Gab-1-mediated IGF-1 Signaling in IRS-1-deficient 3T3 Fibroblasts*

The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and in-sulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-bind-ing protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1-deficient embryos exhibit a 70–80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c -fos and egr-1 but unaltered activation of the mitogen-activated protein kinases extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2. Reconstitution of IRS-1 expression in IRS-1-deficient fibroblasts by retroviral mediated gene trans-duction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration of egr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation. were subjected to immunoprecipitation ( with antiphosphotyrosine (anti- PY monoclonal antibody and subsequently immunoblotted ( IB ) with anti-Gab-1 antiserum. The representative of multiple experiments. No tyrosine phosphorylation of Gab-1 could be detected in all cell lines five minutes after IGF-1 EGF in reduced mobility (**) and the tyrosine phosphorylation of Gab-1 in KO 1 cells. were subjected to immunoprecipitation with antiphosphotyrosine monoclonal antibody and subsequently immuno- blotted with antiphosphotyrosine monoclonal antibody. experiment shown is representative of at least three independent experiments. The IGF-1-stimulated phosphorylation of IRS-1 in KO IRS-1 in and cells could be detected after No de- tectable tyrosine-phosphorylated could be appropriate molecular weight range

The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and insulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-binding protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1-deficient embryos exhibit a 70 -80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases extracellular signalregulated kinase-1 and extracellular signal-regulated kinase-2. Reconstitution of IRS-1 expression in IRS-1deficient fibroblasts by retroviral mediated gene transduction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration of egr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation.
Gab-1 was initially identified in a cDNA library of glioblastoma tumors based upon its ability to bind Grb-2 (18). Gab-1 shares functional and structural homology with the IRS family of proteins, including an N-terminal pleckstrin homology (PH) domain, several tyrosine phosphorylation sites, and two potential SH3 binding proline-rich regions. Several studies have also demonstrated that Gab-1 functions as a substrate and docking protein downstream in the signaling pathways of several receptor tyrosine kinases, including the receptors for epidermal growth factor (EGF), insulin, fibroblast growth factor, nerve growth factor, hepatocyte growth factor, and various cytokines such as interleukin-3, interleukin-6, interferon-␣, and interferon-␥ (18 -21). Like the IRS proteins, tyrosine-phosphorylated Gab-1 has been shown to interact with several Src homologous and collagen-containing adapter molecules, such as the p85 regulatory subunit of PI 3-kinase, SH2 domain containing phosphatase-2, and Grb-2 (18,21,22). In addition, Gab-1 can interact with phospholipase C-␥, which does not interact with IRS-1.
Previous studies in our laboratory have demonstrated a specific role for IRS-1 in insulin and IGF-1-mediated signaling (23). Thus, although there is a high degree of homology and structural similarity among the IRS family members, cells with a homozygous deficiency of IRS-1 show a reduced mitogenic response that cannot be recovered by the overexpression of IRS-2 (23). These data highlight the potential for specific IRS proteins to uniquely mediate distinct downstream biological events. In the present study, we have investigated the role of Gab-1 in mediating IGF-1 signaling by overexpressing Gab-1 in IRS-1-deficient 3T3 fibroblasts by retroviral mediated gene transfer. Our studies revealed that Gab-1 overexpression produces a unique pattern of downstream signals in IRS-1-deficient fibroblasts, despite an absence of detectable IGF-1-stimulated tyrosine phosphorylation, suggesting a novel role and mechanism for this substrate in insulin/IGF-1 action. described previously (24). The cDNA for murine egr-1 was generously provided by B. Spiegleman (Dana-Farber Cancer Institute, Boston, MA). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) reagents were from Bio-Rad; medium and supplements were purchased from Life Technologies, Inc.; fetal bovine serum was from Sigma; and nitrocellulose used for Western blots and nylon membrane used for Northern blots were purchased from Schleicher & Schuell.
