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Cell Growth & Differentiation Vol. 12, 307-318, June 2001
© 2001 American Association for Cancer Research

Identification of Epidermal Growth Factor Receptor- Grb2-associated Binder-1-SHP-2 Complex Formation and Its Functional Loss during Neoplastic Cell Progression

Hideto Kameda, John I. Risinger, Bing-Bing Han, Seung Joon Baek, J. Carl Barrett, Wayne C. Glasgow and Thomas E. Eling1

Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709 [H. K., J. I. R., B-B. H., S. J. B., J. C. B., T. E. E.], and Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207 [W. C. G.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The adaptor protein Grb2-associated binder-1 (Gab1) is known to bind to the SHP-2 tyrosine phosphatase on epidermal growth factor (EGF) receptor stimulation. To clarify the roles of these two proteins in EGF receptor (EGFR) signaling and determine their possible alteration during neoplastic cell progression, we studied these proteins in a Syrian hamster embryo (SHE) cell line model of neoplastic progression. Specifically, we used asbestos-transformed SHE fibroblasts: the 10W+8 clone, which is immortal but nontumorigenic; and the 10W2T clone, which is tumorigenic. Gab1 was detected, and the EGF-dependent formation of the EGFR-Gab1-SHP-2 complex was observed in 10W+8 cells. After cloning hamster Gab1 cDNA, exogenous expression of Gab1 significantly enhanced EGF-dependent mitogenic activity in 10W+8 cells. On the other hand, Gab1 was not detected in 10W2T cells, and the EGF-dependent association of SHP-2 with EGFR was also absent. Exogenous Gab1 expression in transfected 10W2T cells restored the EGF-dependent association of SHP-2 with EGFR, although it only showed a marginal effect on EGF-dependent mitogenic activity. Thus, Gab1 plays a pivotal role in the EGFR signaling pathway via the formation of the EGFR-Gab1-SHP-2 complex, and alteration in the expression and function of Gab1 is implicated in the neoplastic progression of SHE cells.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The progression of a primary cell to a malignant phenotype involves multiple genetic events including the dysregulation of growth control pathways (1 , 2) . Signaling through the EGFR2 pathway is implicated in the tumorigenesis and neoplastic progression of many cancers, including breast and lung cancers (3) . Ligand binding to EGFR is known to initiate receptor dimerization and the activation of an intrinsic tyrosine kinase (4) . Tyrosine phosphorylation of the EGFR is followed by the recruitment of various SH2 domain-containing intracellular proteins to the cytoplasmic domain of EGFR. Formation of a Shc-Grb2-son of sevenless complex and the activation of Ras are key events in the initiation of signals through activated EGFR (5, 6, 7, 8) . These events are followed by the sequential kinase activation of Raf, mitogen-activated protein/extracellular signal-regulated kinase kinase, and MAPK (9) . Linking cytoplasmic events to nuclear activity, MAPK phosphorylates and activates transcription factors required for mitogenesis and other cellular activities. In addition, accumulating evidence suggests that some other proteins such as SHP-2 and Gab1 may play an important role in EGFR signaling (10, 11, 12, 13, 14, 15, 16, 17, 18, 19) .

SHP-2 is a ubiquitously expressed protein tyrosine phosphatase that contains two SH2 domains and an active catalytic domain (20 , 21) . Recent reports suggest that SHP-2 is a positive mediator of the mitogenic signal transduction induced by EGF (10 , 11 , 13 , 14 , 17, 18, 19) and other growth factors (22, 23, 24, 25, 26, 27, 28, 29, 30, 31) . However, the molecular mechanisms regulating the involvement of SHP-2 in the EGFR signaling pathway remain unclarified.

Gab1 is a recently reported docking protein, which originally was discovered as a Grb2-binding protein (12) . Gab1 has been reported to be involved in the signaling through cell surface receptors for EGF (12 , 15, 16, 17, 18, 19 , 32) , insulin (12 , 33) , hepatocyte growth factor (19 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43) , nerve growth factor (44 , 45) , fibroblastic growth factor (46) , PDGF (19 , 47) , granulocyte colony-stimulating factor (48) , erythropoietin (49) , thrombopoietin (47) , stem cell factor (47) , interleukin 3 (47 , 48) , interleukin 6 (19 , 47 , 48) , IFN-{alpha} and -{gamma} (47 , 48) , and T-cell and B-cell receptors (47 , 50) . Gab1 shares amino acid homology and the presence of a PH domain at the NH2 terminus with IRS-1 (12) , and the association of IRS-1 with SHP-2 has been shown to play an important role in insulin receptor signal transduction (22, 23, 24 , 26 , 51, 52, 53, 54) . These findings suggest that the Gab1-SHP-2 complex may potentially be a key functional element in the EGFR signaling pathway.

Our laboratory has developed a carcinogen-treated SHE cell culture system that models specific stages of the neoplastic transformation process (55 , 56) . When SHE cells are treated with asbestos and cultured, they first show morphological transformation and then subsequently acquire immortality (57) . These cells, typified by the 10W+8 strain, still retain a tumor suppressor gene-positive phenotype, as evidenced by their ability to suppress the tumorigenicity of malignant BP6T cells in cell-cell hybrid experiments (56) . During subsequent passages, some cells may lose the tumor suppressor genes and no longer be able to convert tumorigenic cells to nontumorigenic hybrids. The 10W-1 clone represents this tumor suppressor-negative phenotype (56) . Cells like 10W-1 are not yet tumorigenic, as determined by s.c. injection of cells into nude mice, and do not grow in soft agar in the absence of additional growth factor stimulations (56 , 58) . With additional passages, some cells acquire a tumorigenic phenotype like the 10W2T clone.3 Using this in vitro transformation system, we and others have observed altered responses to EGF including mitogenic activity, anchorage-independent growth, and altered modulation of EGFR signaling by specific lipid metabolites during neoplastic progression of SHE cells (55, 56, 57, 58, 59, 60, 61, 62) .

