©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (*)

(Received for publication, July 6, 1995; and in revised form, January 23, 1996)

Toshiyasu Sasaoka (1) W. John Langlois (2) Frances Bai (2) David W. Rose (2) J. Wayne Leitner (4) Stuart J. Decker (3) Alan R. Saltiel (3) Gordon N. Gill (2) Masashi Kobayashi (1) Boris Draznin (4) Jerrold M. Olefsky (2)(§)

From the  (1)First Department of Medicine, Toyama Medical and Pharmaceutical University, Toyama, 930-01, Japan, the (2)Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093, the Veterans Administration Medical Center, Medical Research Service, San Diego, California 92161, the (3)Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105, and the (4)Medical Research Service and the Department of Medicine, Veterans Affairs Medical Center and the University of Colorado Health Sciences Center, Denver, Colorado 80220

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To investigate the mechanisms underlying the enhanced mitogenic activity of the truncated epidermal growth factor receptor (EGFR) lacking the C-terminal autophosphorylation sites (Delta973-EGFR), we studied the intracellular signaling pathways in NR6 cells expressing human wild type EGFR and Delta973-EGFR. Microinjection of dominant/negative p21(N17) completely inhibited EGF-induced DNA synthesis in both cell types. EGF stimulated Shc phosphorylation as well as the formation of wild type EGFRbulletShc complexes. In contrast, EGF stimulated Shc phosphorylation without formation of Delta973-EGFRbulletShc complexes. Tyrosine-phosphorylated Shc formed complexes with Grb2bulletSos, and microinjection of anti-Shc antibody and Shc-SH2 GST fusion protein inhibited EGF stimulation of DNA synthesis in both cell lines. EGF markedly increased ErbB2 tyrosine phosphorylation in wild type EGFR cells. In Delta973-EGFR cells, ErbB2 was tyrosine phosphorylated in the basal state and EGFR stimulated further phosphorylation of ErbB2. In addition to ErbB2, additional proteins were tyrosine phosphorylated in Delta973-EGFR cells, mostly in the molecular mass range of 120-170 kDa. Taken together with our findings indicating coupling of ErbB2 to Shc, these data suggest the importance of an alternative signaling pathway in Delta973-EGFR cells mediated by the formation of heterodimeric structures between the truncated EGFR and ErbB2, followed by coupling through Shc to Grb2bulletSos and the p21 pathway, ultimately leading to mitogenesis.


INTRODUCTION

Epidermal growth factor (EGF) (^1)stimulates the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR)(1) . The activated EGFR phosphorylates its own tyrosine residues as well as intracellular substrate proteins. For EGFR, five autophosphorylation sites (tyrosine 992, 1068, 1086, 1148, and 1173) located at the C terminus are identified as the major binding region for Src homology (SH) 2 domain containing proteins(2, 3) . SH2 and/or phosphotyrosine binding domain containing proteins such as phospholipase C-(1)(4) , GTPase-activating protein of p21(5) , Grb2(6) , and Shc (7, 8) are direct candidates to signal downstream. p21 has been shown to play an important role in EGF-induced mitogenic signaling(9, 10) . p21 is active in its GTP-bound form (11) and this activation is mainly controlled by the translocation of the p21 guanine nucleotide exchange factor (GEF), the Son of Sevenless (Sos) protein, to the cell membrane, where it is in proximity to p21(12, 13, 14, 15, 16, 17) . The proline-rich region of Sos binds to the SH3 domain of Grb2 and preformed Grb2bulletSos complexes exist within unstimulated cells (17, 18, 19, 20, 21) . Through the Grb2-SH2 domain, Grb2bulletSos complexes can bind to tyrosine phosphorylated EGFRs(17, 18, 19, 20, 21, 22, 23) . Alternatively, the SH2 domain of Grb2 can also bind to phosphorylated Shc, which itself can recognize EGFR autophosphorylation motifs via its SH2 and N-terminal phosphotyrosine binding domain(18, 19, 20, 21, 22, 24, 25) . Thus, EGFRbulletGrb2bulletSos and/or EGFRbulletShcbulletGrb2bulletSos provide a mechanism whereby EGF can stimulate p21 GTP formation. Recently, we reported the importance of Shc as an adaptor protein transducing mitogenic signaling from activated EGFR to p21 activation (26, 27) . Although the association of phosphorylated EGFRs with these adaptor proteins provides the mechanism to trigger downstream signals, mutant EGFRs in which the tyrosine phosphorylation sites were replaced with phenylalanine signal to the same extent as wild type EGFRs do (28) . In addition, mutant EGFRs truncated at amino acid residue 973 (Delta973-EGFR) which lack the C-terminal five autophosphorylation sites mediate enhanced cell cycle progression(2, 29, 30) . Since neither Shc nor Grb2, which are important for p21 activation, directly binds to Delta973-EGFR following EGF stimulation, an alternative pathway leading to cell cycle progression bypassing p21 may reside in cells expressing Delta973-EGFRs. Alternatively, since Shc was still tyrosine phosphorylated without association with the EGFR mutant lacking the autophosphorylation sites(3) , the formation of ShcbulletGrb2bulletSos complexes may lead to p21 activation initiated even by the truncated EGFR.

