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Endocrinology Vol. 145, No. 9 4232-4243
Copyright © 2004 by The Endocrine Society

Differential Activation of Epidermal Growth Factor (EGF) Receptor Downstream Signaling Pathways by Betacellulin and EGF

Tsugumichi Saito, Shuichi Okada, Kihachi Ohshima, Eijiro Yamada, Minoru Sato, Yutaka Uehara, Hiroyuki Shimizu, Jeffrey E. Pessin and Masatomo Mori

Department of Medicine and Molecular Science (T.S., S.O., E.Y., M.S., Y.U., H.S., M.M.), Gunma University Graduate School of Medicine, Gunma 371-8511, Japan; Gunma University Health and Medical Center (K.O.), Gunma 371-8510, Japan; and Department of Pharmacological Sciences (J.E.P.), State University of New York-Stony Brook, Stony Brook, New York 11794

Address all correspondence and requests for reprints to: Shuichi Okada, Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma 371-8511, Japan. E-mail: okadash{at}showa.gunma-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine the downstream signaling pathways regulated by betacellulin (BTC) in comparison with epidermal growth factor (EGF), we used Chinese hamster ovary cells overexpressing the human EGF receptor (ErbB1/EGFR). The overall time-dependent activation of EGFR autophosphorylation was identical in cells treated with 1 nM BTC or 1.5 nM EGF. Analysis of site-specific EGFR phosphorylation demonstrated that the BTC and EGF tyrosine phosphorylation of Y1086 was not significantly different. In contrast, the autophosphorylation of Y1173 was markedly reduced in BTC-stimulated cells, compared with EGF stimulation that directly correlated with a reduced BTC stimulation of Shc tyrosine phosphorylation, Ras, and Raf-1 activation. On the other hand, Y1068 phosphorylation was significantly increased after BTC stimulation, compared with EGF in parallel with a greater extent of Erk phosphorylation. Expression of a dominant interfering MEK kinase 1 (MEKK1) and Y1068F EGFR more efficiently blocked the enhanced Erk activation by BTC, compared with EGF. Interestingly BTC had a greater inhibitory effect on apoptosis, compared with EGF, and expression of Y1068F EGFR abolished this enhanced inhibitory effect. Together, these data indicated that although BTC and EGF share overlapping signaling properties, the ability of BTC to enhance Erk activation occurs independent of Ras. The increased BTC activation results from a greater extent of Y1068 EGFR tyrosine phosphorylation and subsequent increased recruitment of the Grb2-MEKK1 complex to the plasma membrane, compared with EGF stimulation. The increased Erk activation by BTC associated with antiapoptotic function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EPIDERMAL GROWTH factor receptor (EGFR) family consists of four related receptors referred to as ErbB1/EGFR, ErbB2, ErbB3, and ErbB4 that can form both homo- and heterodimers. Each of these receptors consists of a large extracellular domain, a single transmembrane domain, and a cytoplasmic domain that exhibits tyrosine kinase activity, with the notable exception of ErbB3 (1). After ligand binding to the extracellular domain, the receptor undergoes dimerization and intermolecular transphosphorylation of several tyrosine residues within the cytoplasmic domains (2). These tyrosine phosphorylated residues then serve specific binding sites for various Src homology 2 (SH2) domains containing adapter molecules such as Grb2 and Shc (3). Shc can also be tyrosine phosphorylated by the EGFR and thereby engage binding site for the SH2 domain of Grb2 (4). In addition, Grb2 also contains an SH3 domain that directly interacts with the guanylnucleotide exchange factor son of sevenless (Sos). Thus, the interaction of the Grb2-Sos or the Shc-Grb2-Sos complexes with the EGFR results in the targeting of Sos to the plasma membrane, which is the location of Ras (3). Subsequently, activated GTP-bound Ras recruits the Raf kinase to the plasma membrane resulting in Raf activation and phosphorylation of its downstream target MAPK/Erk kinase (MEK) (5, 6). MEK is an unusual dual specific kinase that activates Erk through phosphorylation on both tyrosine and threonine residues (7).

Betacellulin (BTC) is one member of the epidermal growth factor (EGF) family that also includes amphiregulin, TGF{alpha}, and heparin binding EGF (8). BTC is predominantly expressed in the pancreas and generally thought to promote proliferation and differentiation of pancreatic ß-cells (9). BTC typically serves as a ligand for ErbB1/EGFR and ErbB4, although the possibility of specific receptor for BTC itself has also been suggested (10, 11, 12). The ability of BTC to induce increase of islet cell mass is surprising because the Ras activation pathway is a potent stimulator of mitogenesis, prevents apoptosis, and does not appear to have a role in cell fate determination. For example, several types of pancreatic, lung, and colon cancers have been attributed to constitutive activation of the EGFR and stimulation of Ras (13). Nevertheless, under certain circumstances EGFR activation can increase pancreatic ß-cell mass and differentiation, a process that is directly opposed to mitogenesis (14, 15). In addition, BTC has been observed to reduce elevated blood glucose levels that occur in pancreatectomized rats, a property not shared by EGF (16).

