Involvement of ErbB2 in the Signaling Pathway Leading to Cell Cycle Progression from a Truncated Epidermal Growth Factor Receptor Lacking the C-terminal Autophosphorylation Sites*

To investigate the mechanisms underlying the en- hanced mitogenic activity of the truncated epidermal growth factor receptor (EGFR) lacking the C-terminal autophosphorylation sites ( (cid:68) 973-EGFR), we studied the intracellular signaling pathways in NR6 cells expressing human wild type EGFR and (cid:68) 973-EGFR. Microinjection of dominant/negative p21 ras (N17) completely inhibited EGF-induced DNA synthesis in both cell types. EGF stimulated Shc phosphorylation as well as the formation of wild type EGFR (cid:122) Shc complexes. In contrast, EGF stimulated Shc phosphorylation without formation of (cid:68) 973-EGFR (cid:122) Shc complexes. Tyrosine-phosphorylated Shc formed complexes with Grb2 (cid:122) Sos, and microinjection of

phorylation sites (3), the formation of Shc⅐Grb2⅐Sos complexes may lead to p21 ras activation initiated even by the truncated EGFR.
In this report, we directly evaluated the importance of p21 ras in ⌬973-EGFR mediated mitogenic signaling using a single cell microinjection assay, and identified an alternative mechanism leading to the p21 ras activation. Here, we show that ErbB2 plays an important role in NR6 cells expressing ⌬973-EGFRs.
Glutathione S-Transferase (GST) SH2 Domain Fusion Protein Preparation-The molecular cloning of GST fusion proteins containing SH2 domains has been described elsewhere (26). Briefly, a cDNA for the human Shc-SH2 domain was amplified by polymerase chain reaction, using oligonucleotides with BamHI(5Ј)-3ЈEcoRI linkers. The purified BamHI-EcoRI DNA fragments from polymerase chain reaction products were ligated into a BamHI/EcoRI-digested pGEX-KT expression vector. A plasmid was generated that encoded the peptide fused to the C terminus of GST. The fusion protein contained residues 378 -471 of the human Shc protein (7). GST fusion proteins were produced in Escherichia coli by isopropyl-1-thio-␤-galactopyranoside induction and purified by affinity chromatography on glutathione-agarose beads (26).
Microinjection-Cells were grown on glass coverslips and rendered quiescent by starvation for 24 h in serum-free DMEM. Antibodies or glutathione S-transferase fusion proteins, which were solubilized in microinjection buffer consisting of 5 mM NaPO 4 and 100 mM KCl, pH 7.4, were then microinjected using glass capillary needles. Approximately 1 ϫ 10 Ϫ14 l of the buffer was introduced into each cell. The injection included about 1 ϫ 10 6 molecules of IgG to permit identification of injected cells. Two hours after microinjection, cells were incubated with BrdU plus either vehicle, 160 nM EGF or 10% fetal calf serum, for 16 h at 37°C. The cells were fixed with acid alcohol (90% ethanol, 5% acetic acid) for 20 min at 22°C and then incubated with mouse monoclonal anti-BrdU antibody for 1 h at 22°C. The cells were then stained by incubation with rhodamine-labeled donkey anti-mouse IgG antibody and fluorescein isothiocyanate-labeled donkey anti-rabbit IgG antibody for 1 h at 22°C. After the coverslips were mounted, the cells were analyzed with an Axiphot fluorescence microscope (Carl Zeiss). Microinjected cell numbers were 250 -300 per coverslip. Immunofluorescent staining of the injected cells indicated that about 75% of the cells were successfully microinjected (26,33).
Western Blotting Studies-Cell monolayers were starved for 24 h in serum-free DMEM. The cells were then treated with various concentrations of EGF for the indicated times at 37°C. Cells were lysed in a buffer containing 30 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM Na 3 VO 4 , pH 7.4. The cell lysates were centrifuged to remove insoluble materials. The supernatants (100 g of protein) were used for immunoprecipitation with the indicated antibodies for 5 h at 4°C, or for 90 min with Shc-SH2 GST fusion protein, which was then precipitated with glutathione-agarose. The precipitates were separated by SDS-PAGE and transferred to Im-mobilon-P using a Bio-Rad Transblot apparatus. The membranes were blocked in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, 2.5% bovine serum albumin, pH 7.5, for 2 h at 20°C. The membranes were then probed with specified antibodies for 2 h at 20°C. After washing the membranes in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5, blots were incubated with horseradish peroxidase-linked second antibody followed by enhanced chemiluminescence detection using the ECL reagent according to the manufacturer's instructions (Amersham Corp.) (21,26,27).