To clarify the underlying molecular mechanisms of the altered responsiveness to EGF during neoplastic progression of SHE cells, we investigated the expression and function of Gab1 in nontumorigenic (10W+8) and tumorigenic (10W2T) SHE cells. We found that the expression of Gab1, which was observed in 10W+8 cells, had been lost in 10W2T cells. Moreover, the cloning and exogenous expression of hamster Gab1 in both 10W+8 and 10W2T cells revealed the different function of Gab1 in these cells, which may explain the altered mitogenic responsiveness to EGF between nontumorigenic and tumorigenic cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Both Gab1 and SHP-2 Associate with Tyrosine-phosphorylated EGFR on EGF Stimulation.
To examine the expression of functional hamster Gab1 protein in nontumorigenic 10W+8 cells, we performed an immunoprecipitation study with anti-Gab1 antibodies using EGF-stimulated (1–15 min) or unstimulated 10W+8 lysates. Separation with SDS-8% PAGE was followed by immunoblotting with anti-p-Tyr antibody (Fig. 1A)Citation . Tyrosine-phosphorylated proteins with molecular weights of approximately 170,000, 110,000, 56,000, and 53,000 were coprecipitated with Gab1, and all of these proteins reached their peaks at 1 min after EGF stimulation and gradually decayed afterward. These signals specifically represented Gab1 and associated proteins because the immunoprecipitation with NRS as a control showed nothing significant except for a faint signal with Mr 56,000 on EGF stimulation (Fig. 1A)Citation . Anti-Gab1 blotting analysis with the same immunoprecipitation samples used in Fig. 1ACitation revealed the expression of hamster Gab1 of around Mr 100,000, which showed a mobility shift to Mr 110,000 after EGF stimulation (Fig. 1B)Citation . The tyrosine-phosphorylated Mr 170,000 protein observed in Fig. 1ACitation , which showed a rapid association with Gab1 on EGF stimulation, was suspected to be EGFR because it showed the same migration distance on SDS-PAGE as authentic human EGFR and hamster EGFR (data not shown). To confirm the association of EGFR with Gab1, we performed an immunoprecipitation study with anti-EGFR using the lysates of 10W+8 stimulated with EGF for 0–15 min, followed by immunoblotting with anti-Gab1 antibodies (Fig. 1C)Citation . Coprecipitation of Gab1 with EGFR was observed clearly at 1 min after EGF stimulation and decayed within 15 min. Therefore, we concluded that Gab1 associated with EGFR after EGF stimulation. However, the population of Gab1 that becomes associated with EGFR seemed to be limited in comparison with its abundant expression shown in Fig. 1BCitation .



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Fig. 1. Gab1 and SHP-2 are expressed in 10W+8 cells and show rapid and temporal association with EGFR on EGF stimulation. 10W+8 cells were challenged with or without 100 ng/ml EGF at 37°C for 1, 3, 5, and 15 min or for up to 120 min (E, bottom panel). Cells were lysed, and Gab1 was immunoprecipitated (IP) with specific antibodies against the COOH-terminal region of human Gab1 (A, B, and D), EGFR was immunoprecipitated (IP) with anti-EGFR antibodies (C and H), or SHP-2 was immunoprecipitated (IP) with anti-SHP-2 (E–G) and analyzed by SDS-PAGE (8%) and immunoblotting with anti-p-Tyr antibody (A and F), anti-Gab1 antibodies (B, C, and E), and anti-SHP-2 antibodies (D, G, and H), respectively, followed by ECL. NRS (5 µl) was used as a control immunoprecipitation to show the specificity of immunoprecipitation of Gab1 and coprecipitation of other tyrosine-phosphorylated proteins with anti-Gab1 antibodies (A). The data shown are representative of three independent experiments.

 
Previous reports suggest that SHP-2 is one of the major proteins associated with Gab1 on EGF stimulation (12 , 17, 18, 19 , 48 , 50) . Therefore, we next examined the EGF-dependent association of Gab1 with SHP-2 in 10W+8. Gab1 was immunoprecipitated from 10W+8 cell lysates, separated by SDS-8% PAGE, and analyzed by immunoblotting with anti-SHP-2 antibodies (Fig. 1D)Citation . SHP-2 with an apparent molecular weight of 66,000 showed a clear association with Gab1 1 min after EGF stimulation, and the association was well sustained over 15 min. To confirm this finding, we then performed a reversed experiment, namely, immunoprecipitation of SHP-2 and Western analysis with anti-Gab1 (Fig. 1E)Citation . Gab1 was found to be associated with SHP-2 just after EGF stimulation, and this association was well sustained over 15 min (Fig. 1ECitation , top panel); indeed, the association could be traced for up to 2 h (Fig. 1. ECitation , bottom panel).

We then investigated the time course of the association of SHP-2 with tyrosine-phosphorylated proteins after EGF stimulation for 0–15 min (Fig. 1F)Citation . SHP-2 was immunoprecipitated from 10W+8 lysates with specific antibodies, separated on SDS-PAGE, and analyzed by anti-p-Tyr immunoblotting. Tyrosine-phosphorylated proteins with molecular weights of approximately 170,000, 110,000, and 56,000 and others were coprecipitated with SHP-2, and all reached their peaks at 1 min after EGF stimulation and gradually decayed afterward. We did not detect a distinct band at Mr 66,000 representing tyrosine-phosphorylated SHP-2 protein itself, which suggested that tyrosine phosphorylation of SHP-2 on EGF stimulation was not intense in 10W+8 cells, although SHP-2 was highly expressed in 10W+8 cells (Fig. 1G)Citation . This finding was consistent with the fact that no apparent mobility shift of SHP-2 was observed on EGF stimulation (Fig. 1G)Citation . To confirm that a tyrosine-phosphorylated Mr 170,000 protein observed in Fig. 1FCitation , which showed a rapid association with SHP-2 on EGF stimulation, represented EGFR, we performed an immunoprecipitation study with anti-EGFR using the lysates of 10W+8 stimulated with EGF for 0–15 min, followed by immunoblotting with anti-SHP-2 antibodies. As shown in Fig. 1HCitation , SHP-2 was coprecipitated with EGFR on EGF stimulation. The peak of association between these two proteins occurred at 1 min after EGF stimulation and then declined rapidly. This time course was consistent with the time course shown in Fig. 1FCitation and strongly supported the previous reports proposing that the association of SHP-2 with EGFR was dependent on the tyrosine phosphorylation of EGFR (20 , 21 , 63) . Taken together, we concluded that a small fraction of the Gab1 and SHP-2 pool showed a rapid and transient association with EGFR on EGF stimulation, whereas the association between Gab1 and SHP-2 was much more abundant and sustained for a longer period of time.

