Determination of Gab1 (Grb2-Associated Binder-1) Interaction with Insulin Receptor-Signaling Molecules

Stéphane Rocchi, Sophie Tartare-Deckert, Joseph Murdaca, Marina Holgado-Madruga, Albert J. Wong and Emmanuel Van Obberghen

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The newly identified insulin receptor (IR) substrate, Gab1 [growth factor receptor bound 2 (Grb2)-associated binder-1] is rapidly phosphorylated on several tyrosine residues by the activated IR. Phosphorylated Gab1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins. These include the regulatory subunit p85 of phosphatidylinositol 3-kinase and phosphotyrosine phosphatase, SHP-2. In this report, using a modified version of the yeast two-hybrid system, we localized which Gab1 phospho-tyrosine residues are required for its interaction with phosphatidylinositol 3-kinase and with SHP-2. Our results demonstrate that to interact with p85 or SHP-2 SH2 domains, Gab1 must be tyrosine phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the major site for association with p85, while tyrosines 447 and 589 are participating in this process. Concerning Gab1/SHP-2 interaction, only mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains, suggesting the occurrence of a monovalent binding event. Finally, we examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR interaction and in Gab1 tyrosine phosphorylation by IR. Using the modified two-hybrid system and in vitro experiments, we found that the Gab1 PH domain is not important for IR/Gab1 interaction and for Gab1 tyrosine phosphorylation. In contrast, in intact mammalian cells, Gab1 PH domain appears to be crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The insulin receptor (IR) is a tyrosine kinase that becomes activated upon hormone binding (1, 2). Known direct targets of IR include the insulin receptor substrate (IRS) proteins (IRS-1, IRS-2, and IRS-3) (3, 4). They serve as multidocking molecules for several proteins, some of which have been recognized as key players in insulin signal transduction. These molecules include Grb2 (growth factor receptor bound 2), the regulatory subunits of phosphatidylinositol 3-kinase (PI3-K), p85 and p55, and the phosphotyrosine phosphatase SHP-2 (SH2-containing protein tyrosine phosphatase) (4, 5, 6). PI3-K is involved in several insulin responses, such as glucose transport, p70 S6 kinase activation, membrane ruffling, and mitogenesis (7, 8, 9, 10, 11). The role of SHP-2 in insulin signaling is likely to be complex as the phosphatase appears to function as a positive effector in the insulin-stimulated mitogen-activated protein kinase pathway and mitogenesis (12, 13, 14, 15), whereas it may also be an "attenuator" of insulin signaling by inducing dephosphorylation of IRS-1 (5, 16).

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-{gamma}, 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In a first series of experiments, we examined, in the yeast two-hybrid system, interaction of Gab1 with insulin-signaling molecules such as IR ß-subunit, insulin-like growth factor-I receptor (IGF-I R) ß-subunit, p85 regulatory subunit of PI3-K, and SH2 domains of phosphotyrosine phosphatase SHP-2. However, we failed to detect interaction between Gab1 and these molecules in our system (data not shown). We hypothesized that in yeast this lack of interaction between Gab1 and p85 or n/c SH2 SHP-2 could be due to absence of Gab1 tyrosine phosphorylation. To test this, we engineered a novel two-hybrid vector in which the IR ß-subunit cDNA was subcloned downstream of the methionine-repressible promoter MET25 (pVJL-HIR-3H). The full-length p85 cDNA or the cDNA corresponding to n/c SH2 domains of SHP-2 cDNA were also subcloned in this vector pVJL-HIR-3H in frame with the DNA-binding domain of lexA (they are called, respectively, pVJL-HIR-p85 and pVJL HIR n/c SH2 SHP-2) (Fig. 1Go). 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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Figure 1. Schematic Representation of the Modified Two-Hybrid System

Yeast is cotransformed with the plasmid pACT-Gab1 encoding GAD-Gab1 in combination with a new two-hybrid vector in which the IR ß cDNA was subcloned downstream of the methionine-repressible promoter MET25 and encoding LDBD-SHP-2 or -p85 constructs. Expression of IR ß is prevented in the presence of methionine, while absence of methionine allows expression of the IR ß.