Unexpectedly we have found that 1 nM BTC is a more potent activator of Erk in ßHC-9 cell, which is a tissue culture model of pancreatic ß-cell, compared with 1 nM EGF (Fig. 1AGo). However, phosphotyrosine immunoblotting suggested us that besides ErbB1/EGFR, ßHC-9 cell expresses ErbB4, which can bind BTC but not EGF and ErbB4 could be also tyrosine phosphorylated. To examine the potential differences between BTC and EGF signaling pathway, we took an advantage of engineered Chinese hamster ovary cell line overexpressing human ErbB1/EGFR that have negligible levels of the ErbB2, ErbB3, and ErbB4 receptors. Surprisingly we found that BTC activated EGFR pathway results in overlapping but different extents of phosphorylation on specific tyrosine residues of ErbB1/EFGR, compared with EGF. In addition, BTC is a more potent activator of Erk but a relatively weaker stimulatory of Shc, Ras, and Raf-1. We also discovered that BTC uses a greater extent of the Grb2-MEK kinase (MEKK)1 pathway than does EGF due to enhanced tyrosine phosphorylation of Y1068 on EGFR. Furthermore, this enhanced Erk activation through the tyrosine phosphorylation of Y1068 on EGFR was found important for a greater antiapoptotic action of BTC. These data underscore selective signaling differences between BTC and EGF ligand activation of ErbB1/EGFR.



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  		FIG. 1. EGF and BTC stimulation of Erk phosphorylation. ßHC-9 cells were treated with 1 nM EGF or 1 nM BTC for 2, 5, and 15 min and whole-cell extracts were separated by SDS-PAGE subjected to immunoblotting (IB) with a phosphotyrosine (PY20), phosphospecific Erk, and Erk2 antibodies (A). Erk2 immunoblotting representing that equal amount of sample was loaded on each lane. CHOIE cells were treated with 1.5 nM EGF or 1 nM BTC for 2, 5, and 15 min and whole-cell extracts were separated by SDS-PAGE subjected to immunoblotting with a phosphotyrosine (py20) (B), phosphospecific Erk, and Erk2-specific antibodies (B). Phosphorylated Erk2 signals were quantified and presented as a bar graph from three independent experiments. Erk2 immunoblotting representing that equal amount of sample was loaded on each lane. EGF, Open bar; BTC, solid bar. *, P < 0.05; **, P < 0.01.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and reagents
Recombinant human BTC was purchased from R&D Systems (Minneapolis, MN). Recombinant human EGF was purchased from Strathman Biotech GMBH (Hannover, Germany). Polyclonal phospho-Erk antibody was purchased from New England BioLabs (Beverly, MA). Polyclonal Grb2, MEKK1, EGFR, and Erk antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal horseradish peroxidase (HRP)-conjugated py20, monoclonal Sos, and polyclonal Shc antibodies were purchased from Transduction Laboratory (Lexington, KY). Anti-EGFR (pY1068, 1086, and 1173) phosphospecific antibodies were purchased from Biosource International (Camarillo, CA). Monoclonal phospho Raf-1 (ser338) antibody and Ras activation assay kit was purchased from Upstate Biotechnology (Lake Placid, NY).

Cell culture
We also used Chinese hamster ovary cells expressing the human EGF and insulin receptor (CHOIE). These cells were cultured in {alpha}-modified Eagle’s medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin plus 20 mM glutamine, under a humidified condition of 95% air and 5% CO2 at 37 C. In the case of ßHC-9 cells DMEM with high-glucose medium was used instead of {alpha}-modified Eagle’s medium. The cells were incubated in serum-free medium at 37 C for 6 h before growth factor stimulation for various time periods as indicated in the figures and legends. The reactions were stopped by rapid removal of medium, and cells were washed immediately with ice-cold PBS three times. After the removal of PBS, the cells were immediately frozen with liquid nitrogen. The frozen cells were stored at –80 C until use.

Immunoprecipitation
Scraped frozen cells were rocked for 10 min at 4 C with Nonidet P-40 lysis buffer [25 mM HEPES (pH7.4), 10% glycerol, 50 mM sodium fluoride, 10 mM sodium phosphate, 137 mM sodium chloride, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml pepstatin, and 5 µg/ml leupeptin]. Insoluble material was separated from the soluble extract by centrifugation for 10 min at 4 C, and the total protein amount in the supernatant was determined by bicinchoninic assay method. After the addition of 4 µg of a Shc polyclonal antibody (Transduction Laboratory) or 2 µg of a Grb2 polyclonal antibody (Santa Cruz Biotechnology) or 4.5 µg of a MEKK1 polyclonal antibody (Santa Cruz Biotechnology) to the whole-cell lysates, samples (typically 2–3 mg lysates) were incubated for 2 h at 4 C. Then 50 µl protein A-agarose (Santa Cruz Biotechnology) was added, and samples were consequently rocked during the next 1 h at 4 C. After the incubation, samples were extensively washed three times with the Nonidet P-40 lysis buffer. The washed samples were resuspended in sodium dodecyl sulfate (SDS) sample buffer [125 mM Tris-HCl (pH6.8), 20% (vol/vol) glycerol, 4% (wt/vol) SDS, 100 mM dithiothreitol, 0.1% (wt/vol) bromophenol blue], and heated at 100 C for 5 min. After a quick spin, entire supernatants were applied on the SDS polyacrylamide gel.