Measurement of GEF Activity in Membranes-Cells were starved for 16 h in serum-free DMEM. The cells were then treated with 160 nM EGF at 37°C for 2 min. The cells were collected in a buffer containing 50 mM Hepes, 150 mM NaCl, 10 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 2 HPO 4 , 1 mM Na 3 VO 4 , 10 g/ml leupeptin, 10 g/ml aprotinin, 1 mM dithiothreitol, pH 7.5. The cells were disrupted by 20 strokes of a tight fitting Dounce homogenizer. The homogenate was centrifuged at 3,000 rpm in an Eppendorf 5402 centrifuge at 4°C for 3 min to remove the nuclear fraction. The supernatants were recentrifuged at 220,000 ϫ g at 4°C for 60 min. The particulate fraction was suspended in a buffer containing 0.05% SDS, 0.1% Triton X-100, 50 mM Hepes, 150 mM NaCl, 10 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 2 HPO 4 , 1 mM Na 3 VO 4 , 10 g/ml leupeptin, 10 g/ml aprotinin, 1 mM dithiothreitol, 100 M GTP, 100 M GDP, pH 7.5, and sonicated at 4°C for 30 s. The GEF activity in the membranes was determined by measuring the dissociation of protein-bound [ 3 H]GDP radioactivity using a nitrocellulose filter binding assay. Purified c-Ha-Ras was incubated with [ 3 H]GDP in a buffer containing 25 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 400 g/ml chain A insulin, pH 7.5, for 15 min at 30°C. The Ras⅐GDP complex was added to the membrane preparations and incubated at 23°C. After 15 min, aliquots were removed and filtered through 0.45-m Millipore nitrocellulose filters. The amount of [ 3 H]GDP radioactivity bound to p21 ras was quantitated by scintillation counting. [ 3 H]GDP binding to p21 ras was confirmed by immunoprecipitation with anti-Ras antibody. Background counts/min were less than 1% of total bound [ 3 H]GDP (27,34).

Effect of Dominant Negative N17-Ras on EGF Signaling-
We and others have shown that p21 ras is involved in EGF stimulation of cell cycle progression (9,10,26). To evaluate the role of p21 ras in the signaling pathway engaged by the truncated ⌬973-EGFR, we performed single cell microinjection studies. Dominant negative p21 ras (N17) was injected into NR6 cells expressing wild type EGFRs or ⌬973-EGFRs. Following microinjection, the cells were stimulated with EGF, and cell cycle progression was monitored by measuring BrdU incorporation into newly synthesized DNA. In the basal state, 3.5 Ϯ 0.3% and 12.1 Ϯ 1.1% of cells incorporated BrdU in wild type and ⌬973-EGFR cells, respectively. EGF stimulated BrdU incorporation into 34.2 Ϯ 0.8% and 50.9 Ϯ 5.6% of the cells, respectively (Fig. 1). Microinjection of preimmune control IgG did not alter this stimulatory effect (data not shown). In contrast, microinjection of N17-Ras markedly inhibited EGF stimulation of DNA synthesis by 91% and 100%, respectively, indicating that p21 ras plays a central role in ⌬973-EGFR as well as wild type EGFR signaling ( Fig. 1).