Cell Culture-Female mice from breeding pairs of IRS-1 ϩ/Ϫ mice were sacrificed at day 16.5 postconception. Embryos were dissected from the uterus, and extra embryonic membranes, as well as viscera and heads, were removed. The embryos were soaked overnight in 0.25% trypsin-EDTA at 4°C. Meanwhile, the genotype of the embryos was determined by using DNA extracted from the removed tissues by polymerase chain reaction as described previously (25). The following day, the tryptic digest was activated at 37°C for 15 min and then inactivated by the addition of 10 ml of Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were then suspended by extensive pipetting, counted, and plated at a density of 10 6 /cm 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Adherent cells were allowed to reach confluency once and then passaged according to the 3T3 protocol of Todaro and Green (26). The reconstitution of IRS-1 in IRS-1 Ϫ/Ϫ 3T3 cell lines was achieved as described previously (23). To overexpress Gab-1 in IRS-1 Ϫ/Ϫ 3T3 cell lines, a retroviral expression vector was constructed by inserting a blunt-ended XbaI and HpaI fragment from pBSGab-1 into the SnaBI site of pBabe. The correct orientation of the insert was monitored by BamHI digestion and partial sequence analysis. BOSC 23 cells were transiently transfected with 20 g of plasmid DNA/10-cm diameter dish. Cells were refed 12-16 h after transfection, and Polybrene (8 g/ml)-supplemented virus-containing supernatant was transferred to the target cells 48 h after transfection. After an overnight infection period, target cells were refed. Selection was begun by using 2.5 g of puromycin (Sigma)/ml 48 h after infection.
Production of Anti-Gab-1 Antibodies-A C-terminal fragment of human Gab-1 (amino acids 600 -694) was expressed as a glutathione S-transferase fusion protein. This purified fragment was then used as antigen for the immunization of rabbits.
Immunoprecipitation and Western Blot Analysis-For stimulation of IGF-1-and EGF-mediated responses, cells were serum-starved overnight in medium containing 0.1% bovine serum albumin and then exposed to IGF-1 or EGF at a final concentration of 10 nM or 20 ng/ml, respectively, unless noted otherwise, in Dulbecco's modified Eagle's medium supplemented with 0.1% bovine serum albumin for the indicated times. Protein extracts were prepared using buffer A (50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM Na 3 VO 4 , 20 mM Na 4 P 2 O 2 , 100 mM NaF, 1% Triton X-100, 2 mM phenylmethylsulphonyl fluoride, 20 g of aprotinin/ml) for 15 min at 4°C, and insoluble protein was removed by centrifugation at 12,000 rpm in a microcentrifuge. Protein content was measured by the method of Bradford. The extract was then resolved directly in SDS-polyacrylamide gels after boiling in Laemmli SDS sample buffer or subjected to immunoprecipitation with the indicated antibodies. For immunoprecipitation, 1 mg of cellular protein was incubated with the indicated antibodies for 2 h at 4°C. Immunocomplexes were collected by adding 50 l of a 50% slurry of protein A-Sepharose (Amersham Pharmacia Biotech) in phosphatebuffered saline for 1 h at 4°C. After two washes in buffer A containing 0.1% SDS, protein complexes were liberated from beads by boiling in SDS sample buffer for 3 min. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose. Immunodetection was performed after the membranes were blocked for 2 h at room temperature in 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 0.05% Tween-20, 3% bovine serum albumin followed by incubation with the appropriate antibody at the indicated concentration for 2 h at room temperature. Specifically bound primary antibodies were detected with 125 I-protein A and autoradiography.
In Vitro Kinase Reactions-Two hundred micrograms of cellular protein was subjected to immunoprecipitation with a polyclonal antiserum to ERK-1 and ERK-2, and immunocomplexes were collected with protein A-Sepharose beads. After two washes in lysis buffer and two washes in 50 mM HEPES, 10 mM MgCl 2 (pH 7.4), beads were resuspended in 30 l of reaction buffer, and the reactions were started by the addition of 2 Ci of [␥]ATP, 100 M ATP, 2 M protein kinase C inhibitor (Sigma), and 5 g of myelin basic protein (Sigma). Reactions were allowed to proceed for 15 min at 30°C and stopped by the addition of 12 l of 5ϫ Laemmli sample buffer; the reaction mixtures were boiled for 5 min, resolved by SDS-PAGE (12% gel), electroblotted onto polyvinylidene difluoride membranes, and exposed to autoradiography. Bands of labeled substrates were quantified with a Molecular Dynamics densitometer.