In this report, we directly evaluated the importance of p21in Delta973-EGFR mediated mitogenic signaling using a single cell microinjection assay, and identified an alternative mechanism leading to the p21 activation. Here, we show that ErbB2 plays an important role in NR6 cells expressing Delta973-EGFRs.


EXPERIMENTAL PROCEDURES

Cell Lines and Materials

NR6 cells expressing 1times10^5 wild type human EGFRs or a similar number of EGFRs truncated at amino acid residue 973 (Delta973) were maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 containing 10% fetal calf serum as described previously(29, 30) . NR6 cells do not express endogenous mouse EGFR(29) . The p21 probe (c-Ha-Ras) was a kind gift from Dr. Alan Wolfman (Cleveland Clinic Foundation). Dominant-negative mutant p21 protein(N17) was a kind gift from Dr. James R. Feramisco (University of California, San Diego, CA). EGF was purchased from Life Technologies, Inc. [^3H]GDP (32 Ci/nmol) was from DuPont NEN. Electrophoresis reagents were from Bio-Rad (Hercules, CA). Characteristics of polyclonal (4-17) and monoclonal (528) anti-EGFR antibodies, and a polyclonal anti-ErbB2 antibody were as described previously(29, 31) . The polyclonal anti-ErbB3 antibody was a kind gift from Dr. Sally A. Prigent (University of California, San Diego, CA) (32) . Bromodeoxyuridine (BrdU), a monoclonal anti-BrdU antibody, and enhanced chemiluminescence reagents were from Amersham Corp. The monoclonal anti-phosphotyrosine antibody (pY20), polyclonal and monoclonal anti-Shc antibodies, and a monoclonal anti-Grb2 antibody were from Transduction Laboratories (Lexington, KY). The polyclonal anti-Grb2 antibody was from Santa Cruz (Santa Cruz, CA). Rabbit IgG, fluorescein isothiocyanate- or rhodamine-conjugated anti-mouse and anti-rabbit IgG antibodies were from Jackson Laboratories (West Grove, NY). All other routine reagents were purchased from Sigma.

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(5`)-3`EcoRI 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-beta-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 times 10 l of the buffer was introduced into each cell. The injection included about 1 times 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 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Na(3)VO(4), pH 7.4. The cell lysates were centrifuged to remove insoluble materials. The supernatants (100 µg 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 Immobilon-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 µg/ml leupeptin, 10 µg/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 times 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 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM dithiothreitol, 100 µM GTP, 100 µM 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 [^3H]GDP radioactivity using a nitrocellulose filter binding assay. Purified c-Ha-Ras was incubated with [^3H]GDP in a buffer containing 25 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 400 µg/ml chain A insulin, pH 7.5, for 15 min at 30 °C. The RasbulletGDP complex was added to the membrane preparations and incubated at 23 °C. After 15 min, aliquots were removed and filtered through 0.45-µm Millipore nitrocellulose filters. The amount of [^3H]GDP radioactivity bound to p21 was quantitated by scintillation counting. [^3H]GDP binding to p21 was confirmed by immunoprecipitation with anti-Ras antibody. Background counts/min were less than 1% of total bound [^3H]GDP(27, 34) .