SHP-2 and Gab1 Form a Complex with the Same EGFR Molecule on EGF Stimulation.
The comparison of SDS-PAGE resolving tyrosine-phosphorylated proteins associated with Gab1 (Fig. 1A)Citation or SHP-2 (Fig. 1F)Citation on EGF stimulation showed a very similar pattern, indicating the possibility that common proteins are associated with Gab1 and SHP-2. The proteins associated with Gab1/SHP-2 might not be associated separately with Gab1 or SHP-2 alone but rather bound to Gab1-SHP-2 complexes. Therefore, we investigated whether Gab1 and SHP-2 associate with the same EGFR molecule to form an EGFR-Gab1-SHP-2 complex. To test this hypothesis, we first removed a specific protein from cell lysate by immunodepletion, and then a subsequent immunoprecipitation of a second protein was performed, followed by measurement of the associated proteins by anti-p-Tyr immunoblotting (Fig. 2)Citation . First, Gab1 was immunodepleted from 10W+8 cell lysate before the immunoprecipitation of SHP-2 (Lane 3). Likewise, we immunodepleted SHP-2 from another cell lysate before the immunoprecipitation of Gab1 (Lane 7). Finally, SHP-2 (Lanes 1–4)- or Gab1 (Lanes 5–8)-associated, tyrosine-phosphorylated proteins were analyzed by SDS-PAGE and anti-p-Tyr immunoblotting. The efficiency of the immunodepletion was 70–80% for either Gab1, SHP-2, or EGFR (data not shown). The immunodepletion of Gab1 resulted in a significant decrease in SHP-2 binding to tyrosine-phosphorylated Mr 170,000 protein and others (compare Lane 2 with Lane 3), and, in turn, the immunodepletion of SHP-2 decreased Gab1 binding to Mr 170,000 protein and others (compare Lane 6 with Lane 7). The immunodepletion of EGFR before the immunoprecipitation of SHP-2 (Lane 4) or Gab1 (Lane 8) specifically reduced the intensity of the Mr 170,000 band, but not that of other bands. This observation finally convinced us that the Mr 170,000 tyrosine-phosphorylated protein represented EGFR, and only a small portion of Gab1-SHP-2 complex was bound to EGFR on EGF stimulation. These results indicate that Gab1 and SHP-2 bind to the same EGFR molecule and form a complex on EGF stimulation.



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Fig. 2. The immunodepletion of Gab1 or SHP-2 causes a marked decrease in the association of EGFR with SHP-2 or Gab1, respectively. 10W+8 cells were unstimulated (Lanes 1 and 5) or stimulated with 100 ng/ml EGF at 37°C for 1 min (Lanes 2–4 and 6–8). Cells were lysed, and SHP-2 (Lanes 1–4) or Gab1 (Lanes 5–8) was immunoprecipitated (IP) with specific antibodies and analyzed by SDS-PAGE and anti-p-Tyr immunoblotting, followed by ECL. Before immunoprecipitation, some of the lysate samples were subjected to two rounds of immunodepletion of Gab1 (Lane 3), SHP-2 (Lane 7), or EGFR (Lanes 4 and 8) as described in "Materials and Methods." The data shown are representative of four independent experiments.

 
cDNA Cloning of Hamster Gab1.
To further address the mechanism of EGFR-Gab1-SHP-2 complex formation, we cloned the cDNA of hamster Gab1 using a 5'- and 3'-RACE strategy for use in subsequent transfection and expression studies designed to ascertain the functional role of Gab1. DNA sequence analysis of the hamster Gab1 cDNA indicated 2085 nucleotide bases for the coding region from ATG to TGA (Fig. 3ACitation ; GenBank accession number AF307847) and shows 84% and 89% homology to human (GenBank accession number U43885) and mouse (GenBank accession number AJ250669) Gab1, respectively (12 , 34) . The deduced amino acid sequence revealed that hamster Gab1 protein is composed of 694 amino acids with very high homology to both human (92%) and mouse (92%) Gab1 (Fig. 3B)Citation . The PH domain (amino acids 14–116) showed completely identical amino acid structure among these three species. Moreover, hamster Gab1 shares core functional domains such as MBD, three binding sites for PI3K, and two binding sites for SHP-2 with human and mouse Gab1.



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Fig. 3. Nucleotide sequences of hamster Gab1 cDNA, its deduced protein sequence, and comparison with mouse and human Gab1. A, nucleotide sequence of hamster cDNA covering whole coding region (1–2085). Translation start codon ATG and stop codon TGA are shaded. B, alignment of hamster, mouse, and human Gab1 amino acid sequences. Hamster Gab1 and human Gab1 lack Pro376 found in mouse Gab1, thus they are composed of 694 amino acids, whereas mouse Gab1 is composed of 695 amino acids. The PH domain and MBD are boxed, and the binding sites for the SH2 or SH3 domain of Grb2 and the SH2 domain of PI3K and SHP-2 are underlined.

 
Overexpression of Gab1 Resulted in a Significant Enhancement of the EGF-dependent Mitogenic Activity in 10W+8.
To examine the involvement of Gab1 in the mitogenic response to EGF in 10W+8 cells, we constructed Gab1-overexpressing 10W+8 as well as mock-transfected 10W+8. The protein expression of Gab1 in the 10W+8 cell pool that stably expresses exogenous Gab1 as well as endogenous Gab1 (Fig. 4ACitation , Lane 3) showed an increased Gab1 expression up to 2-fold of that in parental 10W+8 (Lane 1) or mock-transfected 10W+8 cells (Lane 2). Exogenous Gab1 expression was confirmed by Northern analysis (Fig. 4BCitation , Lane 3). We then examined the effect of Gab1 overexpression on the EGF-dependent mitogenic response in 10W+8 cells. As shown in Fig. 4CCitation , EGF stimulation enhanced [3H]thymidine incorporation up to 2.8-fold of the EGF-negative control value in mock-transfected 10W+8 cells, which was comparable to the EGF effect in parental 10W+8 cells (2.6-fold; see Fig. 7BCitation ). Gab1-overexpressing 10W+8 cells showed a considerably greater response to EGF, with a 4.5-fold increase in [3H]thymidine incorporation compared with EGF-negative control. Thus, the EGF-dependent mitogenic response was amplified with the increase in Gab1 expression in 10W+8 cells.



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Fig. 4. Gab1 overexpression significantly enhances the mitogenic response of 10W+8 to EGF stimulation. A, Gab1 was immunoprecipitated (IP) from the lysates of parental 10W+8 (Lane 1), 10W+8-mock (Lane 2), and 10W+8-Gab1 (Lane 3). The samples were analyzed by SDS-PAGE (8%), followed by anti-Gab1 blotting and detection with the ECL system. Gab1 expression in 10W+8-Gab1 was approximately 2-fold of that in 10W+8 or 10W+8-mock. B, 10 µg of total RNA from 10W+8 (Lane 1), 10W+8-mock (Lane 2), and 10W+8-Gab1 (Lane 3) were examined for the expression of Gab1 mRNA. 10W+8-Gab1 shows exogenous expression of mRNA in addition to endogenous 5.2-kb mRNA (top panel, Lane 3). Equal loading of total RNA is visualized under UV light with an ethidium bromide staining of the gel (bottom panel). C, 10W+8-mock and 10W+8-Gab1 were serum-starved for 16 h and incubated with 1 µCi/well [3H]thymidine in the presence or absence of 50 ng/ml EGF at 37°C for 24 h. The radioactivity of the incorporated [3H]thymidine was measured. Gab1 overexpression resulted in a 2-fold increase in the mitogenic activity of 10W+8 in response to EGF stimulation. The data are the means ± SD of six determinations. *, P < 0.05 versus 10W+8-mock, EGF (+).

