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Endocrinology, doi:10.1210/en.2004-0713
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Endocrinology Vol. 145, No. 11 4976-4984
Copyright © 2004 by The Endocrine Society

Evidence that Basal Activity, But Not Transactivation, of the Epidermal Growth Factor Receptor Tyrosine Kinase Is Required for Insulin-like Growth Factor I-Induced Activation of Extracellular Signal-Regulated Kinase in Oral Carcinoma Cells

Ami Kuribayashi, Keiko Kataoka, Tohru Kurabayashi and Masahiko Miura

Molecular Diagnosis and Therapeutics (A.K., K.K., M.M.) and Oral and Maxillofacial Radiology (A.K., T.K.), Department of Oral Restitution, Graduate School, Tokyo Medical and Dental University, Tokyo 113-8549, Japan

Address all correspondence and requests for reprints to: Dr. Masahiko Miura, Molecular Diagnosis and Therapeutics, Department of Oral Restitution, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail: masa.mdth{at}tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I receptor (IGF-IR) is involved in numerous biological functions via its major downstream signaling molecules, extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase/Akt. The IGF-I-induced activation of ERK, but not that of Akt, is reportedly mediated by the transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase (TK). The mechanism for the EGFR-TK-dependent activation, however, still remains largely unknown. We found that an oral carcinoma cell line overexpressing EGFR, Ca9–22, exhibited IGF-I-induced activation of both Akt and ERK, but that only the latter was significantly decreased by a specific inhibitor of EGFR-TK, tyrphostin AG1478. In this report we provide evidence for the existence in this cell line of a novel mechanism by which IGF-I induces ERK activation in a manner that is dependent on the basal level of EGFR-TK activity, but is independent of receptor transactivation. In addition, we show that c-Raf kinase is likely to be a key regulator of this mechanism. The elucidation of such a unique mechanism involving cross-talk between EGFR and heterologous receptors may shed additional light on the clinical use of EGFR-TK inhibitors in antitumor therapies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I RECEPTOR (IGF-IR), formed by a heterotetramer ({alpha}2ß2), is a type II receptor tyrosine kinase that is highly homologous to the insulin receptor (1). There are many reports that activation of IGF-IR by its ligands, IGF-I and IGF-II, plays a pivotal role in varied biological processes, including cell proliferation, the establishment and maintenance of transformation, antiapoptotic effects, differentiation, and the control of the life span (2, 3, 4, 5, 6). Its downstream signal transduction cascades are also well investigated, and phosphatidylinositol 3'-kinase (PI3-K) and MAPK are known to be major control elements. The former is activated mainly by insulin receptor substrate (IRS) proteins, which directly bind the NPXY motif in the juxtamembrane region of IGF-IR. The activated PI3-K, in turn, leads to the activation of Akt, which is a known survival factor (7). In contrast, the MAPK pathway, particularly through extracellular signal-regulated kinase (ERK), is thought to be activated mainly by Shc, which also binds the NPXY motif as described above, and by the 14-3-3 proteins that bind to the C terminus of IGF-IR (7, 8). Although mutations at either of the binding sites in IGF-IR do not influence ERK activation, mutations at both sites together significantly reduced its activation (9, 10). The ERK pathway is generally considered to promote survival and provide growth-stimulating signals, albeit with exceptions depending on cellular context (11). We have reported that the pathway is a strong contributor to radioresistance in mouse embryo fibroblast (MEF) cell lines (10).

Epidermal growth factor (EGF) receptor (EGFR) is a type I receptor tyrosine kinase that mediates many biological processes, including cell proliferation and protection from apoptosis, in response to ligands such as EGF, TGF-{alpha}, heparin-binding EGF (HB-EGF), or amphiregulin (12, 13). Ligand stimulation results in receptor dimerization, intrinsic tyrosine kinase activation, and autophosphorylation of the receptor at tyrosine residues (14). A variety of docking proteins that contain Src homology-2 and phosphotyrosine binding domains bind activated EGFR and, in turn, activate its downstream pathways, including PI3-K and MAPK (14). Because the majority of tumor cells overexpress EGFR, this tyrosine kinase (TK) has been considered a promising molecular target for tumor therapy; indeed, some drugs designed to target the EGFR-TK have been applied in clinical practice (13).