 
We first verified that the two vectors, pVJL-HIR-p85 and pVJL-HIR-n/c SH2 SHP-2, were incapable by themselves, or in combination with an unrelated GAD (Gal4 activation domain) fusion protein, of activating expression of the two reporter genes (data not shown). We then tested interaction of full-length p85 with Gab1 in the presence or in the absence of IR ß-subunit (Fig. 2AGo). 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. 2BGo). 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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Figure 2. Gab-1 Binding to PI3-Kinase in the Yeast Two-Hybrid System Depends on the IR Kinase

A, The yeast strain L40 was cotransformed with a plasmid encoding GAD-Gab1 and with a plasmid encoding LDBD-p85 and IR ß. Transformants were isolated on selective plates in the absence or in the presence of methionine. Activation of ß-galactosidase was measured using the substrate CPRG, and indicated activities were calculated according to Miller. Values represent the average ± SE of six independent transformants. Similar results were obtained using the filter color assay or growth on SC plates lacking histidine. K1018A corresponds to IR ß in which lysine 1018 is replaced by alanine and is kinase-dead. B, Yeasts cotransformed with different IR ß constructs were grown overnight in 100 ml yeast medium. The yeasts were solubilized, and IR ß was immunoprecipitated with an antibody to hemagglutinin. The immunoprecipitated proteins were separated by SDS-PAGE under reducing conditions and transferred to an Immobilon P membrane. The membrane was probed with antibodies to hemagglutinin (upper part of the figure) or antibodies to phosphotyrosine (lower part of the membrane). A representative experiment is shown.

 
As a whole, these results validate our hypothesis that the IR kinase activity is required for tyrosine phosphorylation of GAB1 and for its association with p85 subunit of PI3-K and with phosphotyrosine phosphatase SHP-2.

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. 3AGo). As shown in Fig. 3AGo, 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. 3BGo). 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. 3BGo). 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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Figure 3. Identification of Gab1 Tyrosine Residues Are Implicated in Interaction with p85

A, Yeast was cotransformed with plasmids encoding the indicated GAD-Gab1 mutants and a plasmid encoding LDBD-p85 and IR ß. Transformants were isolated on selective plates. Activation of ß-galactosidase was measured using CPRG, and indicated activities were calculated according to Miller. Values represent the average ± SE of six independent transformants. Similar results were obtained by the filter color assay and growth on SC plates lacking histidine. Y447F, Y472F, and Y589F are mutated forms of GAD-Gab1 in which tyrosines 447, 472, and 589 were replaced by phenylalanine. B, Insulin-stimulated PI3-K activity associated with Gab1 mutants expressed in 293 EBNA cells. 293 EBNA cells transfected with plasmids expressing the various Gab1 forms were incubated for 5 min at 37 C in the absence or presence of 10-7 M insulin. The cells were solubilized, and Gab1 was immunoprecipitated with antibodies to Gab1. The PI3-K activity associated with Gab1 was measured as described in Materials and Methods and analyzed by TLC and autoradiography. [32P]Phosphate incorporation into PI3-P was quantified using a Molecular Imager (Bio-Rad). Results are presented as a percentage of the insulin-dependent increase in PI3-K activity associated with the Gab1 WT after subtraction of the basal activity. A representative experiment is shown.

 
We next studied interaction between Gab1 and phosphotyrosine phosphatase SHP-2. For several proteins, it has been shown that YXXL or YXXI motifs are potential binding sites for SHP-2 SH2 domains. In Gab1, two tyrosines are contained in a YXXL motif, i.e. tyrosines 183 and 627. Hence we constructed mutants of the yeast two-hybrid construct, GAD-Gab1, in which tyrosines 183 and 627 were replaced by phenylalanine (GAD-Gab1 Y183F and GAD-Gab1 Y627F). Using a modified yeast two-hybrid system, we tested their ability to interact with LDBD-n/c SH2 SHP-2 in the presence of IR ß (Fig. 4AGo). 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. 4BGo). 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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Figure 4. Identification of Gab1 Tyrosine Residues Are Implicated in Interaction with SHP-2

A, Yeast was cotransformed with plasmids encoding indicated GAD-Gab1 mutated forms in combination with a plasmid encoding LDBD-n/c SH2 SHP-2 and IR ß. Transformants were isolated on selective plates. Activation of ß-galactosidase was measured as described in legend to Fig. 3Go. Y183F and Y627F are mutated forms of GAD-Gab1 in which tyrosines 183 and 627 were replaced by phenylalanine. B, 293 EBNA cells were transfected with plasmids expressing Gab1 WT or Y627F. Then, cells were incubated with 10 mM sodium orthovanadate for 10 min at 37 C and thereafter for 5 min at 37 C in the absence or presence of 10-7 M insulin. The cells were solubilized, and the extracted proteins were subjected to immunoprecipitation with antibodies to Gab1. The immunoprecipitated proteins were separated by SDS-PAGE under reducing conditions and transferred to an Immobilon P membrane. The membrane was probed with antibodies to Gab1 (upper part of the membrane) and antibodies to SHP-2 (lower part of the membrane). A representative experiment is shown.