 


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Fig. 7. Introduction of Gab1 into 10W2T restores the EGF-dependent association of SHP-2 with EGFR. 10W2T-Gab1 (clone 1; A) or 10W2T-none (clone 7; B) cells were treated with or without 100 ng/ml EGF at 37°C for 1 min. Cells were lysed, and the immunoprecipitation (IP) was performed with specific antibodies for EGFR (Lanes 3 and 4), Gab1 (Lanes 5 and 6), or SHP-2 (Lanes 7 and 8) or with NRS as controls (Lanes 1 and 2). Samples were analyzed by SDS-PAGE and immunoblotting with anti-p-Tyr antibody, followed by ECL. Tyrosine-phosphorylated Gab1 of approximately Mr 110,000 was detected in 10W2T-Gab1 as well as associated with EGFR (A, Lane 6). Tyrosine-phosphorylated proteins of Mr 90,000–100,000 were found to be associated with SHP-2 in EGF-dependent (Mr 90,000) and EGF-independent (Mr 100,000) manners in both 10W2T-Gab1 (A, Lanes 5 and 6) and 10W2T-none (B, Lanes 5 and 6). However, the coprecipitation of tyrosine-phosphorylated EGFR (Mr 170,000) with SHP-2 on EGF stimulation was exclusively observed in 10W2T-Gab1 (A, Lane 8) and not in 10W2T-none (B, Lane 8). The data shown are representative of three independent experiments.

 
Gab1 Is Not Expressed in Tumorigenic SHE Cell Clone 10W2T in Which Activated EGFR Fails to Recruit SHP-2.
Next, to examine the role of Gab1 in SHE cells in an advanced stage of neoplastic progression, we investigated the expression of Gab1 in 10W2T, a tumorigenic cell clone. To our surprise, this cell clone lacks expression of Gab1 as determined by immunoprecipitation analysis (Fig. 5Citation , compare Lanes 1 and 2 with Lanes 5 and 6) as well as Western (see Fig. 6ACitation ) and Northern (data not shown) analyses. The expression of EGFR and SHP-2 in 10W2T cells was comparable to that in 10W+8 cells, which was confirmed by immunoblot analysis of equal amounts of cell lysate (data not shown). Moreover, we could not detect a tyrosine-phosphorylated protein of Mr 170,000 (EGFR) associated with SHP-2 after EGF stimulation (Fig. 5Citation , Lanes 3 and 4), although tyrosine phosphorylation of EGFR was observed in 10W2T cells (Fig. 5Citation , Lane 8), which was similar to the EGF-dependent tyrosine phosphorylation of EGFR in 10W+8 cells. Interestingly, uncharacterized tyrosine-phosphorylated protein(s) of around Mr 100,000 was (were) constitutively detected in both unstimulated (Fig. 5Citation , Lane 3) and EGF-stimulated (Fig. 5Citation , Lane 4) 10W2T cells, and additional intense signal at Mr 85,000–90,000 developed on EGF stimulation (Fig. 5Citation , Lane 4).



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Fig. 5. Tumorigenic 10W2T cells lack Gab1 expression and fail to recruit SHP-2 to activated EGFR. 10W2T cells were treated with or without 100 ng/ml EGF at 37°C for 1 min. Cells were lysed, and the immunoprecipitation (IP) was performed with specific antibodies for SHP-2 (Lanes 3 and 4), Gab1 (Lanes 5 and 6), EGFR (Lanes 7 and 8), or with NRS as controls (Lanes 1 and 2). Samples were analyzed by SDS-PAGE and immunoblotting with anti-p-Tyr antibody, followed by ECL. The data shown are representative of three independent experiments.

 


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Fig. 6. Selection of 10W2T-derived cell clones with differential expressions of exogenous Gab1. A, eight clones of Gab1-transfected 10W2T (clones 1–3 and 6–10) were examined for the expression of Gab1 by Western analysis. Fifty µg of protein were loaded to each lane, separated by SDS-PAGE (8%), and analyzed by anti-Gab1 blotting and ECL. The highest expression of Gab1 was observed in clone 1, and Gab1 expression was negative in clone 7. Low but significant expression of Gab1 was detected in clone 10, and moderate expression was observed in clone 9. B, 10W2T-none (clone 7) and three clones of 10W2T-Gab1 (clones 1, 9, and 10) were cultured for 4 days with 10% FBS, and the proliferation curves were compared among those cells. The data are the means of six determinations.

 
Exogenous Gab1 Expression in 10W2T Cells Restored the Association of EGFR with SHP-2 on EGF Stimulation.
Next, we constructed 10W2T exogenously expressing Gab1. Because the pilot studies using a Gab1-expressing 10W2T cell pool failed due to small expression of Gab1 as a whole (data not shown), we moved to assays with single cell clones. Ten cell clones that showed resistance to G418 were selected and examined for Gab1 expression by Western analysis, with the exception of clones 4 and 5, which did not grow well after selection (Fig. 6A)Citation . Clones 7, 10, 9, and 1 were determined to have no, low, moderate, and high expression of Gab1, respectively, and were used in the following studies. These cell clones were morphologically similar. First, we examined the proliferation rate of these cells in response to 10% FBS. Exogenous Gab1 expression did not considerably enhance the proliferative activity of 10W2T cells (Fig. 6B)Citation ; even a clone with the highest Gab1 expression (clone 1) only showed ~30% increase compared with a Gab1-negative clone (clone 7). Next, to determine whether exogenously expressed Gab1 retained the function of EGF-dependent complex formation with EGFR and SHP-2 in 10W2T cells, we examined the phosphorylation of Gab1 and the association of EGFR with Gab1 and SHP-2 on EGF stimulation in a clone of 10W2T exogenously expressing Gab1 (Fig. 7A)Citation . Exogenously expressed Gab1 retained EGF-dependent phosphorylation (Mr 110,000) and EGFR (Mr 170,000) association (Lane 6). Interestingly, the expression of Gab1 restored the association of SHP-2 with EGFR on EGF stimulation as well (Lane 8). On the other hand, 10W2T-none (clone 7; Fig. 7BCitation ) showed results similar to those for parental 10W2T (Fig. 5)Citation . Specifically, the association of SHP-2 with EGFR was not observed (Fig. 7BCitation , Lanes 7 and 8). The phosphorylation of EGFR and the amount of tyrosine-phosphorylated proteins (Mr 90,000 and Mr 100,000) associated with SHP-2 on EGF stimulation were similar in 10W2T-Gab1 (Fig. 7ACitation , Lane 4) and 10W2T-none (Fig. 7BCitation , Lane 4). Consistent with these results, the expression of EGFR and SHP-2 in 10W2T cells was not affected by the exogenous expression of Gab1 (data not shown). Therefore, it is suggested that SHP-2 is recruited to activated EGFR mainly via Gab1 on EGF stimulation in SHE cells. This model is consistent with the previous results shown in Fig. 2Citation , which demonstrated that SHP-2 and Gab1 were bound to the same EGFR molecules. Moreover, it is indicated that 10W2T cells retain the ability to form an EGFR-Gab1-SHP-2 complex on EGF stimulation.