The traditional view of growth factor receptors is that a specific ligand directly binds its cognate receptor. Recently, however, many reports have demonstrated that the activation of heterologous receptors can indirectly induce transactivation of EGFR, subsequently leading to MAPK activation. For example, some G protein-coupled receptor ligands activate Src and matrix metalloproteinases (MMPs), which results in shedding of the extracellular domains of ligands such as HB-EGF, which, in turn, bind EGFR and stimulate its tyrosine kinase (15, 16, 17). This mechanism has also been reported for IGF-I-induced ERK activation in COS-7 cells; IGF-I stimulates MMPs, and HB-EGF is released, which then activates EGFR. Interestingly, only ERK activation, not PI3-K/Akt activation, was strongly dependent on EGFR-TK activity upon IGF-I stimulation (18). Similar results were reported using mammary epithelial cells, showing that IGF-I-induced ERK activation was dependent on EGFR transactivation, but was not mediated by HB-EGF shedding (19). Very recently, Ahmad et al. (20) reported that IGF-IR forms a complex with EGFR and that IGF-I transphosphorylates EGFR, presumably through this interaction, which then leads to ERK activation. Because only these few reports address this phenomenon of EGFR transactivation after IGF-I stimulation, the mechanism of its dependence on EGFR-TK remains largely unknown. Considering that IGF-IR possesses its own means of activating ERK pathways, we examined whether EGFR transactivation is indeed required, and whether it is the true source of ERK activation.

In this study we used an oral carcinoma cell line that overexpresses EGFR, Ca9–22 (21, 22), because we reasoned that it would facilitate the detection of EGFR autophosphorylation in response to IGF-I. Using this cell line, we identified a novel mechanism by which IGF-I-induced ERK activation depends on the presence of basal EGFR-TK activity, but not on EGFR-TK transactivation beyond the basal activity level. Furthermore, we show that c-Raf kinase is likely to be a key regulator for this mechanism. We understand that extrapolations of the results from only a single cell line should be examined with caution, but we believe that our findings may have clinical implications for antitumor therapies using EGFR-TK inhibitors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Antibodies against IGF-IR ß-subunits, c-Raf-1, ERK2, mouse IgG conjugated with horseradish peroxidase (HRP), rabbit IgG-HRP, and Protein A/G Plus-agarose were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA). Antibodies against phosphotyrosine (PY20), SHC, and GRB2 were purchased from Transduction Laboratories (Lexington, KY). Antibodies against protein kinase B/AKT pSer473 and EGFR pTyr1068 were obtained from BioSource International (Camarillo, CA). The anti-ACTIVE MAPK (ERK2 pThr183/ pTyr185) antibody was purchased from Promega Corp. (Madison, WI), and the anti-ß-actin antibody was obtained from Chemicon International (Temecula, CA). The enhanced chemiluminescence Western blotting analysis system and [{gamma}-32P]ATP were purchased from Amersham Biosciences (Arlington Heights, IL). Recombinant human IGF-I and EGF were purchased from Invitrogen Life Technologies (Gaithersburg, MD). Tyrphostin AG1478 was purchased from Alexis Biochemicals (Lausen, Switzerland). [Glu52]Diphtheria toxin (CRM197), 1,10-phenanthroline, and PD98059 were purchased from Sigma-Aldrich Corp. (St. Louis, MO). The EZ-Detect Ras Activation Kit was purchased from Pierce Chemical Co. (Rockford, IL). The Raf-1 Kinase Cascade Assay Kit was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).

Cell lines and culture conditions
Ca9–22 cells were obtained from Health Science Research Resources Bank (Sendai, Japan) and were maintained in RPMI 1640 containing 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin, supplemented with 10% fetal bovine serum. MEFs with a targeted disruption of the IGF-IR gene (R–; gift from Dr. Renato Baserga, Thomas Jefferson University, Philadelphia, PA) (2) and MEFs overexpressing human IGF-IR [wild type (WT)] (10) were maintained in Eagle’s MEM containing 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin, supplemented with 10% fetal bovine serum. All cell lines were maintained at 37 C in a humidified atmosphere containing 5% CO2.