RESULTS

Effect of Dominant Negative N17-Ras on EGF Signaling

We and others have shown that p21 is involved in EGF stimulation of cell cycle progression(9, 10, 26) . To evaluate the role of p21 in the signaling pathway engaged by the truncated Delta973-EGFR, we performed single cell microinjection studies. Dominant negative p21(N17) was injected into NR6 cells expressing wild type EGFRs or Delta973-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 ± 0.3% and 12.1 ± 1.1% of cells incorporated BrdU in wild type and Delta973-EGFR cells, respectively. EGF stimulated BrdU incorporation into 34.2 ± 0.8% and 50.9 ± 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 plays a central role in Delta973-EGFR as well as wild type EGFR signaling (Fig. 1).


Figure 1: Inhibition of DNA synthesis by microinjection of dominant/negative p21(N17). Serum-starved wild type EGFR (panel A) and Delta973-EGFR (panel B) cells were microinjected with 2 mg/ml N17-Ras containing 5 mg/ml rabbit IgG. BrdU incorporation stimulated by 160 nM EGF, 10% serum or neither in the injected cells (open bars) and uninjected cells (solid bars) on the same coverslip was determined as described under ``Experimental Procedures.'' Cumulative data are shown and results are the means ± S.E. of three experiments.



Shc Phosphorylation

Previous studies suggest that the predominant mechanism whereby EGF activates p21 is by inducing the formation of ShcbulletGrb2bulletSos 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 Delta973-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 Delta973-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 Delta973-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 Delta973 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).


Figure 2: EGF-stimulated tyrosine phosphorylation of Shc. A, time course of Shc phosphorylation. Serum-starved cells were treated with 160 nM EGF for the indicated times. B, dose-response of Shc phosphorylation. Serum-starved cells were treated with the indicated concentrations of EGF for 5 min. After treatment, cell lysates were immunoprecipitated with anti-Shc antibody. The immunoprecipitates were separated by SDS-PAGE and transferred to Immobilon-P. Immunoblots were performed with anti-phosphotyrosine antibody. Molecular masses of predominant Shc isoform (52 kDa), EGFR (170 kDa), and Shc-associated proteins pp145 (145 kDa) are shown by arrows. Results are representative of three separate experiments.



Microinjection of anti-Shc Antibody

To evaluate the functional role of Shc in EGF induced mitogenic signaling in Delta973-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 Delta973-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 Delta973 EGFR.


Figure 3: Inhibition of DNA synthesis by microinjection of anti-Shc antibody. Serum-starved wild type EGFR (panel A) and Delta973-EGFR (panel B) cells were microinjected with 5 mg/ml anti-Shc antibody. EGF (160 nM) or serum (10%) were then added, and BrdU incorporation in the injected cells (open bars) and uninjected cells (solid bars) on the same coverslip was determined as described under ``Experimental Procedures.'' Cumulative data are shown and results are the means ± S.E. of four experiments.



Microinjection of Shc-SH2 GST Fusion Protein

Shc binds to the phosphorylated EGFR at least partially through its SH2 domain(18, 19, 20, 21, 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-SH2bulletEGFR interaction in these cells (Fig. 4). Interestingly, microinjection of the Shc-SH2 GST fusion protein significantly blocked EGF stimulation of cell cycle progression in Delta973-EGFR cells (Fig. 4), despite the lack of any demonstrable co-precipitation of Delta973-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.