Shc Phosphorylation-Previous studies suggest that the predominant mechanism whereby EGF activates p21 ras is by inducing the formation of Shc⅐Grb2⅐Sos complexes (27,35). To assess Shc tyrosine phosphorylation, cells were stimulated with EGF for the indicated times. The cell lysates were immunoprecipitated with a polyclonal anti-Shc antibody, and the immunoprecipitates were immunoblotted with a monoclonal anti-phosphotyrosine antibody. EGF stimulated Shc phosphorylation in a dose-and time-dependent manner in wild type EGFR cells (Fig. 2). Shc was tyrosine phosphorylated in a similar fashion in ⌬973-EGFR cells, even though the C-terminal EGFR autophosphorylation sites which bind to Shc are absent in the truncated receptor. As evidenced in Fig. 2, EGF treatment of wild type EGFR cells stimulated the association of Shc with two phosphoproteins, pp170 and pp145. pp170 is the EGFR which was confirmed by immunoblotting with anti-EGFR antibody (data not shown). In the case of ⌬973-EGFR cells, pp145 was also found to associate with Shc in an EGF dependent manner. Furthermore, there was much more tyrosine phosphorylated pp145 associated with Shc in the ⌬973-EGFR cells than in the wild type EGFR cells. pp145 is not the truncated EGFR, since it was seen in the wild type EGFR cells also, was not detected by reprobing the membrane for EGFR (data not shown), and because the ⌬973 EGFR has previously been shown not to become tyrosine-phosphorylated (36). In addition, a tyrosine phosphoprotein of pp185 was seen after EGF stimulation in the Shc precipitates (data not shown).
Microinjection of anti-Shc Antibody-To evaluate the functional role of Shc in EGF induced mitogenic signaling in ⌬973-EGFR cells, anti-Shc antibody was microinjected into quiescent cells. Microinjection of anti-Shc antibody did not have any effect on ligand independent DNA synthesis, since basal BrdU incorporation in both uninjected and antibody-injected cells was comparable (Fig. 3). Stimulation of quiescent cells with EGF induced a marked increase in DNA synthesis. Microinjection of anti-Shc antibody reduced the ability of EGF to stimulate DNA synthesis by 80% in wild type EGFR cells. Microinjection of anti-Shc antibody also inhibited DNA synthesis in ⌬973-EGFR cells, although the inhibition was less (48%) than that seen in wild type EGFR cells. Interestingly, microinjection of anti-Shc antibody did not block serum-induced DNA synthesis in cells expressing either wild type or ⌬973 EGFR.
Microinjection of Shc-SH2 GST Fusion Protein-Shc binds to the phosphorylated EGFR at least partially through its SH2 domain (18 -22, 24, 25). Microinjection of Shc-SH2 GST fusion protein inhibited EGF-induced DNA synthesis by 80% in wild type EGFR cells, indicating the importance of Shc-SH2⅐EGFR interaction in these cells (Fig. 4). Interestingly, microinjection of the Shc-SH2 GST fusion protein significantly blocked EGF stimulation of cell cycle progression in ⌬973-EGFR cells (Fig.  4), despite the lack of any demonstrable co-precipitation of ⌬973-EGFR and Shc (Fig. 2). As seen with microinjection of anti-Shc antibody, the Shc-SH2 GST fusion protein did not block serum-induced DNA synthesis in either cell type. These results contrast with N17 dominant negative ras, where microinjection inhibited both EGF-and serum-induced DNA synthesis.
EGF Stimulation of Complex Formation in Wild Type EGFR Cells-It has been shown that the EGFR, Grb2, and Shc form complexes after EGF stimulation (18 -21). To assess their association more quantitatively, EGF-treated cell lysates were immunoprecipitated with anti-Shc, anti-EGFR, or anti-Grb2 antibody, and the immunoprecipitates were immunoblotted with anti-phosphotyrosine, anti-Shc, or anti-Grb2 antibody as shown in Fig. 5, panels A, B, and C. Panel A illustrates that the EGFR underwent tyrosine phosphorylation in response to EGF. Further, the presence of the EGFR in anti-Shc and anti-Grb2 immunoprecipitates demonstrates that Shc and Grb2 associate with the phosphorylated EGFR. Panels B and C indicate the EGF stimulation caused association of Shc with Grb2, confirming that an EGFR⅐Shc⅐Grb2⅐Sos signaling complex was formed. Precipitation of cell lysates with a Shc-SH2 GST fusion protein revealed that a small amount of pp145 was precipitated after EGF stimulation (Fig. 6, lanes 1 and 2).