Northern Blot Analysis-Northern blot analysis was performed according to standard techniques in denaturing formaldehyde containing agarose gels. After stimulation with 10 nM IGF-1 for the indicated time, total cellular RNA was isolated by using RNAzol B; 10 -20 g of RNA was subjected to electrophoresis in 1% agarose gels. Ethidium bromide staining of the gels confirmed equal loading and integrity of the RNA. After transfer to nylon membrane, blots were hybridized with the indicated probe. A probe specific for IRS-1 was generated by the directional cloning of an EcoRI/SalI fragment corresponding to the C-terminal 1010 base pairs of murine IRS-1 into pBluescript. A probe specific for IRS-2 was generated by polymerase chain reaction amplification of the C-terminal 970 base pairs (3100 -4070) of murine IRS-2 and subsequent cloning into the PCRII cloning vector. A Gab-1-specific probe corresponding to the first 1679 base pairs of human Gab-1 was constructed by XhoI digestion of pBSGab-1 and subsequent vector religation. Egr-1 expression was assessed using a full-length cDNA to the human egr-1 gene. All probes used for Northern blot analysis were labeled using a random primed labeling kit (Amersham Pharmacia Biotech). After an overnight incubation at 42°C, blots were washed three times in 1ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% SDS for 20 min each time at room temperature and for 30 min in 0.1ϫ SSC, 0.1%SDS at 52°C. Filters were air dried and subjected to autoradiography.
[ 3 H]Thymidine Incorporation into DNA-Cells were plated at a density of 2 ϫ 10 5 /well in 24-well dishes. After 1 day, the medium was changed to Dulbecco's modified Eagle's medium with 0.1% insulin-free bovine serum albumin for 72 h. The cells were then stimulated with the indicated concentrations of IGF-1 for 16 h and pulsed with 2 Ci of [methyl-3 H]thymidine (NEN Life Science Products )/well for 1 h at 37°C. After two washes with ice-cold phosphate-buffered saline, cells were lysed for 1 h in 0.1% SDS. Trichloroacetate-precipitable DNA was then counted for incorporated radioactivity. All assays were performed in triplicate.

Overexpression of Gab-1 and Reconstitution of IRS-1-IRS-
1-deficient 3T3 cells were established from embryonic fibroblasts derived from IRS-1-deficient mice (23). As described previously, these cells have a reduced response to IGF-1-stimulated mitogenesis and early gene expression (23). To investigate the potential role of Gab-1 in IGF-1-mediated signaling, we overexpressed Gab-1 (KO ϩGab-1 ) or IRS-1 (KO ϩIRS-1 ) in these IRS-1 Ϫ/Ϫ 3T3 cell lines by retroviral mediated gene transduction. By Northern blot analysis, no IRS-1 message could be detected in IRS-1 KO or KO ϩGab-1 cells using a probe specific for IRS-1, whereas in KO cells reconstituted with IRS-1 using retroviral mediated gene transfer, a transcript of 9.0 kilobases corresponding to the human IRS-1 mRNA could be easily detected (Fig. 1A). Northern blot analysis using an IRS-2-specific probe indicated that all cell lines expressed equivalent levels of IRS-2 message. On parallel blots of total RNA, no Gab-1 message was detected in IRS-1 Ϫ/Ϫ cells either before or after IRS-1 expression using a probe specific for human Gab-1. By contrast, a 4.2-kilobase transcript corresponding to Gab-1 mRNA was easily detected in KO cells transfected with Gab-1.
The level of protein expression of IRS-1, IRS-2, and Gab-1 was assessed by Western blot analysis on cell lysates of the same cells. Consistent with Northern blot analysis, immunoblotting using an antibody specific to the C-terminal region of IRS-1 confirmed the absence of IRS-1 protein in KO and KO ϩGab-1 cells as well as the reconstitution of IRS-1 in KO ϩIRS-1 cell lines (Fig. 1B). By contrast, all cell lines expressed equivalent levels of IRS-2 protein, as determined by Western blot analysis with anti-IRS-2 antibodies. Immunoblots probed with an antibody raised against the C terminus of Gab-1 indicated the presence of low endogenous expression of Gab-1 in KO and KO ϩIRS-1 cells and a 3.4-fold overexpression of human Gab-1 when compared with KO and KO ϩIRS-1 cell lines. These data confirm the reconstitution of IRS-1 and the overexpression of Gab-1 in KO ϩIRS-1 and KO ϩGab-1 cell lines, respectively, and indicate that no alterations in the level of IRS-2 protein expression resulted from the reconstitution of IRS-1 or overexpression of Gab-1.