Immunoblotting
Samples (typically 20 µg whole-cell lysates per each lane) were separated by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The samples were immunoblotted with monoclonal or polyclonal specific antibody as indicated in the figures and legends. The primary monoclonal and polyclonal antibodies (except for PY20-HRP) for immunoblots were detected with HRP-conjugated antirabbit or antimouse IgG (Pierce, Rockford, IL) antibodies. Immunoblotting was performed as detailed by the methodologies provided in the manufacturer’s instructions. Specific signals were visualized by enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK). Each band was scanned and quantified by Molecular Imager FX (Bio-Rad Laboratories, Hercules, CA). Briefly, the same size rectangle was used to surround each band, and the band intensity was determined using the Molecular Imager software. Background intensity was determined in blank region of the gel and was subtracted form each of the specific bands.

Apoptosis assay
Electroplated or nontransfected cells were serum starved for 5 h at 37 C and incubated with 100 nM staurosporine in the absence or presence of either BTC or EGF for an additional 3 h at 37 C. Cell extracts were then prepared by treatment with cell lysis buffer (MBL, Nagoya, Japan) for 10 min on ice, and 200 µg of extract proteins were reacted with 200 µM aspartate-glutamate-valine-aspartate (DEVD)-p-nitroanilide (pNA) substrate for 2 h at 37 C. The released amount of pNA from DEVD was measured by spectrophotometer at 400 nm.

Statistical analysis
All data are expressed as mean ± SD. Data were evaluated for statistical significance by ANOVA or t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BTC is a more effective activator of Erk despite equal extents of total ErbB1/EGFR tyrosine phosphorylation
To search for a unique signal property and find mechanisms to increase islet cell mass of BTC, compared with EGF, we stimulated ßHC-9 cells with either 1 nM EGF or 1 nM BTC for 2, 5, and 15 min. Phosphotyrosine immunoblotting resulted in a single band with increased signal intensity at the approximate molecular weight of the EGFR (Fig. 1AGo). However, because BTC can bind to both the ErbB4 and ErbB1/EGFR, it is not possible to conclude whether the BTC stimulated band represents one and/or both of these receptors. We also noticed that BTC was a more potent activator of Erk phosphorylation, compared with EGF (Fig. 1AGo).

To specifically examine the relative signaling properties between BTC and EGF ligand activation of the ErbB1/EGFR, we took advantage of the CHOIE cell line that overexpresses the human EGFR but does have very low levels of the ErbB2, ErbB3, and ErbB4 receptors. Preliminary dose-response studies demonstrated that stimulation of these cells with 1 nM BTC and 1.5 nM EGF resulted in a similar overall extent of ErbB1/EGFR tyrosine autophosphorylation. This was further confirmed by comparing the initial time course of total ErbB1/EGFR tyrosine phosphorylation by phosphotyrosine immunoblotting (Fig. 1BGo). Under these conditions there was no significant difference in the overall extent in ErbB1/EGFR tyrosine autophosphorylation. Because maximal stimulation of ErbB1/EGFR tyrosine phosphorylation was 20 nM each for BTC and EGF, our working condition was submaximal concentration. Surprisingly, however, BTC was a significantly more effective agonist in the activation/phosphorylation of Erk2, compared with EGF (Fig. 1BGo). Analysis of several immunoblottings demonstrated that, compared with EGF, BTC increased Erk2 phosphorylation 18% at 2 min, 27% at 5 min, and 27% at 15 min. Because Erk2 activation started to diminish after 15 min stimulation in our CHOIE cells as previously described (17), we elected to analyze early time points (2, 5, and 15 min) in this study. It should also be noted that the apparent differences in Erk activation between the ßHC-9 and CHOIE cell lines is likely due to the relative content of ErbB receptor family members and/or downstream signaling components.

EGF and BTC stimulate site-specific differences in ErbB1/EGFR autophosphorylation
Previous studies have hypothesized that the tyrosine autophosphorylation of ErbB1/EGFR may not be identical after EGF vs. BTC stimulation, although an assessment of individual tyrosine acceptor sites was not determined (18). Therefore, to determine the effect of EGF and BTC on individual tyrosine residues, we used ErbB1/EGFR site-specific phosphotyrosine antibodies. As observed in Fig. 1BGo, stimulation with 1.5 nM EGF or 1 nM BTC resulted in the same extent of total ErbB1/EGFR tyrosine phosphorylation. Similarly, the phosphorylation of tyrosine 1086 (Y1086) was essentially identical after EGF and BTC stimulation at all the time points examined (Fig. 2AGo). In contrast, BTC was significantly less effective in stimulating the phosphorylation of Y1173, compared with EGF (Fig. 2BGo). The extent of phosphorylation of Y1173 was greater at 2 and 5 min after EGF stimulation, compared with BTC. However, by 15 min this difference was not statistically significant because the EGF-induced stimulation appeared to decrease by this time point. On the other hand, BTC was a more effective stimulation of Y1068 phosphorylation, compared with EGF (Fig. 2CGo). The difference of Y1068 phosphorylation was readily detected at 2 and 5 min with a 31 and 23% increase over EGF stimulation, although it was not statistically different by 15 min.