EGF Stimulation of Complex Formation in ⌬973 EGFR Cells-Co-immunoprecipitation studies also showed complex formation in ⌬973-EGFR cells, as seen in Fig. 5, panels D, E, and F. Phosphotyrosine immunoblotting in panel D revealed that EGF stimulated the association of tyrosine-phosphorylated pp145 with both Shc and Grb2, while phosphorylated pp185 was precipitated by the EGFR antibody. Fig. 6, lanes 3 and 4, indicate that much more pp145 is precipitated by the Shc-SH2 GST fusion protein after EGF stimulation of the ⌬973-EGFR cells than by the wild type EGFR cells. Thus, phosphorylation of pp145 seems to be up-regulated in the ⌬973-EGFR cells. Panels E and F illustrate that EGF induced the association of Shc with Grb2. The absence of Shc and Grb2 in the anti-EGFR immunoprecipitates in panels E and F indicates that neither Shc nor Grb2 associates with the ⌬973-EGFR, and this was expected due to its lack of autophosphorylation sites for SH2 domain recognition. Thus, EGF induced the formation of Shc⅐Grb2 complexes, and at least some of these complexes were associated with phosphorylated pp145.
Comparison of GEF Activity in Cells Expressing Wild Type and ⌬973 EGFR-Sos is a GEF for p21 ras which is translocated from the cytosol to membrane fractions following EGF stimulation (18,27,37). GEF activity was measured in the membrane fractions from wild type and ⌬973-EGFR cells. Membrane associated GEF activity in the basal state was 16.5 Ϯ 0.4% and 28.6 Ϯ 3.0% in wild type EGFR and ⌬973-EGFR cells, respectively. EGF increased membrane GEF activity to 46.8 Ϯ 1.0% and 51.5 Ϯ 4.0%, respectively (Fig. 7). Thus, both basal and EGF-stimulated GEF activity were higher in ⌬973-EGFR cells than in wild type EGFR cells, but EGF caused translocation to the membrane in both cell types.
EGF-stimulated ErbB2 Phosphorylation-Ligand mediated interactions between EGFR and ErbB2 have been previously reported (38 -42). The presence of an EGF-stimulable, tyrosine-phosphorylated pp185 in anti-EGFR immunoprecipitates in the ⌬973-EGFR cells (Fig. 5) suggested the possibility that the 185-kDa ErbB2 might be involved in EGF signaling in the ⌬973-EGFR cells. To assess this, cell lysates from EGF-treated cells were immunoprecipitated with a specific anti-ErbB2 antibody, and the immunoprecipitates were blotted with antiphosphotyrosine antibody (Fig. 8). In wild type EGFR cells, minimal tyrosine phosphorylation was observed in the basal state, and EGF markedly increased ErbB2 phosphorylation. Since EGF is not a direct ligand for ErbB2, the EGF dependence of ErbB2 phosphorylation probably represents heterophosphorylation of ErbB2 by the EGFR. Importantly, ErbB2 was tyrosine-phosphorylated even in the basal state, and EGFR stimulation induced further tyrosine phosphorylation of ErbB2 in ⌬973-EGFR cells. In addition to ErbB2, additional proteins were tyrosine-phosphorylated in ⌬973-EGFR cells, mostly in the molecular mass range of 120 -170-kDa. DISCUSSION EGF stimulates mitogenesis by activating the tyrosine kinase activity of the EGFR which then sets in motion a signaling cascade eventually leading to cell cycle progression (1). Although not completely understood, many elements of this signaling pathway have been defined. One of the initial steps in EGF action involves binding of various SH2 and phosphotyrosine binding domain-containing molecules to the phosphoty-  1 and 2) and ⌬973-EGFR cells (lanes 3 and 4) were stimulated without (lanes 1  and 3) or with (lanes 2 and 4) 160 nM EGF for 2 min at 37°C. After treatment, cell lysates were affinity-precipitated with Shc-SH2 GST fusion protein. The precipitates were separated by SDS-PAGE and transferred to Immobilon-P, and immunoblots were performed with anti-phosphotyrosine antibody. Molecular masses of EGFR (170 kDa), and ErbB2 (185 kDa), and Shc-associated protein pp145 (145 kDa) are shown by arrows. Results are representative of two separate experiments.