Growth factor stimulation and inhibitor treatments
Cells grown on plastic dishes in growth medium were washed with PBS and incubated in serum-free medium containing 1 mg/ml BSA for 6 h. Cells were then stimulated with the indicated doses of IGF-I or EGF for the indicated times. Cells were treated with either the inhibitor tyrphostin AG1478 (EGFR tyrosine kinase inhibitor) or PD98059 [MAPK kinase (MEK) inhibitor] 30 min before stimulation with growth factors. For CRM197 (catalytically inactive Glu52 mutant of diphtheria toxin) or phenanthroline (pan-MMPs inhibitor) treatment, cells were treated 1 h before growth factor stimulation.

Western blotting and immunoprecipitation
For Western blotting, cells were first digested in lysis buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM sodium orthovanadate (NaVO4), 100 mM sodium fluoride (NaF), 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1 µg/ml aprotinin]. Next, equal amounts of the cell lysate were separated using SDS-PAGE, and the proteins were transferred to nitrocellulose membranes in Tris-glycine buffer containing 20% methanol. The membrane was blocked in 5% nonfat milk in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 (TBST). Filters were probed with primary antibodies against target proteins at 4 C overnight. Filters were washed three times in TBST, then incubated with secondary antibodies conjugated to HRP in TBST at 37 C for 1 h and washed three more times in TBST. Proteins were visualized using the enhanced chemiluminescence system. For immunoprecipitation, cells were first lysed in RIPA buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 µM NaVO4, 1 mM NaF, 1 mM PMSF, and 10 µg/ml aprotinin], and equal amounts of the lysate were immunoprecipitated with 10-µl mixtures of the appropriate primary antibody bound to Protein A/G Plus-agarose (1:1) at 4 C for 1 h. The immune complexes were washed three times with ice-cold RIPA buffer and eluted by boiling the beads in Laemmli sample buffer for 5 min. Samples were subjected to SDS-PAGE, then immunoblotted as described above. All Western blotting experiments were repeated at least three times, and a representative blot is shown. NIH Image was used to quantitate the intensity of the bands.

Ras activity assay
The activation of Ras (Ras-GTP) was determined using the EZ-Detect Ras Activation Kit (Pierce Chemical Co.) according to the manufacturer’s instructions. Treated cells were first lysed in lysis/binding/wash buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 1 mM dithiothreitol (DTT), 5% glycerol, 1 µg/ml aprotinin, and 1 mM PMSF]. The cell lysates were incubated with a glutathione-S-transferase (GST) fusion protein containing the Ras binding domain (RBD) of Raf-1 [2.7 mg/ml GST-Raf1-Ras-RBD, 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.5% Triton X-100, 5 mM MgCl2, 1 mM DTT, and 10% glycerol] in spin columns at 4 C for 1 h, then captured on Swell Gel Immobilized Glutathione Discs (Pierce Chemical Co.) containing 50 µl resin. The resin was washed three times with lysis/binding/wash buffer, and 52.5 µl 2x SDS sample buffer [125 mM Tris-HCl (pH 6.8), 2% glycerol, 4% SDS, 0.05% bromophenol blue, and 5% 2-mercaptoethanol] were added. The spin columns were centrifuged at 7200 x g for 2 min, and the collected solution was boiled for 5 min before being subjected to SDS-PAGE. Ras was detected by Western blotting as described using an anti-Ras antibody (Pierce Chemical Co.) as the primary antibody.