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  		FIG. 2. EGF and BTC stimulation of ErbB1/EGFR autophosphorylation. CHOIE cells were treated with 1.5 nM EGF or 1 nM BTC for 2, 5, and 15 min and whole-cell extracts were separated by SDS-PAGE subjected to immunoblotting (IB) with site-specific phosphotyrosine antibodies (py1068, py1086, and py1173) and EGFR antibody as described in Materials and Methods. A, Y1086-specific phosphotyrosine immunoblotting is represented. B, Y1173-specific phosphotyrosine immunoblotting is represented. C, Y1068-specific phosphotyrosine immunoblotting is represented. Each phosphorylated signal (py1086, py1173, and py1068) was quantified and presented as a bar graph from three independent experiments. EGFR immunoblotting representing the equal amount of sample was loaded on each lane. EGF, Open bar; BTC, solid bar. *, P < 0.05; **, P < 0.01.

 
EGF is a more potent activator of Shc tyrosine phosphorylation that BTC
It has been established that the phosphorylation of Y1068 and Y1086 provide direct binding sites for Grb2, whereas Y1173 is a major interaction site for Shc (3, 19, 20). Immunoprecipitation of Shc after EGF stimulation demonstrated the tyrosine phosphorylation of the 66-, 52-, and 46-kDa Shc isoforms (Fig. 3AGo). Consistent with the decreased Y1173 phosphorylation after BTC stimulation, the extent of Shc tyrosine phosphorylation was significantly reduced, compared with EGF-stimulated cells. This decrease in tyrosine phosphorylation was apparent in all three Shc isoforms. Consistent with this reduction in Shc tyrosine phosphorylation, the amount of ErbB1/EGFR that coimmunoprecipitated with Shc was also reduced in BTC- compared with EGF-stimulated cells (Fig. 3BGo).



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  		FIG. 3. EGF and BTC stimulation of ErbB1/EGFR binding to Shc and Grb2. A, CHOIE cells were either left unstimulated or stimulated with 1.5 nM EGF or 1 nM BTC for the indicated times followed by immunoprecipitation with a Shc antibody and immunoblotting (IB) with the py20 phosphotyrosine antibody for the 66-, 52-, and 46-kDa Shc isoforms. Tyrosine-phosphorylated 52-kDa Shc amounts were quantified and presented as a bar graph from three independent experiments. EGF, Open bar; BTC, solid bar. *, P < 0.05, **, P < 0.01. B, After Shc immunoprecipitation the samples were also immunoblotted with the py20 phosphotyrosine antibody for ErbB1/EGFR and a Shc antibody. The amount of phospho-ErbB1/EGFR associated with three isoforms of Shc was quantified and presented as a bar graph from three independent experiments. EGF, Open bar; BTC, solid bar. *, P < 0.05. C, CHOIE cells were either left unstimulated or stimulated with 1.5 nM EGF or 1 nM BTC for the indicated times followed by immunoprecipitation with a Grb2 antibody and immunoblotting with the py20 phosphotyrosine antibody for ErbB1/EGFR or with a Grb2 antibody. The amount of tyrosine-phosphorylated ErbB1/EGFR immunoprecipitated with Grb2 was quantified from three independent experiments and presented as a bar graph. Grb2 immunoblotting representing that immunoprecipitation efficiency was equal in each sample. EGF, Open bar; BTC, solid bar.

 
Grb2 can bind to ErbB1/EGFR by direct interaction of the Grb2 SH2 domain with phosphorylated Y1068 and Y1086 of the ErbB1/EGFR or indirectly through Shc at phosphorylated Y1173. To assess the overall extent of Grb2 interaction with ErbB1/EGFR, Grb2 was immunoprecipitated from EGF- and BTC-stimulated cells followed by phosphotyrosine immunoblotting (Fig. 3CGo). As seen in Fig. 3CGo, the amount of ErbB1/EGFR immunoprecipitated with Grb2 was not significantly different between EGF- and BTC-stimulated cells. Because Shc association was reduced after BTC stimulation, this suggests that Grb2 primarily interacts with ErbB1/EGFR at Y1068 and Y1086 and not indirectly through Shc at Y1173 (see Figs. 7Go, 8AGo, and 10Go). However, there might exist an unidentified Grb2 binding site on EGFR besides Y1068 and Y1086, which contributes to activate Erk.



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  		FIG. 7. EGFR/Grb2 complex mediating through phosphorylated Y1068. CHOIE cells were treated with 1.5 nM EGF or 1 nM BTC for 2, 5, and 15 min, and whole-cell lysates were subjected to immunoprecipitation with a Grb2 antibody and immunoblotting (IB) with phosphospecific py1068 and Grb2 antibodies. The amount of pY1068 immunoprecipitated with Grb2 was quantified from three independent experiments and presented as a bar graph. Grb2 immunoblotting representing that immunoprecipitation efficiency was equal in each sample. EGF, Open bar; BTC, solid bar. *, P < 0.05, **, P < 0.01.