rosine motifs within the autophosphorylated EGFR. These proteins, such as Grb2, Shc, and phospholipase C-␥, then further propagate the biologic signals initiated by autophosphorylation of the EGFR (4 -7). The five EGFR autophosphorylation sites are contained within the receptor C terminus, and it has recently been shown that cells expressing mutant EGFRs in which the five autophosphorylation sites have been deleted by C-terminal truncation are normally responsive to EGF, with respect to stimulation of DNA synthesis (2,29,30). This is surprising, given the presumed importance of EGFR phosphotyrosine motifs in docking SH2 containing signaling proteins to the EGFR. This suggested the presence of an alternate pathway for EGF action in cells expressing these mutant receptors. To evaluate this possibility, we have studied the intracellular signaling mechanisms in transfected cells overexpressing a truncated EGFR (⌬973-EGFR) from which all five tyrosine autophosphorylation sites are deleted.
An important step in normal EGF action is formation of Shc⅐Grb2⅐Sos complexes which lead to the generation of p21 ras GTP and subsequent activation of the MAP kinase pathway (26,27). To determine whether p21 ras GTP was critical to EGF action in ⌬973-EGFR cells, we conducted single cell microinjection studies using dominant negative Ras (N17) protein. As expected, inhibition of p21 ras completely prevented the ability of EGF to stimulate DNA synthesis in wild type EGFR cells. Interestingly, the same result was observed in ⌬973-EGFR cells. Thus, despite the fact that the ⌬973-EGFR contains no autophosphorylation sites, and, therefore, cannot bind to Grb2 or Shc directly (2, 3), p21 ras is still necessary for mitogenic signaling in these cells.
Given these results, we next explored potential upstream elements connecting the ⌬973-EGFR to p21 ras . We have previously shown that EGF mediated formation of Shc⅐Grb2⅐Sos complexes is the predominant mechanism coupling wild type EGFRs to the Ras pathway (27). The current studies confirm these results, but also show that EGF stimulation of ⌬973-EGFR cells leads to comparable dose and time dependent tyrosine phosphorylation of Shc, as compared to wild type EGFR cells. Interestingly, when Shc immunoprecipitates from wild type and ⌬973-EGFR cells were probed with anti-phosphotyrosine antibody, the predominant phosphoprotein co-precipitating with Shc in wild type EGFR cells was the EGFR, whereas, in ⌬973-EGFR cells, a pp145 and pp185 protein were the only ones visualized. pp185 was found to be ErbB2. The identity of pp145 remains unknown, although it is not the truncated EGFR. Interestingly, when lysates from EGF stimulated ⌬973-EGFR cells were precipitated with a Shc-SH2 GST fusion protein, pp145 and pp185 were readily precipitated and identified (Fig. 6). Thus, despite the absence of any autophosphorylation sites, the ⌬973-EGFR was normally capable of mediating ligand induced Shc tyrosine phosphorylation. Since this truncated receptor did not coprecipitate with either intact Shc protein or the Shc-SH2 GST fusion protein, these results suggest an alternate pathway coupling the truncated EGFR to Shc.
To determine whether Shc was a functionally significant molecule conveying signals between ⌬973-EGFR and p21 ras , we conducted microinjection studies using anti-Shc antibodies and a Shc-SH2 GST fusion protein. Microinjection of either reagent inhibited EGF-induced DNA synthesis by 80 -90% in wild type EGFR cells. While inhibition was clearly demonstrated in ⌬973-EGFR cells, the magnitude of this inhibition was less than in wild type EGFR cells. Thus, although Shc is clearly an important signaling molecule mediating EGF stimulated DNA synthesis in ⌬973-EGFR cells, the partial inhibition by the microinjected reagents suggests that alternative pathways are also operative. The failure of either anti-Shc antibody or Shc-SH2 GST fusion protein to block serum-stimulated DNA synthesis confirms that additional mechanisms to activate p21 ras exist. In all cases the amount of anti-Shc antibody or Shc-SH2 GST fusion protein microinjected into the cells was optimal, because when higher concentrations of these re- agents were microinjected, no additional inhibition was observed (data not shown). Some component of mitogenic signaling could partially bypass Shc to activate p21 ras in ⌬973-EGFR cells. In this regard, a predominant pp145 kDa protein was phosphorylated in response to EGF in these cells, whereas, this protein was only minimally phosphorylated in wild type EGFR cells. The identity and function of this protein are unknown, but it is possible that pp145 represents a new signaling molecule which provides an input to p21 ras , independent of the Shc⅐Grb2⅐Sos pathway. Furthermore, EGF stimulation led to a much broader spectrum of tyrosine phosphorylation of substrate proteins in ⌬973-EGFR cells compared to wild type EGFR cells, confirming a broader array of substrate specificities in this cell line (36).