 
Finally, we investigated the role of the Gab1 PH domain in tyrosine phosphorylation of Gab1 by IR. It has been shown previously that the IRS-1 PH domain is essential for insulin-stimulated tyrosine phosphorylation of IRS-1, IRS-1-associated PI3-K activity, and subsequent p70 S6 kinase phosphorylation (23, 24). By analogy, we hypothesized that the Gab1 PH domain could play a role in the Gab1/IR interaction. The Gab1 PH domain was deleted, and the ability of GAD-Gab1 {Delta}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. 5AGo). In contrast, in the presence of IR, interaction was detectable with GAD-Gab1 as well as with GAD-Gab1 {Delta}PH. No significant difference was observed between GAD-Gab1 {Delta}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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Figure 5. Analysis of the Role of the Gab1 PH Domain in Interaction of Gab1 with the IR Using a Modified Yeast Two-Hybrid System and in Vitro Phosphorylation

A, The yeast strain L40 was cotransformed with a plasmid encoding GAD-Gab1 or Gab1 {Delta}PH and with a plasmid encoding LDBD-p85 and the IR ß. Transformants were isolated on selective plates in the absence or in the presence of methionine. Activation of ß-galactosidase was measured using CPRG, and indicated activities were calculated according to Miller. Values represent the average ± SE of six independent transformants. B, 293 cells were transfected with plasmid expressing various HA-tagged forms of Gab1. After cell lysis, HA-Gab1 was immunoprecipitated with a specific antibody to hemagglutinin. In parallel, WGA-purified IRs were incubated in the absence or presence of insulin. The receptors were added to pellets containing immunoprecipitated Gab1. Tyrosine phosphorylation reaction was initiated by addition of phosphorylation buffer and was stopped after 30 min. Phosphorylated proteins were separated by SDS-PAGE under reducing conditions and transferred to an Immobilon P membrane. The membrane was probed with antibodies to phosphotyrosine. A representative experiment is shown.

 
To confirm this, we examined the role of the PH domain in Gab1 in vitro tyrosine phosphorylation by partially purified IR. To do this, WT and {Delta}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 {Delta}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 5BGo shows that Gab1 WT and Gab1 {Delta}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 {Delta}PH and their hormone-induced association with SHP-2. Transfected cells expressing WT or {Delta}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 {Delta}PH Gab1 were similar (data not shown). As shown in Fig. 6Go, 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 {Delta}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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Figure 6. Gab1 Tyrosine Phosphorylation and Association of Gab1 with SHP-2 in Intact Cells

293 EBNA cells were transfected with plasmids expressing various HA-Gab1 forms. Then, cells were incubated with 10 mM sodium orthovanadate for 20 min at 37 °C and thereafter for 5 min at 37 °C in the absence or presence of 10-7 M insulin. Cells were solubilized and extracted proteins were subjected to immunoprecipitation with antibodies to hemagglutinin. The immunoprecipitated proteins were separated by SDS-PAGE under reducing conditions and transferred to an Immobilon P membrane. The membrane was probed with antibodies to phosphotyrosine (upper part of the membrane) and by antibodies to SHP-2 (lower part of the membrane). A representative experiment is shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Upon hormone binding, the IR undergoes multisite autophosphorylation on tyrosine residues, including the juxtamembrane tyrosine 960, which becomes a key binding site for the receptor substrates, IRS-1, IRS-2, IRS-3, and Shc (3, 19, 20, 21, 22). Recently, Gab1 has been added to the list of IR substrates (17). This newly identified protein contains in its N terminus a PH domain that is highly homologous to the IRS-1 PH domain. Its C-terminal part carries several potential tyrosine phosphorylation sites included in motifs that are expected to function as binding sites for SH2 domain-containing proteins. In contrast with IRS-1, Gab1 does not possess a PTB domain.

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The yeast strain L40 and the plasmid encoding the Gal4 activation domain GAD-Raf were provided by A. Votjek (Seattle, WA) (30); the yeast expression plasmid pACTII was from S. Elledge (Houston, TX) (31). The pECE/HA-tagged expression vector and the antibodies to HA were a gift from J. Pouysségur (Nice, France) (32). The pVJL9 3H yeast expression plasmid was a gift from J. Camonis (Paris, France). This plasmid has been described (33). Human SHP-2 cDNA was obtained from E. Fischer (Seattle, WA). p85{alpha} 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.


    ACKNOWLEDGMENTS
 
We thank A. Vojtek and S. Elledge for L40 strain and yeast plasmids, E. Fischer for SHP-2 cDNA, M. F. White and C. R. Kahn for rat IRS-1 cDNA, A. Ullrich for human IR cDNA, J. E. Pessin for p85{alpha} 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
 
Address requests for reprints to: Emmanuel Van Obberghen, INSERM U145, Faculté de Médecine, avenue de Valombrose, 06107 Nice Cédex 2, France. E-mail: vanobbeg{at}unice.fr

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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 MATERIALS AND METHODS
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