 


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  		FIG. 8. Grb2-MEKK1 pathway contributes to different activation status of Erk in EGF and BTC. A, CHOIE cells were treated with 1.5 nM EGF or 1 nM BTC for 2, 5, and 15 min and whole-cell lysates were subjected to immunoprecipitation with a MEKK1 antibody and immunoblotting (IB) with MEKK1, Grb2, and phosphotyrosine py1068 antibodies. The amount of pY1068 immunoprecipitated with MEKK1 was quantified from three independent experiments and presented as a bar graph. MEKK1 immunoblotting representing that immunoprecipitation efficiency was almost identical in each sample. Interestingly, Grb2 started to associate with MEKK1 during EGF stimulation, but Grb2 started to dissociate from MEKK1 during BTC stimulation. Thus, there is a possibility that the enhanced pY1068-EGFR/Grb2-MEKK1 interaction was related to the change of Grb2-MEKK1 complex amount. EGF, Open bar; BTC, solid bar. *, P < 0.05. B, Dominant-interfering MEKK1 (MEKK1-KM) form was overexpressed before 1.5 nM EGF or 1 nM BTC stimulation. Then cells were treated with 1.5 nM EGF or 1 nM BTC for 2, 5, and 15 min and whole-cell lysates were subjected to immunoblotting with a phosphospecific Erk and Erk2-specific antibody. The extent of Erk2 phosphorylation with dominant-interfering MEKK1 (MEKK1 KM) was corrected with the extent of Erk2 phosphorylation without dominant-interfering MEKK1 (MEKK1 KM) and represented as an inhibitory effect of MEKK1 KM. Those inhibitory effects were quantified and presented as a bar graph from three independent experiments. Erk2 immunoblotting representing that equal amount of sample was loaded on each lane. EGF, Open bar; BTC, solid bar. *, P < 0.05, **, P < 0.01.

 


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  		FIG. 10. The different signaling pathways between EGF and BTC emanating from ErbB1/EGFR that were analyzed in the present study. EGF predominantly phosphorylates Y1173 (pY1173) of ErbB1/EGFR, whereas BTC predominantly phosphorylates Y1068 (pY1068) of ErbB1/EGFR. Subsequently BTC recruits more Grb2/MEKK1 complex toward pY1068 on ErbB1/EGFR and leads to enhanced MEK/Erk activation through pY1068, which is Ras-independent pathway, resulting in increased antiapoptotic function. In the case of BTC, conventional Ras/Raf pathway is less active, compared with EGF, because Y1173 of ErbB1/EGFR is less tyrosine phosphorylated, compared with EGF. Also in the case of BTC signal pathway, there might exist an unidentified EGFR/Grb2 pathway to activate Erk, which is a Ras/Raf-independent signal. This was shown as ? in this figure. N-Ter, N-Terminal; TM, transmembrane.

 
EGF and BTC have no significant effect on the assembly state of the Grb2-Sos complex
Erk1/2 are downstream targets of the Ras/Raf/MEK pathway and EGF activation of Ras occurs through the recruitment of the Grb2-Sos complex to the plasma membrane by the ErbB1/EGFR (21, 22, 23). In addition, insulin stimulation results in the desensitization of Ras activation through the uncoupling of the Grb2-Sos complex, whereas the Grb2-Sos complex remains persistent after ErbB1/EGFR activation (24). Consistent with these previous studies, insulin stimulation resulted in the dissociation of the Grb2-Sos complex (Fig. 4AGo). In contrast, both EGF and BTC stimulation had no effect on the assembly state of the Grb2-Sos complex (Fig. 4AGo). Furthermore, there was no significant time-dependent dissociation of the Grb2-Sos complex with either EGF or BTC stimulation (Fig. 4BGo).



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  		FIG. 4. Effect of insulin, EGF, and BTC on the Grb2-Sos complex. A, CHOIE cells were either left unstimulated or stimulated with 100 nM insulin, 1.5 nM EGF, or 1 nM BTC for 5 min followed by immunoprecipitation with a Grb2 antibody and immunoblotting (IB) with a Sos or Grb2 antibody as described in Materials and Methods. B, CHOIE cells were either left unstimulated or stimulated with 1.5 nM EGF or 1 nM BTC for the indicated times followed by immunoprecipitation with a Grb2 antibody and immunoblotting with a Grb2 or Sos antibody. The amount of Sos immunoprecipitated with Grb2 was quantified from three independent experiments and presented as a bar graph. Grb2 immunoblotting representing that immunoprecipitation efficiency was equal in each sample. EGF, Open bar; BTC, solid bar.

 
BTC is a weaker stimulator of both Ras activation and Raf kinase activity
To date, there does not appear to be any specific difference in the signal role played by the 52- and 46-kDa Shc isoforms. Both species, when tyrosine phosphorylated, associated with Grb2 and thereby presumably link Sos to the activation of Ras (24). According to our results shown in Fig. 3AGo, BTC predominantly reduced both 52- and 46-kDa Shc tyrosine phosphorylation, compared with EGF. Therefore, we reasoned to check Ras/Raf activation status by BTC or EGF.