Absence of IGF-1-stimulated Gab-1 Tyrosine Phosphorylation-To determine whether Gab-1 is capable of becoming phosphorylated on tyrosine residues in response to IGF-1 stimulation, whole cell lysates from IGF-1 and EGF stimulated KO; KO ϩIRS-1 and KO ϩGab-1 cell lines were subjected to immunoprecipitation using antiphosphotyrosine antibody and immunoblotting with anti-Gab-1 antibodies. In KO ϩGab-1 cells, some basal tyrosine phosphorylation of Gab-1 could be observed ( Fig.  2A). Stimulation with EGF rapidly and markedly increased the level of Gab-1 phosphorylation (18,19). In addition, this phosphorylation resulted in decreased mobility of the protein on SDS-PAGE. In contrast, the level of tyrosine phosphorylation was not augmented in response to IGF-1 stimulation, and no mobility shift of the protein was observed. Similar results were obtained when whole cell lysates prepared from IGF-1-stimulated cell lines were subjected to immunoprecipitation with anti-Gab-1 antibody followed by immunoblotting with antiphosphotyrosine (data not shown). Consistent with these findings, IGF-1 stimulation failed to increase the association of Gab-1 with SH2 domain containing adapter proteins such as Grb-2 and the p85 regulatory subunit of PI 3-kinase (data not shown).
To determine the level of IGF-1-stimulated IRS-1 and IRS-2 tyrosine phosphorylation and to confirm the absence of tyrosine-phosphorylated Gab-1 in response to IGF-1, whole cell lysates from IGF-1 stimulated KO; KO ϩIRS-1 and KO ϩGab-1 cells were subjected to immunoprecipitation and immunoblotting using antiphosphotyrosine antibody. As previously observed, KO cells lack IRS-1 tyrosine phoshorylation and the only high molecular weight phosphoprotein detected corresponded to IRS-2, as indicated by its relatively retarded mobility on SDS-gels. In KO ϩIRS-1 cells, IGF-1 stimulation resulted in a marked increase in IRS-1 tyrosine phosphorylation and lower levels of IRS-2 tyrosine phosphorylation. In KO ϩGab-1 cells, IGF-1 stimulation resulted in the tyrosine phosphorylation of IRS-2 comparable to that seen in KO cells, but no tyrosine phosphorylated proteins were detected in the appropriate molecular weight range of Gab-1 (Fig. 2B). Thus, although Gab-1 is capable of undergoing tyrosine phosphorylation in response to EGF stimulation in 3T3 cells, it does not undergo tyrosine phosphorylation in response to IGF-1 stimulation. In addition, these data indicate that the overexpression of Gab-1 does not alter the extent or ability of IRS-2 to become tyrosine phosphorylated in response to IGF-1 stimulation, i.e. it does not appear to be a competitive substrate for the IGF-1 receptor.
MAP Kinase Activation Is Unaltered in KO ϩ Gab-1 Cells-An important pathway activated by IGF-1 stimulation and involved in gene expression and the mitogenic response is the activation of the MAP kinase signaling cascade. Upon receptor activation, phosphorylated IRS-1, IRS-2, and SHC bind to the SH2 domain of the adapter molecular Grb-2, which in turn binds the guanine nucleotide exchange factor SOS, allowing for activation of Ras and initiation of a signaling cascade of serine/ threonine kinases, including Raf, mitogen-activated protein kinase kinase, ERK-1, and ERK-2. To assess the potential impact of Gab-1 in mediating signaling via this pathway, we performed in vitro kinase assays using anti-MAP kinase immunoprecipitates and myelin basic protein as a substrate. As can be seen in Fig. 3, the MAP kinases ERK-1 and ERK-2 were activated to the same extent and also followed the same time course in response to IGF-1 in all cell lines. These data indicate that both Gab-1 and IRS-1, although capable of binding Grb-2 and activating the MAP kinases, had no apparent effect on the level and time course of IGF-1-stimulated activation of ERK-1 and ERK-2.