The Expression of Gab1 Did Not Add to the Mitogenic Response of 10W2T Cells to EGF.
Finally, we examined the effect of Gab1 expression on the mitogenic response to EGF in 10W2T cells. The basal (before EGF stimulation) incorporation of [3H]thymidine into 10W2T-derived cells was much higher than that in 10W+8 cells, which was consistent with decreased growth factor requirements of the cells in an advanced stage of neoplastic progression. In contrast to the results with 10W+8 cells (Fig. 4C)Citation , exogenous Gab1 expression in 10W2T cells did not enhance the EGF-dependent [3H]thymidine incorporation; values were 1.8- and 2.1-fold of control values (no EGF stimulation) in 10W2T-none (clone 7) and 10W2T-Gab1 (clone 1), respectively (Fig. 8)Citation . These results were consistent with the modest effect of Gab1 expression on serum-stimulated cell proliferation (Fig. 6B)Citation .



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Fig. 8. Effect of Gab1 expression on EGF-dependent mitogenesis in 10W2T cells. 10W2T-none (clone 7), 10W2T-Gab1 (clone 1), and 10W+8 cells were serum-starved for 16 h and incubated with 1 µCi/well [3H]thymidine in the presence or absence of 50 ng/ml EGF at 37°C for 24 h. The radioactivity of the incorporated [3H]thymidine was measured. Exogenous Gab1 expression did not enhance the mitogenic activity of 10W2T cells in response to EGF stimulation. The data are the means ± SD of six determinations.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
This is the first report demonstrating the formation of an EGFR-Gab1-SHP-2 complex on EGF stimulation as well as its functional significance in EGF-dependent mitogenic activity of the cells. Furthermore, this is the first study to investigate the differences in Gab1 expression as well as its altered function in the EGFR signaling using nontumorigenic and tumorigenic cells.

Although Gab1 has been reported to associate with the c-Met receptor [hepatocyte growth factor receptor (34, 35, 36, 37, 38, 39, 40, 41) ], the binding of Gab1 to EGFR was not observed by a yeast two-hybrid screen (34) . However, while this work was in progress, several relevant findings to the results presented here have been published. First, Gab1 was identified as a high-affinity substrate for the EGFR with the major tyrosine phosphorylation site Tyr657 in the COOH terminus (16) . Second, coprecipitation of EGFR with Gab1 on EGF stimulation was reported using A431 cells (64) , in which abundant EGFR expression is observed [2.6 x 106 sites/cell (65) ] as compared with the lower EGFR expression in 10W+8 cells [0.7 x 105 sites/cell (58) ]. The fact that the population of Gab1 that associates with EGFR on EGF stimulation seems to be limited (Fig. 1)Citation may reflect the relatively low EGFR expression in 10W+8 cells. This association requires MBD of Gab1 as well as p-Tyr1068/p-Tyr1086 of EGFR, which are the binding sites for the SH2 domain of Grb2 (64 , 66) . Thus, the present demonstration of EGFR-Gab1-SHP-2 complex formation provides a new insight into understanding the role of Gab1 in the transduction of EGF-dependent signaling.

The association of SHP-2 with EGFR occurred rapidly, with a peak association at 1 min after EGF stimulation, and soon declined in 10W+8 cells (Fig. 1, F and H)Citation . Reports in the literature have also demonstrated SHP-2 binding to EGFR after tyrosine phosphorylation of EGFR (20 , 21 , 27 , 63 , 67) . However, in these reports, the association of SHP-2 with EGFR was less than that with PDGF receptor (20 , 21) , which was consistent with the 2observation that SHP-2 acted as an adaptor between PDGF receptor, not EGFR, and the Grb2-son of sevenless complex (68) . In human lung fibroblasts, SHP-2 was found to coprecipitate with a Mr 170,000 tyrosyl phosphoprotein on EGF stimulation (63) . The Mr 170,000 protein comigrated with EGFR, although Lechleider et al. (63) could not successfully detect it by anti-EGFR immunoblotting. Moreover, activation of the EGFR resulted in a relatively low level (<1%) of the total SHP-2 pool associated with the tyrosine-phosphorylated EGFR using EGFR-overexpressing NIH3T3 fibroblasts and double immunoprecipitation methods (67) . Taken together, these findings indicate that a small fraction of the total pools of Gab1 and SHP-2 become associated with EGFR on EGF stimulation. Alternatively, the association duration of each molecule of Gab1 or SHP-2 with EGFR may be very brief, which makes it hard to precisely estimate these association rates.

In contrast to the transient and small fraction of association between EGFR and Gab1/SHP-2, Gab1-SHP-2 association was sustained over 2 h after EGF treatment in 10W+8 cells (Fig. 1E)Citation . SHP-2 appears to be the major binding partner of Gab1 on EGF stimulation because ~15% of the endogenous SHP-2 associates with Gab1 (16) . These data suggest that a Gab1-SHP-2 complex may interact with other downstream proteins after the dissociation from EGFR and transduce the downstream signaling. Very recently, the interaction of Gab1 through MBD with phosphorylated extracellular signal-regulated kinase 2 has been reported (69) .