Because Ras is a primary downstream target of the Sos guanlynucleotide exchange activity, we assessed Ras activation by precipitation of GTP-bound Ras using the effector binding domain (RBD) of Raf (25). Precipitation of cell extracts with equal amounts of recombinant Raf-RBD followed by Ras immunoblotting demonstrated a rapid activation of Ras from the GDP to GTP-bound state (Fig. 5AGo). The extent of Ras activation was similar between EGF and BTC stimulation at 2 min. Although Ras activation is transient and begins to decrease after 2 min, the rate of inactivation was greater in the BTC- compared with EGF-stimulated cells. In parallel with the changes in Ras activation, the serine phosphorylation of Raf was significantly reduced in BTC-stimulated cells in comparison with EGF (Fig. 5BGo). Thus, these data demonstrate that the relative reduced phosphorylation and ErbB1/EGFR association of Shc after BTC stimulation directly correlated with a reduction in Ras-GTP loading and Raf activation.



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  		FIG. 5. EGF and BTC stimulation of Ras activation and Raf phosphorylation. A, CHOIE cells were either left unstimulated or stimulated with 1.5 nM EGF or 1 nM BTC for the indicated times followed by precipitation with a GST-RBD fusion protein and immunoblotting (IB) with a Ras antibody. These data were quantified and presented as a bar graph from three independent experiments. EGF, Open bar; BTC, solid bar. *, P < 0.05. B, Whole-cell lysates were used for this experiment. Serine338 phosphorylation of Raf-1 was detected with a phosphospecific Raf-1 antibody, and Raf-1 protein amount was estimated by immunoblotting with a Raf-1-specific antibody. Phosphorylated Raf-1 amounts were quantified and presented as a bar graph from three independent experiments. Raf immunoblotting representing that equal amount of sample was loaded on each lane. EGF, Open bar; BTC, solid bar. *, P < 0.05.

 
MEK dependency of EGF and BTC on Erk activation
The immediate upstream activator of Erk1/2 is the MAPK kinases, MEK1 and MEK2 (7, 26, 27); however, several recent reports suggest that a MEK-independent pathway can also activate Erk (28, 29). Because BTC resulted in a weaker Ras and Raf activation but greater Erk activation, compared with EGF, we next assessed the presence of a MEK-independent activation of Erk (Fig. 6Go). As observed in Fig. 6Go, BTC stimulation for 5 min resulted in a greater extent of Erk1/2 phosphorylation than EGF stimulation. Incubation with the specific MEK1/2 inhibitor U0126 completely blocked Erk1/2 activation by both BTC and EGF. These data indicate that MEK1/2 is the primary mechanism accounting for Erk2 activation after BTC and EGF stimulation.



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  		FIG. 6. EGF and BTC stimulation of Erk1/2 phosphorylation is MEK dependent. CHOIE cells were either left untreated or incubated with the MEK-specific inhibitor U0126 (100 nM) for 30 min. The cells were then either unstimulated or stimulated with 1.5 nM EGF or 1 nM BTC for 5 min followed by immunoblotting (IB) with the phosphospecific Erk antibody and Erk-specific antibody. Erk2 immunoblotting representing that equal amount of sample was loaded on each lane.

 
Phosphorylation of Y1068 induces the interaction of ErbB1/EGFR with Grb2
As previously described, Y1068 on the ErbB1/EFGR is one target site for Grb2 binding. In addition, BTC increased the extent of Y1068 phosphorylation, compared with EGF (Fig. 2Go). To assess the site-specific association of Grb2 with the ErbB1/EGF receptor, we next immunoprecipitated Grb2 followed by immunoblotting with the ErbB1/EGFR Y1068 phosphospecific antibody (Fig. 7Go). Consistent with increased Y1068 phosphorylation, BTC stimulation resulted in an increase in the amount of phosphoY1068 coimmunoprecipitated with Grb2, compared with EGF-stimulated cells (Fig. 7Go).

Grb2-MEKK1 pathway is responsible for the enhanced BTC activation of Erk
In addition to the Grb2-Sos complex, Grb2 can also interact with MEKK1 and form a ternary complex with the ErbB1/EGFR (30). In the basal state, Grb2 was coimmunoprecipitated with MEKK1 demonstrating the presence of a stable Grb2-MEKK1 complex (Fig. 8AGo). However, both EGF and BTC stimulation resulted in a greater association of MEKK1 with Grb2. Importantly, BTC stimulation resulted in a greater rate of Grb2-MEKK1 association with the ErbB1/EGF receptor, compared with EGF. This was more dramatic at the earlier time point (2 min) but was not statistically significant at the latter time points (5 and 15 min). Consistent with this result, expression of a dominant interfering MEKK1 mutant had no significant effect on the phosphorylation of Erk2 in response to EGF (Fig. 8BGo). In contrast, the dominant interfering MEKK1 mutant partially blocked BTC stimulation of Erk phosphorylation preferentially at the early time points (2 and 5 min). Surprisingly, however, BTC stimulation resulted in enhanced Erk2 phosphorylation at 15 min. In any case, these data indicated that in addition to the Ras/Raf/MEK pathway, BTC can also use a Grb2-MEKK1 pathway to stimulate Erk activation at particularly early time point.