Immediate-Early Gene Expression Deficit in KO Cells Is Restored by Gab-1-Mice made IRS-1 deficient by targeted gene knockout exhibit intrauterine and postnatal growth retardation because of resistance to the actions of IGF-1 and insulin. In culture, IRS-1-deficient 3T3 cells also exhibit a decrease in IGF-1-mediated egr-1 and c-fos expression (23). To assess the potential role for Gab-1 in mediating immediate-early gene expression, IGF-1-stimulated expression of the immediateearly gene egr-1 was assessed by Northern blot analysis. As previously reported, KO cells showed a markedly reduced expression of egr-1 in response to IGF-1 stimulation over a 60min time course (Fig. 4A). This deficit in egr-1 expression could be restored by the reconstitution of IRS-1 in KO ϩIRS-1 cells. Quantification of three independent experiments revealed a 50 -60% reduction of egr-1 induction in KO cells when compared with KO ϩIRS-1 cells. Interestingly, Gab-1 overexpression was also capable of restoring the defects in egr-1 expression observed in KO cells to levels 80 -90% that of KO ϩIRS-1 cells. These data indicate the potential existence of a common pathway(s) mediated by both IRS-1 and Gab-1, which is necessary for IGF-1-stimulated activation of the immediate-early gene egr-1 (23).
Expression of Exogenous Gab-1 Restores S-Phase Entry in IRS-1-deficient Cells-Finally, to address whether overexpression of Gab-1 was capable of restoring IGF-1-stimulated cell cycle progression, we determined the level of IGF-1-stimulated incorporation of [methyl-3 H]thymidine into DNA in KO, KO ϩIRS-1 , and KO ϩGab-1 cell lines. As previously observed, [methyl-3 H]thymidine labeling in response to IGF-1 was decreased by 70% in KO cells, and reconstitution of IRS-1 restored the potential to enter S phase (Fig. 5). Surprisingly, overexpression of Gab-1 markedly increased IGF-1-stimulated S-phase progression at every point in the dose-response curve. At the higher concentrations of IGF-1 tested, KO ϩGab-1 cells exhibited a 4.2-fold higher incorporation of [methyl-3 H]thymidine labeling as compared with KO ϩIRS-1 cells (Fig. 5) and by 3-fold when compared with results obtained from wild type 3T3 fibroblast cell lines (data not shown). Thus, Gab-1 is uniquely capable of potently stimulating cell cycle progression. DISCUSSION A variety of studies have demonstrated that Gab-1 acts downstream of several receptor protein-tyrosine kinases, including the insulin and EGF receptors, by binding SH2 domain-containing signaling molecules following receptor-medi-ated phosphorylation. These studies indicate that Gab-1 functions as a substrate-signaling platform, like other members of the IRS family of proteins; however, the potential role of Gab-1 vis à vis other IRS proteins in mediating the pleiotropic effects of insulin and IGF-1 has not been investigated. In the current study, we have attempted to determine the potential role of Gab-1 in IGF-1-mediated signaling, taking advantage of cells in which this pathway is reduced by the absence of IRS-1 (23).
The tyrosine phosphorylation of IRS proteins requires a twofold mechanism for the selective interaction with activated insulin and IGF-1 receptors. Analysis of IRS-1 using the yeast two-hybrid system has implicated the N-terminal PH and PTB domains of IRS-1 in mediating receptor recognition, and this has been confirmed in mutagenesis studies in which these domains have been deleted (20,27). Based on studies in 32D cells, Myers et al. (27) have concluded that of these two domains, the PH domain is required for sensitive receptor coupling. These studies have also demonstrated that this domain is critical for insulin-stimulated phosphorylation of IRS-1 and the activation of downstream signaling pathways including the activation of PI 3-kinase and the phosphorylation of p70 s6 kinase (27). Although the exact mechanism by which the PH domain functions in insulin receptor-IRS protein recognition is unclear, the data suggest that the PH domain of IRS proteins may mediate protein-protein interactions or bind phospholipids in the membrane, which may target IRS proteins to the plasma membrane and coordinate receptor interaction (27)(28)(29). Although the PH domain of Gab-1 appears to function homologously to the PH domains of other IRS proteins based upon its ability to couple chimeric IRS-1 phosphorylation to the insulin receptor, in the context of Gab-1 the PH domain alone does not appear to be sufficient to mediate recognition by the insulin and IGF-1 receptors (20, 30 -32). This hypothesis is supported by recent data that suggest that the PH and PTB domains of IRS proteins function cooperatively to mediate receptor recognition (33). One of the first steps in insulin signaling involves the autoactivation of the receptor by phosphorylation of tyrosines 1146, 1150, and 1151 in its catalytic cleft, and phosphorylation of tyrosine 960 in its juxtamembrane region (34,35). The latter phosphorylation leads to recruitment of IRS-1 to the activated receptor and the interaction of the PTB domain of IRS-1 with phosphorylated NPXY motifs in the juxtamembrane region of the receptor. The IGF-1 receptor contains an analogous NPXY motif in its intracellular subunits, suggesting that PTB domain interaction at least partially mediates the association with this receptor. The lack of tyrosine phosphorylation of Gab-1 in response to IGF-1 stimulation in the current study suggests that the absence of a conventional PTB domain in Gab-1 may not allow for its recognition by the IGF-1 receptor. This hypothesis is supported by the failure of the yeast two-hybrid system to detect the interaction of Gab-1 with the insulin receptor (36). Although Gab-1 lacks a conventional PTB domain, Gab-1 may potentially interact with receptor tyrosine kinases via two other mechanisms. Recently, a proline-rich interaction domain in Gab-1 has been identified that mediates the interaction with the c-Met receptor (36). In addition, because the SH3 domains of Grb-2 can bind Gab-1, Grb-2 may also serve to recruit Gab-1 to the activated receptor (37,38).