Our studies indicate that one mechanism explaining the alternative signaling pathway in cells expressing truncated EGFRs involves activation of ErbB2. Four members of the EGFR related family of receptor tyrosine kinases have been identified including ErbB1/EGFR (1), ErbB2 (41), ErbB3 (38,39), and ErbB4 (40) and it has been demonstrated that activated ErbB2 or ErbB3 can lead to tyrosine phosphorylation of Shc and stimulation of p21 ras GTP formation (32,(43)(44)(45). Expression of ErbB3 and ErbB4 is limited to certain epithelial cell types, and they are not expressed in fibroblasts, whereas, ErbB2 has been identified in fibroblast cell lines (46). Taken together with the fact that the NR6 cells used in the current studies are derived from fibroblasts, ErbB2 is a logical candidate signaling molecule in these cells. Furthermore, we demonstrated that no significant tyrosine phosphorylation of ErbB3 was observed in either wild type EGFR or ⌬973-EGFR cells (data not shown). It has been demonstrated that ErbB2 can complement with the EGFR in mediating EGF induced signals (47,48). Although the ligand for ErbB2 has not been identified, the ErbB2 protein can be phosphorylated on tyrosine residues by the activated EGFR (49,50). This may be due to intermolecular cross phosphorylation of ErbB2 by the EGFR (49), or intramolecular transphosphorylation within heterodimers formed between ErbB2 and EGFR molecules (42,50,51). Since a kinase deficient mutant ErbB2 can suppress normal EGFR signaling in a dominant/negative fashion (52), heterodimer formation between EGFR and ErbB2 appears to be the more important mechanism. In the current studies, ErbB2 was heavily tyrosine phosphorylated in basal ⌬973-EGFR cells, whereas basal ErbB2 phosphorylation was undetected in wild type EGFR cells. Following EGF stimulation, an increase in ErbB2 phosphorylation was seen in both cell types. Importantly, co-precipitation studies demonstrated that ErbB2 was associated with Sos, Shc, and Grb2 in both basal and EGF stimulated ⌬973-EGFR cells (data not shown). Taken together with the fact that the ⌬973-EGFR can not interact directly with SH2-containing adaptor proteins such as Shc and Grb2, it would appear that ErbB2 plays a major role in EGF induced mitogenic signaling in ⌬973-EGFR cells. This would occur by transphosphorylation of ErbB2 by the ⌬973-EGFR within heterodimer complexes. Tyrosine phosphorylated ErbB2 may also lead to activation of signaling pathways by other mechanisms. For example, a broad array of tyrosine phosphorylated proteins was detected in ErbB2 precipitates from ⌬973-EGFR cells, and, in particular, a predominant 145 kDa tyrosine phosphorylated protein, whose phosphorylation state was EGF dependent in ⌬973-EGFR cells, was readily identified. Whether pp145 or some other signaling molecule participates in an alternate signaling pathway coupling ⌬973-EGFRs to p21 ras , independent of Shc⅐Grb2⅐Sos, remains to be elucidated.
In summary, our results demonstrate the importance of p21 ras in the mitogenic signal transduction pathway mediated by ⌬973-EGFR. Although the molecular coupling of EGFR⅐Shc⅐Grb2⅐Sos is important as a common mechanism to activate p21 ras , an alternative pathway resides in ⌬973-EGFR cells. Our evidence suggests that ErbB2 plays an important role in coupling the truncated EGFR to p21 ras activation. These results indicate a novel signal transduction mechanism involving ErbB2 in ⌬973-EGFR cells.