One important question is whether Gab1 and SHP-2 are bound to the same EGFR molecule. Our immunodepletion study (Fig. 2)Citation revealed that both proteins could bind to the same EGFR and form an EGFR-Gab1-SHP-2 complex. This finding raises the further question of how these three proteins form a complex on EGF stimulation. SHP-2 can bind directly via its SH2 domain to activated, tyrosine-phosphorylated EGFR (20 , 63) , preferably to p-Tyr992 of EGFR (70) or indirectly through Grb2 via its COOH-terminal SH3 domain (71) . Now that the EGFR-Gab1 association has been demonstrated by us and others (16 , 64) , it is also possible that SHP-2 binds to EGFR indirectly through Gab1 via several binding sites shown in Fig. 3BCitation . This model mimics the association of Gab1 with the c-Met receptor. Gab1 is considered to be recruited to the c-Met receptor predominantly indirectly via Grb2 through the interaction with the COOH-terminal SH3 domain of Grb2 and the association of the Grb2 SH2 domain with p-Tyr1356 in c-Met receptor (35, 36, 37) , in addition to the direct interaction (34 , 36) . Likewise, our results suggest that SHP-2 binds indirectly to EGFR via Gab1 through the interaction with SHP-2-binding sites in COOH-terminal p-Tyr627 and p-Tyr659 of Gab1(Fig. 3BCitation and Fig. 7ACitation ), in addition to the direct interaction described above.

Although accumulating evidence suggests that SHP-2 positively regulates mitogenic signaling through EGFR (10 , 11 , 13 , 14 , 17, 18, 19) , its precise role remains uncharacterized. Activated EGFR is believed to be dephosphorylated by SHP-1, not by SHP-2 (20 , 21 , 27 , 72) . Our working hypothesis is that Gab1 directs the intracellular traffic pathway of SHP-2 in EGFR signaling. On EGF stimulation, SHP-2 associates with the EGFR mainly through an indirect mechanism by binding to Gab1. After the formation of a complex with EGFR and SHP-2, Gab1 appears to dissociate from EGFR together with SHP-2. This Gab1-SHP-2 complex may modulate the activity of Ras, PI3K, or MAPKs, just like IRS-1-SHP-2 complex does on insulin receptor activation (22 , 24, 25, 26 , 53 , 73) , and positively regulate EGFR signaling. Although Gab1-overexpressing NIH3T3 clones showed a decreased MAPK activation on EGF stimulation (12) , several subsequent studies suggest an enhanced MAPK activation by Gab1 overexpression (34 , 48 , 64) . Other studies report a reduced MAPK activation in Gab1-deficient mouse embryonic fibroblasts (19) , and the expression of Gab1 mutant lacking the SHP-2 binding site has been shown to block extracellular signal-regulated kinase 2 activation by EGF (18) . Moreover, Gab1 may activate SHP-2 as SHP-2 is activated by binding of its SH2 domains to p-Tyr1172 and p-Tyr1222 in IRS-1 (52 , 54) . In addition, Gab1 may be a substrate of SHP-2 (17 , 47) , and a rapid tyrosine phosphorylation by activated EGFR and subsequent dephosphorylation by SHP-2 may be required for the appropriate signal transduction.

In this report, we also focused on the altered expression and function of Gab1 during the neoplastic progression of SHE cells. Alteration in the role of growth factors in cell proliferation can develop as a result of the following: (a) altered activation of growth factor receptors, including the constitutive activation of receptor tyrosine kinases; (b) altered expression of growth factors and/or growth factor receptors; and (c) alterations in regulatory proteins or signaling pathways (1, 2, 3 , 59 , 74, 75, 76) . During SHE cell in vitro transformation, decreased mitogenic response of transformed SHE cell lineage to EGF was frequently observed among cell lines treated with various carcinogens (59) . Our 10W series of cell lines transformed by asbestos also showed decreased responsiveness to EGF during neoplastic progression (58 , 60) . The expression and the EGF-induced tyrosine phosphorylation of EGFR were similar in 10W+8 and 10W2T cells. Therefore, based on the hypothesis that the altered expression of signal regulatory molecules like an adaptor protein Gab1 is responsible for the altered responsiveness to growth factors during SHE cell transformation, we examined the expression of Gab1 in 10W2T and found no detectable expression (Figs. 5Citation and 6ACitation ). Moreover, BP6T cells, a benzo(a)pyrene-transformed, tumorigenic hamster fibrosarcoma cell line (55 , 56) , also showed no detectable expression of Gab1 (data not shown). To elucidate the involvement of Gab1 expression in the altered responsiveness to EGF during SHE cell transformation, we compared the EGF-dependent mitogenic responses and the effect of Gab1 overexpression on EGF signal transduction in 10W+8 and 10W2T cells. We found that (a) 10W2T cells showed less mitogenic response to EGF stimulation than 10W+8 cells (Fig. 8)Citation ; (b) overexpression of Gab1 enhanced the mitogenic response to EGF in 10W+8 cells (Fig. 4C)Citation but not in 10W2T cells (Fig. 8)Citation ; and (c) uncharacterized tyrosine-phosphorylated proteins were coprecipitated with SHP-2 exclusively in 10W2T cells in the absence (Mr ~100,000) or presence (Mr ~90,000) of EGF stimulation (Figs. 5Citation and 7, A and BCitation ), but not (or much less) in 10W+8 cells (Fig. 1F)Citation .

In the present report, we demonstrated that Gab1 overexpression up-regulates mitogenic response to EGF. It is noteworthy that we used a 10W+8 cell pool with an overall 2-fold increased expression of Gab1 instead of 8- or 13-fold Gab1-overexpressing NIH3T3 cells that showed enhanced anchorage-dependent and independent growth (12) . Therefore, an extreme up-regulation of Gab1 expression may not be required for the enhanced mitogenic activity in response to growth factors. In contrast, we did not observe any effects of Gab1 expression on the mitogenic activity of 10W2T cells in response to EGF. Moreover, exogenous Gab1 expression did not affect the growth of 10W2T cells in soft agar with or without EGF (50 ng/ml) stimulation (data not shown). Therefore, 10W2T was considered to have developed Gab1 independence in EGFR signaling with its loss of Gab1 expression.

The presence of uncharacterized SHP-2 associated proteins showed an interesting correlation with the altered mitogenic response of 10W2T cells to EGF. Namely, a tyrosine-phosphorylated protein of approximately Mr 100,000 that associated with SHP-2 in 10W2T cells even before EGF stimulation (Figs. 5Citation and 7, A and BCitation ) was not detected in 10W+8 cells (Fig. 1F)Citation . The expression of this protein appears to be related to the increased mitogenic activity of 10W2T cells when compared with 10W+8 cells under the serum-starved condition (Fig. 8)Citation . Likewise, tyrosine-phosphorylated proteins of around Mr 90,000 that exclusively coprecipitated with SHP-2 on EGF stimulation (Figs. 5Citation and Fig. 7BCitation ) may be responsible for the EGF-dependent mitogenic activity of 10W2T cells (Fig. 8)Citation . Potentially, these uncharacterized proteins may functionally replace Gab1 in 10W2T cells, which in turn leads to altered responsiveness to EGF. These uncharacterized proteins do not appear to be Gab1-related proteins such as degradation products or alternative splicing products because neither immunoprecipitation/Western analysis with anti-Gab1 antibodies against the COOH-terminal portion (Figs. 5Citation and 6ACitation ) nor Northern analysis with a probe against the middle portion of coding region (data not shown) gave any detectable signal in the 10W2T cell line.