Figure 4: Inhibition of DNA synthesis by microinjection of Shc-SH2 GST fusion protein. Serum-starved wild type EGFR (panel A) and Delta973-EGFR (panel B) cells were microinjected with 5 mg/ml Shc-SH2 GST fusion protein containing 5 mg/ml rabbit IgG. Cells were treated with EGF (160 nM) or serum (10%), and BrdU incorporation in the injected cells (open bars) and uninjected cells (solid bars) on the same coverslip was determined as described under ``Experimental Procedures.'' Cumulative data are shown and results are the means ± S.E. of three experiments.



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, 19, 20, 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 EGFRbulletShcbulletGrb2bulletSos 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).


Figure 5: EGF stimulated complex formation. Serum-starved wild type EGFR cells (panels A, B, and C) and Delta973-EGFR cells (panels D, E, and F) were treated without or with 160 nM EGF for 2 min at 37 °C. Cell lysates were immunoprecipitated with control preimmune IgG, anti-Shc, anti-EGFR, or anti-Grb2 antibody. The immunoprecipitates were separated by SDS-PAGE and transferred to Immobilon-P. Immunoblots were performed with anti-phosphotyrosine (panels A and D), anti-Shc (panels B and E), or anti-Grb2 (panels C and F) antibody. Results are representative of three separate experiments.




Figure 6: EGF-stimulated complex formation with Shc-SH2 GST fusion protein. Serum-starved wild type EGFR cells (lanes 1 and 2) and Delta973-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.



EGF Stimulation of Complex Formation in Delta973 EGFR Cells

Co-immunoprecipitation studies also showed complex formation in Delta973-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 Delta973-EGFR cells than by the wild type EGFR cells. Thus, phosphorylation of pp145 seems to be up-regulated in the Delta973-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 Delta973-EGFR, and this was expected due to its lack of autophosphorylation sites for SH2 domain recognition. Thus, EGF induced the formation of ShcbulletGrb2 complexes, and at least some of these complexes were associated with phosphorylated pp145.

Comparison of GEF Activity in Cells Expressing Wild Type and Delta973 EGFR

Sos is a GEF for p21 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 Delta973-EGFR cells. Membrane associated GEF activity in the basal state was 16.5 ± 0.4% and 28.6 ± 3.0% in wild type EGFR and Delta973-EGFR cells, respectively. EGF increased membrane GEF activity to 46.8 ± 1.0% and 51.5 ± 4.0%, respectively (Fig. 7). Thus, both basal and EGF-stimulated GEF activity were higher in Delta973-EGFR cells than in wild type EGFR cells, but EGF caused translocation to the membrane in both cell types.


Figure 7: EGF stimulation of GEF activity in membrane fraction. Serum-starved cells were stimulated without or with 160 nM EGF for 2 min at 37 °C. A membrane fraction was then prepared and GEF activity was determined by measuring the dissociation of protein-bound [^3H] GDP radioactivity. Results in wild type EGFR (closed bars) cells and Delta973-EGFR cells (open bars) are expressed as the percentage of [^3H]GDP released in 15 min, and are shown as the mean ± S.E. of three separate experiments.



EGF-stimulated ErbB2 Phosphorylation

Ligand mediated interactions between EGFR and ErbB2 have been previously reported (38, 39, 40, 41, 42) . The presence of an EGF-stimulable, tyrosine-phosphorylated pp185 in anti-EGFR immunoprecipitates in the Delta973-EGFR cells (Fig. 5) suggested the possibility that the 185-kDa ErbB2 might be involved in EGF signaling in the Delta973-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 anti-phosphotyrosine 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 Delta973-EGFR cells. In addition to ErbB2, additional proteins were tyrosine-phosphorylated in Delta973-EGFR cells, mostly in the molecular mass range of 120-170-kDa.


Figure 8: EGF stimulation of ErbB2 phosphorylation. Serum-starved cells were stimulated without or with 160 nM EGF for 2 min at 37 °C. After treatment, cell lysates were immunoprecipitated with anti-ErbB2 specific antibody. The immunoprecipitates were separated by SDS-PAGE and transferred to Immobilon-P. Immunoblots were performed with anti-phosphotyrosine antibody. Molecular mass of ErbB2 (185 kDa) is shown by an arrow. Results are representative of three separate experiments.