c-Raf kinase assay
The activity of c-Raf kinase was determined by a linked MEK/ERK kinase assay using myelin basic protein (MBP) as a substrate in the Raf-1 Kinase Cascade Assay Kit (Upstate Biotechnology, Inc.). The treated cells were lysed in lysis buffer A [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% 2-mercaptoethanol, 1% Triton X, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 0.1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin]. The prepared cell lysates were then immunoprecipitated with 50% protein G-agarose, to which the anti-c-Raf-1 antibody had previously been allowed to bind by incubation at 4 C for 2 h. The pellets were washed twice in ice-cold lysis buffer A and once in ice-cold 20 mM 4-morpholinepropanesulfonic acid (pH 7.2), 25 mM ß-glycerophosphate, 5 mM EGTA, 1 mM NaVO4, and 1 mM DTT (ADBI). The kinase reaction was initiated by incubating the pellets at 30 C for 30 min in a mixture of 20 µl ADBI, 10 µl magnesium/ATP cocktail (500 µM ATP and 75 mM magnesium chloride in ADBI), 1.6 µl MEK1 solution [12.5 µg MEK1, 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.03% Brij-35, 0.1% 2-mercaptoethanol, 270 mM sucrose, 150 mM NaCl, 0.2 mM PMSF, and 1 mM benzamidine], and 4 µl MAPK2/ERK2 solution [50 µg enzyme, 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.03% Brij-35, 0.1% 2-mercaptoethanol, 0.1 mM PMSF, 1 mM benzamidine, and 50% glycerol]. After centrifugation at 20,000 x g for 15 sec, the MBP phosphorylation reaction was initiated by adding 4 µl of the supernatant to a mixture of 10 µl ADBI, 10 µl MBP substrate [50 µg MBP, 10 mM 4-morpholinepropanesulfonic acid (pH 7.0), and 0.05% sodium azide], and 1 µCi [{gamma}-32P]ATP for each assay reaction. The reaction was allowed to proceed for 10 min at 30 C and then was terminated by spotting onto p81 paper. The paper was washed three times in 0.75% phosphoric acid and once in acetone. The amount of incorporated radioactivity was quantitated by a scintillation counter.

Statistical analysis
Statistical comparisons of mean values were performed by one-way ANOVA, followed by calculation of Fisher’s protected least significant difference. Differences with P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of IGF-I- and EGF-induced activation of Akt and ERK in WT and Ca9–22 cells
We first characterized the activation kinetics of Akt and ERK upon IGF-I and EGF stimulation in WT and Ca9–22 cells. The WT cells used were from a cell line (~106 receptors/cell) that overexpresses human IGF-IR due to the transfection of human IGF-IR cDNA into R–cells (10). R– cells are MEFs derived from the littermates of mice with a targeted disruption of the IGF-IR gene (2). Ca9–22 cells are an oral squamous cell carcinoma cell line that overexpresses EGFR (~106 receptors/cell); the capability of EGFR biosynthesis has been well established in this cell line (21, 22). Levels of IGF-IR and EGFR were confirmed by Western blotting (Fig. 1AGo). To assess the ability of each receptor to stimulate their downstream signaling pathways, serum-deprived cells were stimulated with 50 ng/ml IGF-I or EGF, and the activated forms of Akt and ERK were detected by Western blotting at the indicated times after stimulation using antibodies that specifically recognize phosphorylated forms. The time-course experiments (Fig. 1BGo) show that Akt activation occurs rapidly after IGF-I stimulation (~1 min). In contrast, ERK activation becomes apparent somewhat later (~5 min) than Akt activation in both cell lines. Even after 30 min of stimulation, both kinases still exhibited activation, albeit with some variation in intensity. Similar timing of the appearance of activation was observed when cells were stimulated with EGF. At a concentration of 50 ng/ml for both ligands, the intensity of ERK activation by EGF stimulation tended to be higher than that by IGF-I stimulation, and conversely, the intensity of Akt activation was higher by IGF-I stimulation than by EGF stimulation.



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  		FIG. 1. Characterization of the activation of the MAPK and PI3-K pathways after IGF-I or EGF stimulation in Ca9–22 and WT cells. A, Expression levels of EGFR and IGF-IR in both cell lines. Cell lysates were subjected to SDS-PAGE, followed by EGFR and IGF-IR visualization as described in Materials and Methods. ß-Actin was used as a loading control. B, Time course of activation of Akt and ERK after IGF-I or EGF stimulation. Serum-deprived cells were stimulated with 50 ng/ml IGF-I or EGF for the indicated time periods, then the cells were lysed and processed for Western blotting as described in Materials and Methods.