BTC is a more effective inhibitor of apoptosis
To establish the physiological significance of our findings, we compared antiapoptotic action between BTC and EGF stimulation. Under various dosages tested, BTC consistently represented a greater inhibitory effect on apoptosis, compared with EGF (Fig. 9AGo). To explore the mechanism, we expressed Y1068F EGFR mutant and found that expression of Y1068F EGFR significantly reduced this inhibitory effect (Fig. 9BGo). Furthermore, expression of Y1068F EGFR diminished the enhanced activation of Erk2 by BTC (Fig. 9CGo). These data indicated that tyrosine phosphorylation of Y1068 on EGFR is important for the enhanced antiapoptotic action of BTC that occurs through the activation of the EGFR/Erk pathway.



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  		FIG. 9. BTC has a greater antiapoptotic effect through enhanced tyrosine phosphorylation of Y1068 in EGFR, compared with BTC. A, CHOIE cells were treated with 100 nM staurosporine and either EGF or BTC under various concentrations (conc; 0.1, 1, or 10 nM) for 3 h. Caspase-3 activity was determined by measuring cleaved pNA amount from DEVD as described in Materials and Methods. Each value is expressed as a fold of caspase-3 activity, compared with untreated cells with staurosporine. Caspase-3 activity is quantitated as a line graph from three independent experiments. EGF, Open square; BTC, solid square. *, P < 0.05. B, CHOIE cells expressing Y1068F EGFR were treated with 100 nM staurosporine and either 1 nM EGF or BTC for 3 h. Quantitative values are expressed as a fold of caspase-3 activity, compared with untreated cells with staurosporine. Each bar graph is from three independent experiments. EGF, Open bar; BTC, solid bar. C, Y1068F EGFR mutant was overexpressed before 1.5 nM EGF or 1 nM BTC stimulation. Then cells were treated with 1.5 nM EGF or 1 nM BTC for 2, 5, and 15 min and whole-cell lysates were subjected to immunoblotting (IB) with a phosphospecific Erk and Erk2-specific antibody. The inhibitory effect of Y1068F EGFR on Erk phosphorylation is quantitated as a bar graph from three independent experiments. Erk2 immunoblotting representing that equal amount of sample was loaded on each lane. EGF, Open bar; BTC, solid bar. **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The EGF regulation of tyrosine autophosphorylation and subsequent downstream signaling of the ErbB1/EGFR has been extensively investigated in numerous cell types (31, 32, 33). More recently, three other members of this receptor tyrosine kinase family have been identified that can form various homo- and heterodimers. These interactions are further complicated because multiple receptor ligands have also been shown to display relative selectivity for the different receptor combinations (11, 34). Thus, subsequent downstream signaling events appear to be dependent on both the relative expressions of the different receptor isoforms and the specific activating ligands (35, 36). In this regard, BTC is a relatively new member of the EGF ligand family that was originally isolated from the conditioned medium of a mouse pancreatic ß-cell tumor (insulinoma) and can promote the proliferation of epithelial and vascular smooth muscle cells (8). However, BTC administration was found to reverse elevated blood glucose level by inducing ß-cell regeneration in pancreatectomized rats (16). This latter property is inconsistent with a simple mitogenic function after ErbB1/EGFR activation.

BTC displays higher affinity and specificity for ErbB1/EGFR and ErbB4 but can also bind to ErbB1/ErbB2, ErbB1/ErbB3, ErbB1/ErbB4, ErbB2/ErbB3, and ErbB2/ErbB4 heterodimers (37). In our CHOIE cell line, neither BTC nor EGF was able to induce any detectable tyrosine phosphorylation of ErbB4 consistent with these engineered cells primarily expressing ErbB1/EGFR. In addition, we found that BTC and EGF display identical patterns of Erk2 activation in ßHC-9 cells (Fig. 1AGo), which do not express appreciable amounts of ErbB2 or ErbB3. Because it has been suggested that BTC and EGF may have overlapping but slightly different signaling properties, we reasoned that the CHOIE cells would provide a suitable model system to examine the relative effects of these ligands on the same receptor.

Unlike the other receptor kinases, receptor phosphorylation site or sites in the EGFR are not required for full enzymatic activity. However, EGFR autophosphorylation provides many binding and targeting sites for various molecules such as Grb2 and Shc and regulates numerous signaling pathways (19, 20). Five autophosphorylation sites have so far been identified in the EGFR, all of which are clustered at the extreme C-terminal 194 amino acids. Among these sites, Tyr1068, 1148, and 1173 are major sites, whereas Tyr992 and 1086 are minor sites. It was reported that in the human glioma cell line, dextran-conjugated EGF showed weaker phosphorylation of Y992 and Y1173 and led to lower PLC{gamma} phosphorylation (38). Thus, it is known that differential phosphorylation of ErbB1/EGFR affects downstream signaling.

In our study, indeed, EGF stimulation resulted in the phosphorylation of the ErbB1/EGFR resides Y1068, Y1086, and Y1173. Although the extent of Y1086 phosphorylation producing a direct Grb2 binding site was similar, BTC induced an enhanced tyrosine phosphorylation of Y1068 concomitant with a reduced phosphorylation of Y1173. Because Y1173 is the major Shc selective binding site (3, 39), this was associated with a decrease in ErbB1/EGFR-Shc association and Shc tyrosine phosphorylation. In parallel, BTC stimulation resulted in a greater extent of Grb2 association with the phosphorylated Y1068 residue because the phosphorylated Y1068 serves as a direct binding site for Grb2 (3, 40). Thus, there is a possibility that BTC-enhanced Y1068 phosphorylation recruits more Grb2 ternary complexes such as Grb2-MEKK1 and Grb2-Sos to EGFR, compared with EGF (see Fig. 10Go, A and B). According to our results, BTC presumably recruits Grb2-MEKK1 complex much more than Grb2-Sos to the phosphorylated Y1068 of EGFR.