The cloning of hamster Gab1 cDNA and sequence analysis revealed that the core functional sequences of Gab1 have been well conserved among human, mouse, and hamster (Fig. 3B)Citation . Amino acid sequences of the PH domain including a binding site for the SH2 domain of Grb2 and sequences of two sites for the SH3 domains of Grb2, three sites for PI3K, and two sites for SHP-2 were completely identical among those three species. These findings further suggest pivotal roles of Gab1 in growth factor/cytokine signaling among various species. The results strongly indicate that EGFR-Gab1-SHP-2 complex formation and the altered expression and/or function of Gab1 during neoplastic cell progression described in this report may model similar cellular events in human tissues and tumors.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Materials.
IBR, cmf-PBS, trypsin-EDTA, and gentamicin were from Life Technologies, Inc. FBS was purchased from HyClone Laboratories. Nonfat dry milk was purchased from Bio-Rad. BCA protein assay reagent was from Pierce. EGF was obtained from Collaborative Research Associates. Acrylamide and bisacrylamide were purchased from Amresco. The nitrocellulose membrane was obtained from Schleicher & Schuell. The immunoglobulin conjugates, Hyperfilm, and ECL reagents were purchased from Amersham. The anti-EGFR (SC-03), anti-SHP-2 (SC-280), and anti-p-Tyr antibodies (PY99) were obtained from Santa Cruz Biotechnology, whereas another anti-EGFR antibody (SE-5) was a generous gift from Dr. G. C. Clark [National Institute of Environmental Health Sciences, Research Triangle Park, NC (61) ]. Anti-Gab1 (06-579; against amino acids 664–694) was from Upstate Biotechnology Inc., and another anti-Gab1 (162D-124-3; against amino acids 192–205) was a generous gift from Dr. Richard P. DiAugustine (National Institute of Environmental Health Sciences). The protein A-Sepharose beads and all other reagents were obtained from Sigma Chemical Co.

Cell Culture and Transfections.
The experiments were performed with asbestos-transformed SHE cell lines: 10W+8, which retains tumor suppressor genes and has a nontumorigenic phenotype (55 , 56 , 58 , 60 , 61) , and 10W2T, which has lost tumor suppressor genes and obtained a tumorigenic phenotype. SHE 83-9 cells, which have not been treated with carcinogens, were used for hamster cDNA cloning. Cells were cultured in IBR containing 10% FBS and gentamicin (10 µg/ml) at 37°C in a humidified 5% CO2/95% air atmosphere. Trypsin-EDTA (0.05%) was used to subculture the cells. 10W+8 cells under passage 11–17 were used in these experiments. Cell transfections were performed using the LipofectAMINE Plus reagent (Life Technologies, Inc.) following the manufacturer’s instructions.

Immunoprecipitation and Immunodepletion.
After reaching 50–60% confluence on 150-mm dishes, cells were serum-deprived for 16–20 h to synchronize cell cycles in G0 (60) . At least 1 h after changing the medium to fresh IBR, cells were further incubated at 37°C with or without 100 ng/ml EGF for the indicated times. The dishes were then placed on ice, and the cells were rinsed twice with 10 ml of ice-cold cmf-PBS and lysed with 0.4 ml of ice-cold lysis buffer [1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 2 mM sodium orthovanadate, 100 µM p-nitrophenylphosphate, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 10 mM sodium phosphate, 10 µg/ml aprotinin, and 10 µg/ml leupeptin]. After scraping the cells, the lysate was collected, sonicated on ice for 7 s twice, and centrifuged at 2,000 x g for 10 min at 4°C. The supernatant was transferred to a new tube, and an aliquot was removed to measure the protein concentration by BCA protein assay reagent. Two mg of cellular protein from approximately 1 x 107 cells were precleared with 5 µl of NRS and 100 µl of protein A-Sepharose bead slurry. The mixture was tumbled at 4°C for 1 h and centrifuged at 10,000 x g for 2 min, and the supernatant was transferred to a new tube. Specific antibodies (anti-EGFR, 2–3 µg; anti-SHP-2, 2 µg; and anti-Gab1, 2–4 µg) were added to the supernatant, the samples were tumbled overnight at 4°C, and 100 µl of protein A-Sepharose bead slurry were added and tumbled for an additional 2 h. After centrifugation at 10,000 x g for 2 min, the pellet was washed three times with 1 ml of 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol. Protein sample buffer (2x) was added to the final pellet; the sample was boiled for 8 min and centrifuged at 10,000 x g for 10 min. The supernatant was used immediately or frozen at -70°C for later use on SDS-PAGE (8% acrylamide gel). For immunodepletion, 2 mg of cellular protein were incubated with specific antibodies at 4°C for 2 h, and 100 µl of protein A-Sepharose bead slurry were added and tumbled for an additional 3 h. After centrifugation at 10,000 x g for 2 min, the supernatant was transferred to a new tube and provided for the second round of immunodepletion. After that, the supernatant was transferred to a new tube for immunoprecipitation assay.