 
A specific EGFR-TK inhibitor, tyrphostin AG1478, abrogates IGF-I-induced ERK activation in Ca9–22, but not in WT cells
Roudabush et al. (18) reported that IGF-I induces ERK activation through the transactivation of EGFR in COS-7 cells. In fact, the specific EGFR-TK inhibitor, tyrphostin AG1478, abrogated IGF-I-induced ERK activation, but not Akt activation, in this study. Using the WT and Ca9–22 cell lines, we examined whether such a phenomenon was universally observed. In WT cells, as in COS-7 cells, Akt activation was not affected by AG1478 treatment; however, contrary to previous reports, ERK activation was also not much affected upon IGF-I stimulation (Fig. 2AGo). Upon EGF stimulation in the presence of AG1478, the activity of both kinases was significantly reduced in a dose-dependent manner, confirming that the AG1478 used was fully functional. The findings for COS-7 cells, therefore, are not necessarily representative of all cell types; the IGF-IR in WT cells, indeed, seems to possess the ability to activate ERK independently of EGFR-TK. In contrast, Ca9–22 cells, which overexpress EGFR, exhibited a very similar phenotype as COS-7 cells; IGF-I-induced ERK activation was almost eliminated by AG1478 treatment at even lower concentrations than required for abrogation of EGF-induced ERK activation. Akt activation was not affected (Fig. 2Go, A and B). A similar effect was observed when using ZD1839 (data not shown), another EGFR-TK inhibitor. We also examined the effect of AG1478 on the autophosphorylation of EGFR and IGF-IR in Ca9–22 cells after EGF or IGF-I stimulation, respectively. As expected, only EGFR autophosphorylation was abrogated; IGF-IR autophosphorylation was not affected (Fig. 2CGo).



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  		FIG. 2. Effect of tyrphostin AG1478 on IGF-I- or EGF-induced activation of Akt and ERK in WT and Ca9–22 cells. A, Dose dependence of AG1478 inhibition of Akt or ERK activation after IGF-I or EGF stimulation. Serum-deprived cells were stimulated with 50 ng/ml IGF-I or EGF for 10 min after a 30-min incubation in the presence of graded concentrations of AG1478 (0.1, 1, and 10 µM). Cells were processed for Western blotting as described in Materials and Methods. B, Quantitative analysis of the activation of Akt and ERK after IGF-I or EGF stimulation in Ca9–22 cells. The intensities of the bands in A were quantitated using NIH Image, and the results were expressed as a percentage of the maximal response to IGF-I or EGF. C, Specificity of the effect of AG1478 action. Serum-deprived cells were incubated in the presence of 1 µM AG1478 for 30 min and stimulated, or not, with 50 ng/ml IGF-I or EGF for 10 min. Cells were lysed and immunoprecipitated with antibodies against either EGFR or the IGF-IR ß-subunit. Immunoprecipitates were subjected to SDS-PAGE, and the separated proteins were probed with PY20. Phosphorylated forms of each protein were visualized as described in Materials and Methods.

 
IGF-I neither activates HB-EGF shedding nor increases global EGFR tyrosine phosphorylation in Ca9–22 cells
A plausible explanation for the phenomena observed in Ca9–22 cells would be that IGF-I transactivates EGFR, as has been reported by other groups (18, 19). One reported mechanism for the transactivation of EGFR by IGF-I is the IGF-I-induced shedding of the extracellular domain of pro-HB-EGF through the activation of MMPs. The released HB-EGF binds EGFR and then activates EGFR-TK (18). This mechanism is thought to be a common pathway, also used by G protein-coupled receptor ligands to activate ERK (15, 16, 17). To examine whether the same mechanism is functional in Ca9–22 cells, a pan-MMP inhibitor (phenanthroline) and an HB-EGF scavenger (CRM-197) were used. Interestingly, neither inhibitor prevented IGF-I-induced ERK activation (Fig. 3AGo). It is possible that EGFR can be transactivated using other mechanisms, so we next set out to directly detect the level of EGFR autophosphorylation after stimulation with IGF-I. Serum-deprived cells were stimulated with IGF-I or EGF, and the cell lysates were immunoprecipitated with an anti-EGFR antibody. Unexpectedly, Western blotting analysis using an antiphosphotyrosine antibody revealed that EGFR autophosphorylation was detected only when cells were stimulated with EGF; no signals were identified in IGF-I-treated cells or untreated cells (Fig. 3BGo, left panel). Similar results were obtained when cells were immunoprecipitated with an anti-Erb B2 antibody, which is a member of the EGFR family that can be phosphorylated when heterodimerized with EGFR (23) (Fig. 3BGo, left panel). We also analyzed the level of autophosphorylation using an antibody that recognizes phosphorylated forms of EGFR (Tyr1068). When cells were stimulated with increasing doses of IGF-I (5–100 ng/ml), no activation beyond unstimulated basal levels was detected (Fig. 3BGo, right panel). Significant ERK activation was detectable upon application of more than approximately 25 ng/ml IGF-I after reprobing the same blot with an antibody that recognizes phosphorylated ERK (Fig. 3BGo, right panel).