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 phosphotyrosine 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, 5, 6, 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 (Delta973-EGFR) from which all five tyrosine autophosphorylation sites are deleted.

An important step in normal EGF action is formation of ShcbulletGrb2bulletSos complexes which lead to the generation of p21 GTP and subsequent activation of the MAP kinase pathway(26, 27) . To determine whether p21 GTP was critical to EGF action in Delta973-EGFR cells, we conducted single cell microinjection studies using dominant negative Ras(N17) protein. As expected, inhibition of p21 completely prevented the ability of EGF to stimulate DNA synthesis in wild type EGFR cells. Interestingly, the same result was observed in Delta973-EGFR cells. Thus, despite the fact that the Delta973-EGFR contains no autophosphorylation sites, and, therefore, cannot bind to Grb2 or Shc directly(2, 3) , p21 is still necessary for mitogenic signaling in these cells.

Given these results, we next explored potential upstream elements connecting the Delta973-EGFR to p21. We have previously shown that EGF mediated formation of ShcbulletGrb2bulletSos 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 Delta973-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 Delta973-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 Delta973-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 Delta973-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 Delta973-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 Delta973-EGFR and p21, 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 Delta973-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 Delta973-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 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 reagents were microinjected, no additional inhibition was observed (data not shown). Some component of mitogenic signaling could partially bypass Shc to activate p21 in Delta973-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, independent of the ShcbulletGrb2bulletSos pathway. Furthermore, EGF stimulation led to a much broader spectrum of tyrosine phosphorylation of substrate proteins in Delta973-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 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 Delta973-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 Delta973-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 Delta973-EGFR cells (data not shown). Taken together with the fact that the Delta973-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 Delta973-EGFR cells. This would occur by transphosphorylation of ErbB2 by the Delta973-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 Delta973-EGFR cells, and, in particular, a predominant 145 kDa tyrosine phosphorylated protein, whose phosphorylation state was EGF dependent in Delta973-EGFR cells, was readily identified. Whether pp145 or some other signaling molecule participates in an alternate signaling pathway coupling Delta973-EGFRs to p21, independent of ShcbulletGrb2bulletSos, remains to be elucidated.

In summary, our results demonstrate the importance of p21 in the mitogenic signal transduction pathway mediated by Delta973-EGFR. Although the molecular coupling of EGFRbulletShcbulletGrb2bulletSos is important as a common mechanism to activate p21, an alternative pathway resides in Delta973-EGFR cells. Our evidence suggests that ErbB2 plays an important role in coupling the truncated EGFR to p21 activation. These results indicate a novel signal transduction mechanism involving ErbB2 in Delta973-EGFR cells.


FOOTNOTES

*
This work was supported in part by National Institutes of Health, NIDDK Grant DK33651, by the Veterans Administration Medical Research Service, by the Sankyo Diabetes Research Fund (to J. M. O.), by National Institutes of Health, NIDDK Grant DK13149 (to G. N. G.), by the grant for intractable disease from the Ministry of Health and Welfare, by the Grant-in-Aid from the Ministry of Education, Science, and Culture, and by the grant for Diabetes Research from Otsuka Pharmaceutical Co. Ltd., Japan (to M. K.), and by a Medical Research Council of Canada Fellowship Award (to W. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medicine (0673), University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0673. Tel.: 619-534-6651; Fax: 619-534-6653.

(^1)
The abbreviations used are: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; SH, Src homology; GST, glutathione S-transferase; GEF, guanine nucleotide exchange factor; BrdU, bromodeoxyuridine; Sos, son of sevenless; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium.


ACKNOWLEDGEMENTS

We thank Dr. Sally A. Prigent for the kind gift of an anti-ErbB3 antibody. We are grateful to Elizabeth Hansen for her assistance in the preparation of this manuscript.


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