Western Blot Analysis.
The samples obtained by immunoprecipitation were loaded and run on SDS-PAGE (8% acrylamide gel) and electrophoretically transferred to a nitrocellulose membrane in 25 mM Tris, 192 mM glycine, 20% methanol, and 0.1% SDS using semidry electrophoresis equipment (Hoefer). Membranes were blocked in TBST with 5% BSA at 4°C overnight or in 3% nonfat milk in cmf-PBS for 20 min at room temperature (for anti-Gab1). The blots were then incubated with anti-SHP-2 antibody (1:2000) for 1 h, anti-p-Tyr antibody (1:1000) with 1% BSA + TBST at room temperature for 1 h, or anti-Gab1 antibody (1:2000) with 3% nonfat milk in cmf-PBS at 4°C overnight. The blots were washed five times in TBST or water (for anti-Gab1) and then incubated with horseradish peroxidase-conjugated antirabbit immunoglobulin (1:5000) or antimouse immunoglobulin (1:5000, for anti-p-Tyr antibody) in 1% BSA + TBST at room temperature for 1 h or with 3% nonfat milk in cmf-PBS at room temperature for 1.5 h (for anti-Gab1). The blots were again washed five times in TBST or water, washed once with 0.05% Tween 20 + cmf-PBS (for anti-Gab1), and visualized using the Amersham ECL system. For stripping and reprobing membranes, the membrane was submerged in stripping buffer [100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7)] and incubated at 50°C for 30 min with agitation according to the manufacturer’s protocol for the Amersham ECL system. The membrane was washed twice in TBST for 10 min at room temperature and reprobed with specific antibodies after blocking as described above. The intensities of the bands of interest were subjected to densitometry analysis using a Personal Densitometer SI (Molecular Dynamics).

cDNA Cloning and Plasmid Construction.
mRNA was isolated from a nontransformed SHE cell line (SHE83-9) using a mRNA separator kit (Clontech), and 5'- and 3'-RACE strategies with the SMART RACE cDNA amplification kit (Clontech) were applied for cDNA cloning of the hamster Gab1 homologue. First, one set of primers, 5'-CCTTTATAACCTGCCCAGGAGT-3' (GSP-F1) and 3'-GCAG/ATCTTGAGAACTAGCATCT-5' (GSP-R1), was developed according to human and mouse Gab1 sequences (GenBank accession numbers U43885 and AJ250669, respectively), and a cDNA product of 0.4 kb corresponding to the middle portion of human/mouse Gab1 was amplified. This fragment was also used as a template for a DNA probe used in Northern analysis. 3'-RACE PCR with the same 5'-primer gave a 2.7-kb cDNA product. To obtain the upstream sequences, 5'-ACCATGAGCGGTGGTGAAGTGGTC-3' (GSP-F2), which contains the ATG initiation codon (underlined above), was used in PCR with GSP-R1 primer, and a product of 0.9 kb was amplified. Then, 5'-RACE PCR with 3'-GTAAGAGGGAGGTAGTGTGACTGGAGAGGCTT-5' (GSP-R2) was performed using the Advantage-GC cDNA Polymerase Mix (Clontech), and a 1-kb cDNA product was obtained. Sequence analysis of PCR products by dRhodamine Terminator Cycle Sequencing Ready kit (Perkin-Elmer Applied Biosystems) was performed in each step both before and after subcloning into cytomegalovirus promoter-driven mammalian expression vector pCR3.1 using the Eukaryotic TA Cloning kit (Invitrogen), which confirmed that these products were hamster Gab1 homologues. Plasmids were purified with Plasmid Purification kit (Qiagen). For overexpression of hamster Gab1, the 2.2-kb cDNA product amplified by GSP-F2 and 3'-CAGTGGCAACGGCGTGTCTTCACTTTACGCTCTTG-5' (GSP-R3), which contains 5'-TGA-3' stop codon (underlined above),was used.

Northern Blot Analysis.
Total RNA was isolated with Trizol reagent (Life Technologies, Inc.), and 10 µg of total RNA samples were separated by electrophoresis in 1% agarose-formaldehyde gel and transferred to nylon membranes (Hybond-N+; Amersham) by capillary blotting. The membranes were cross-linked by UV radiation. cDNA probes amplified with GSP-F1 and GSP-R1 were labeled with [32P]dCTP with the Prime-It RT Random-Primer Labeling kit (Stratagene). Blots were prehybridized in Rapid-Hyb buffer (Amersham) at 65°C for 2 h followed by hybridization at 65°C overnight. The blots were then washed once in 2x SSC, 0.1% SDS at room temperature and then washed twice in 0.1x SSC, 0.1% SDS at 65°C. The membrane was exposed to Hyperfilm (Amersham) for 24–48 h.

[3H]Thymidine Incorporation Assay.
A [3H]thymidine incorporation assay was used to measure DNA synthesis as described previously (60) . Briefly, 1 x 103 cells were seeded in 96-well plates (Costar) and cultured until 60–70% confluence. Cells were then serum-deprived for 16 h and incubated in the presence of 1 µCi/well [3H]thymidine (ICN Pharmaceuticals, Inc.) with or without 50 ng/ml EGF at 37°C for 24 h. Cells were rinsed twice with ice-cold cmf-PBS and then treated with ice-cold 5% trichloroacetic acid for at least 30 min at 4°C. Cells were rinsed three times with water and lysed with 0.2 N NaOH, 0.1% SDS at room temperature for at least 1 h. Samples were neutralized with hydrochloric acid, and radiation was quantitated in a Packard 2000CA scintillation counter. All incubations were performed in sextuplicate.

Cell Proliferation Assay.
The proliferation rate of cells was determined by using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit (Promega) according to the manufacturer’s instructions. Briefly, 1 x 103 cells were seeded onto 96-well plates, and after 6 h, 20 µl of the 1:20 combined solution of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeththoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium and phenazine methosulfate were added to each well. After a 1-h incubation at 37°C in a humidified 5% CO2 atmosphere, the absorbance at 490 nm was measured using an ELISA plate reader as a control value (day 0). These values represent the quantity of the aqueous soluble formazan product converted from 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeththoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium by dehydrogenase enzymes and are directly proportional to the number of living cells in culture. The absorbance at 490 nm was then measured on the next 4 days (day 1–4), and the values were divided by the control values (day 0). All incubations were performed in sextuplicate.


    Acknowledgments
 
We thank Drs. Richard P. DiAugustine, B. Alex Merrick, and John P. O’Bryan for critical reading of the manuscript and Julie Angerman-Stewart, Mark Geller, and Leigh Wilson for laboratory assistance.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, P. O. Box 12233, NC 27709. Phone: (919) 541-3911; Fax: (919) 541-0146; E-mail: Eling{at}niehs.nih.gov Back

2 The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; Gab1, Grb2-associated binder-1; SHE, Syrian hamster embryo; SH, Src homology; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; IRS-1, insulin receptor substrate 1; ECL, enhanced chemiluminescence; FBS, fetal bovine serum; cmf-PBS, calcium/magnesium-free PBS; IBR, IBR modified Dulbecco’s modified medium; RACE, rapid amplification of cDNA ends; MBD, Met-binding domain; NRS, normal rabbit serum; PH, pleckstrin homology; p-Tyr, phosphotyrosine; TBST, Tris-buffered saline containing 0.1% Tween 20. Back

3 H. Kameda and T. E. Eling, unpublished data. Back

Received for publication 9/29/00. Revision received 4/13/01. Accepted for publication 4/16/01.


    References
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 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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