ErbB1/EGFR activation of Ras can occur through either the ErbB1/EGFR-Shc-Grb2-Sos-Ras or ErbB1/EGFR-Grb2-Sos-Ras pathways. However, our data demonstrated that BTC was a weaker Ras activator, compared with EGF. The reduction in Ras activation was further confirmed by the reduced extent of Raf activation, an immediate downstream target of activated Ras. This relative decrease in Ras/Raf activation is consistent with the assembly of the Shc-Grb2-Sos complex to the ErbB1/EGFR as the major pathway responsible for Ras activation. This is consistent with the relative decrease in Y1173 phosphorylation after BTC stimulation. Although it remains possible that the direct interaction of Grb2-Sos with ErbB1/EGFR also contributes to Ras activation, this pathway would not account for the reduction in Ras activation of BTC because there was increased phosphorylation of Y1068 and no significant difference in Y1086 phosphorylation.

Although these data are consistent with the reduced tyrosine phosphorylation of Y1173, we were surprised to find that BTC was a more potent activator of Erk, compared with EGF. It has been well established that Raf activation functions to stimulate MEK, which in turn phosphorylates and activates Erk (7). However, it has also been postulated that Erk activation can occur independent of MEK activation (28, 29). Nevertheless, both BTC and EGF stimulation of Erk phosphorylation was completely blocked by a specific MEK inhibitor.

In any case, how do we account for the discrepancy between reduced Ras/Raf activation but enhanced Erk phosphorylation after BTC stimulation? Because both BTC and EGF stimulation of Erk require MEK function, we hypothesized that BTC can activate another MEK kinase that functions upstream of MEK. Previous studies have suggested that EGF can activate Erk through ErbB1/EGFR/Grb2/MEKK1/MEK1 pathway (30, 41). Consistent with this latter pathway, BTC enhanced Y1068 phosphorylation and increased the extent of Grb2/MEKK1 recruitment to the ErbB1/EGFR. Moreover, expression of a dominant-interfering MEKK1 mutant had a greater effect on BTC-stimulated Erk activation than it did on EGF stimulation. In addition, as presented in Fig. 9CGo, expression of Y1068F EGFR mutant significantly diminished the enhanced activation of Erk2 by BTC. Thus, the enhanced BTC stimulated phosphorylation of Y1068 provides an additional signal that recruits a greater extent of the Grb2/MEKK1 complex and thereby enhanced MEK and Erk activation.

Cellular apoptosis is induced by UV irradiation and reduced after EGF stimulation through the activation of the Erk pathway (42). The antiapoptotic action of EGF occurs from an inhibition of caspase-9 activation due to an Erk1/2 phosphorylate at Thr 125 (43). To estimate the effects of BTC and EGF on apoptosis, we assessed caspase-3 activation by determining the production of pNA from the DEVD substrate. As expected, both BTC and EGF treatment resulted in a dramatic reduction of caspase-3 activation. However, BTC was significantly more effective than EGF in reducing caspase-3 activation, suggesting that BTC has greater antiapoptotic capacity than EGF. Furthermore, phosphorylation of Y1068 in the EGFR is critical for the enhanced antiapoptotic effect of BTC because expression of the Y1068F EGFR mutant was significantly less effective. Because expression of Y1068F EGFR abolished the BTC-enhanced Erk2 activation, these data directly demonstrate that the enhanced antiapoptotic actions of BTC are due to increased EGFR phosphorylation of Y1068.

In conclusion, these data support a model in which ligand-dependent differences in receptor autophosphorylation patterns result in the assembly and engagement of overlapping but distinct signaling pathways. This appears to be at least one mechanism that can account for the ligand-dependent differences in signal strength and duration. Further studies are now needed to determine the signaling properties and relative patterns of receptor autophosphorylation for the other members and combinations of the ErbB family.


   Acknowledgments
 
We thank for Dr. Shigeo Ohno (Department of Molecular Biology, Yokohama City University School of Medicine) for providing MEKK1-negative dominant construct and Dr. Yoshiaki Kido (Department of Clinical Molecular Medicine, Division of Diabetes, Digestive and Kidney Diseases, Kobe University School of Medicine) for providing Y1068F EGFR construct.


   Footnotes
 
Abbreviations: BTC, Betacellulin; CHOIE, Chinese hamster ovary cells expressing the human EGF and insulin receptor; DEVD-pNA, aspartate-glutamate-valine-aspartate p-nitroanalide; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HRP, horseradish peroxidase; MEK, MAP kinase/Erk kinase; MEKK, MEK kinase; RBD, Ras using the effector binding domain; SDS, sodium dodecyl sulfate; SH2, Src homology 2; Sos, son of sevenless.

Received March 31, 2004.

Accepted for publication June 1, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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