Despite the lack of apparent tyrosine phosphorylation, Gab-1 can participate in downstream signaling from the IGF-1 receptor. Immediate-early gene products often encode transcription factors that have been implicated in cellular growth and differentiation. Waters et al. (40) have shown that expression of IRS-1 antisense RNA causes a reduction of insulin-stimulated activity of a luciferase gene under the control of the c-fos serum response element, the same element that has been implicated in conferring insulin sensitivity to the c-fos promoter (39,40). Our previous data in the IRS-1-deficient fibroblasts also provide evidence that expression of the c-fos and egr-1 genes depend on IRS-1 (23). In addition, the restoration of egr-1 expression in Gab-1 overexpressing cells indicates that Gab-1 is capable of regulating immediate-early gene expression. These data are of even greater interest considering that the IGF-1stimulated activation of the MAP kinases is normal in IRS-1deficient and Gab-1-overexpressing cells. Our data provide evidence for an IRS-1-or Gab-1-dependent pathway activating the egr-1 promoter despite the normal activation of the MAP kinases ERK-1 and ERK-2. Thus the cell lines described here provide a unique system with which to further define the path-ways for Gab-1-dependent egr-1 expression.
Gab-1 also appears to be uniquely capable of enhancing IGF-1-stimulated mitogenesis. These results are supported by data that indicate that the overexpression of Gab-1 is capable of enhancing anchorage-dependent and -independent growth rates (18). In addition, Holgado-Madruga et al. (22) have shown that PC-12 cells overexpressing Gab-1 had a reduced requirement for the amount of nerve growth factor necessary to prevent apoptosis. Mutants of Gab-1 lacking PI 3-kinase binding sites exhibited increased levels of apoptosis, indicating that Gab-1-associated PI 3-kinase activity was required for the inhibition of apoptosis. In addition, the overexpression of Gab-1 in several cell systems leads to increased transforming potential indicating that Gab-1 may have a profound effect on regulation of the cell cycle. Taken together, these data indicate that Gab-1 is capable of both enhancing cell growth and promoting cell survival through the prevention of apoptosis.
The absence of alterations in MAP kinase activity in KO, KO ϩIRS-1 , and KO ϩGab-1 fibroblasts suggests that many IRS family members may not be required for the normal activation of the Ras/MAP kinase pathway. The role of Gab-1 in MAP kinase activation may differ depending on the cell type. Several groups have reported differential activation of the MAP kinases in several cell systems (18,21,36). For example, Holgado-Madruga et al. (18) have found that Gab-1 and SOS form separate complexes with Grb-2 indicating that Gab-1 may compete with SOS for Grb-2 binding. In this way, Gab-1 may potentially regulate the activation of the MAP kinase pathway. The absence of IGF-1-stimulated Gab-1 phosphorylation may potentially negate this effect and allow for the normal activation of the MAP kinase pathway. Alternatively, the level of overexpression of Gab-1 in our system is not sufficient to compete with SOS for Grb-2 binding, and therefore, no alterations in MAP kinase activation are observed.
In conclusion, we have demonstrated that Gab-1 is not phoshorylated by the IGF-1 receptor, but overexpression of Gab-1 in IRS-1-deficient cells can restore the IGF-1-stimulated expression of the immediate-early gene egr-1 and markedly enhances the ability of the cells to enter S-phase. Further studies are required to determine the exact mechanism by which Gab-1 participates in the regulation of these biological end points.