C. Lacombe, and P. Mayeux Erythropoietin Induces the Tyrosine Phosphorylation of GAB1 and Its Association With SHC, SHP2, SHIP, and Phosphatidylinositol 3-Kinase Blood, April 15, 1999; 93(8): 2578 - 2585. [Abstract] [Full Text] [PDF]
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M. P. Czech and S. Corvera Signaling Mechanisms That Regulate Glucose Transport J. Biol. Chem., January 22, 1999; 274(4): 1865 - 1868. [Full Text] [PDF]
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L. Delahaye, I. Mothe-Satney, M. G. Myers, M. F. White, and E. Van Obberghen Interaction of Insulin Receptor Substrate-1 (IRS-1) with Phosphatidylinositol 3-Kinase: Effect of Substitution of Serine for Alanine in Potential IRS-1 Serine Phosphorylation Sites Endocrinology, December 1, 1998; 139(12): 4911 - 4919. [Abstract] [Full Text] [PDF]
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A. Janez, D. S. Worrall, T. Imamura, P. M. Sharma, and J. M. Olefsky The Osmotic Shock-induced Glucose Transport Pathway in 3T3-L1 Adipocytes Is Mediated by Gab-1 and Requires Gab-1-associated Phosphatidylinositol 3-Kinase Activity for Full Activation J. Biol. Chem., August 25, 2000; 275(35): 26870 - 26876. [Abstract] [Full Text] [PDF]
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S. Ali and S. Ali Recruitment of the Protein-tyrosine Phosphatase SHP-2 to the C-terminal Tyrosine of the Prolactin Receptor and to the Adaptor Protein Gab2 J. Biol. Chem., December 8, 2000; 275(50): 39073 - 39080. [Abstract] [Full Text] [PDF]
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J. M. Cunnick, L. Mei, C. A. Doupnik, and J. Wu Phosphotyrosines 627 and 659 of Gab1 Constitute a Bisphosphoryl Tyrosine-based Activation Motif (BTAM) Conferring Binding and Activation of SHP2 J. Biol. Chem., June 22, 2001; 276(26): 24380 - 24387. [Abstract] [Full Text] [PDF]
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R. J. Ingham, L. Santos, M. Dang-Lawson, M. Holgado-Madruga, P. Dudek, C. R. Maroun, A. J. Wong, L. Matsuuchi, and M. R. Gold The Gab1 Docking Protein Links the B Cell Antigen Receptor to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway and to the SHP2 Tyrosine Phosphatase J. Biol. Chem., April 6, 2001; 276(15): 12257 - 12265. [Abstract] [Full Text] [PDF]
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P. Peraldi, C. Filloux, B. Emanuelli, D. J. Hilton, and E. Van Obberghen Insulin Induces Suppressor of Cytokine Signaling-3 Tyrosine Phosphorylation through Janus-activated Kinase J. Biol. Chem., June 29, 2001; 276(27): 24614 - 24620. [Abstract] [Full Text] [PDF]
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C. F. Yu, B. Roshan, Z.-X. Liu, and L. G. Cantley ERK Regulates the Hepatocyte Growth Factor-mediated Interaction of Gab1 and the Phosphatidylinositol 3-Kinase J. Biol. Chem., August 24, 2001; 276(35): 32552 - 32558. [Abstract] [Full Text] [PDF]
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K. Almind, L. Delahaye, T. Hansen, E. Van Obberghen, O. Pedersen, and C. R. Kahn Characterization of the Met326Ile variant of phosphatidylinositol 3-kinase p85alpha PNAS, February 19, 2002; 99(4): 2124 - 2128. [Abstract] [Full Text] [PDF]
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