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  		FIG. 3. Global EGFR autophosphorylation does not occur upon IGF-I stimulation in Ca9–22 cells. A, Effect of inhibition of HB-EGF shedding on IGF-I-induced ERK activation. Serum-deprived cells were incubated in the presence of 10 µg/ml CRM197 (CRM; HB-EGF scavenger) or 300 µM phenanthroline (Phe; pan-MMP inhibitor) for 1 h and stimulated, or not, with 50 ng/ml IGF-I or EGF for 10 min. Cells were lysed and processed for Western blotting. B, Autophosphorylation of EGFR after IGF-I or EGF stimulation. Serum-deprived cells were stimulated, or not, with 50 ng/ml IGF-I or EGF for 10 min, and cell lysates were prepared for immunoprecipitation using antibodies specific for EGFR or Erb B2. Immunoprecipitates were subjected to SDS-PAGE, and the autophosphorylation of each receptor was visualized using PY20 (left panel), as described in Materials and Methods. Serum-deprived cells were stimulated with graded concentrations of IGF-I (5, 25, 50, and 100 ng/ml) or 50 ng/ml EGF for 10 min, and cells were subjected to SDS-PAGE and Western blotting using an antibody that recognizes phosphorylated forms of EGFR (PY1068). The same blot filters were reprobed with an antibody against phosphorylated forms of ERK (right panel). C, Comparison of the sensitivity of signal detection between antibodies to phosphorylated forms of EGFR and ERK. Serum-deprived cells were stimulated with graded concentrations of EGF (0.001, 0.01, 0.1, 1, 10, and 50 ng/ml) or 50 ng/ml IGF-I for 10 min and processed for Western blotting (left panel). A quantitative analysis of these results is shown in the right panel. {square}, EGFR; {blacksquare}, ERK.

 
It remains possible that small increases in EGFR activation were not detectable under these conditions due to the low sensitivity of the antibodies. To assess this possibility, cells were stimulated with increasing doses of EGF, from extremely low levels, and the activation kinetics of both EGFR and ERK were compared using the same blot filters (Fig. 3CGo, left panel). The result of quantitative analysis is shown in the right panel. Activation of EGFR was apparent at an EGF dose of more than approximately 0.01 ng/ml, and activation intensity increased in response to doses up to 50 ng/ml in a dose-dependent manner. In contrast, the activation of ERK became apparent at a dose of 1 ng/ml and reached a plateau at 10 ng/ml. The ERK activation intensity in response to a 50 ng/ml dose of IGF-I was comparable to that found in response to between a 1 and 10 ng/ml dose of EGF; nevertheless, EGFR activation was not detected. These results clearly indicate that in response to EGF stimulation the antibody for activated EGFR is able to detect more sensitively a signal change than the antibody for activated ERK. Thus, the observation that ERK activation, but not EGFR activation, is detectable after IGF-I stimulation, as shown in Fig. 3BGo, strongly suggests that the transactivation of EGFR is unlikely to be responsible for ERK activation.

IGF-IR coprecipitates phosphorylated EGFR upon EGF stimulation, but not upon IGF-I stimulation
Two different methods, using different antibodies to detect EGFR autophosphorylation, indicated that global EGFR activation is not detectable after IGF-I stimulation. The possibility cannot be ruled out, however, that only a very small number of EGFRs are activated, which would not be detectable under the current conditions. Very recently, it has been reported that IGF-IR forms a complex with EGFR, and that IGF-I transactivates EGFR through this binding interaction (20). We therefore tested the possibility of a direct interaction between IGF-IR and EGFR (Fig. 4AGo). Cells were stimulated with IGF-I or EGF, and the cell lysates were immunoprecipitated with IGF-IR. Western analysis revealed that after EGF stimulation, IGF-IR could coprecipitate high molecular weight, tyrosine-phosphorylated proteins (~170 and ~120 kDa) that were not detectable after stimulation with IGF-I. When the blot was reprobed with an antibody for phosphorylated EGFR, the approximately 170-kDa protein was identified as phosphorylated EGFR. The protein represented by the band at approximately 120 kDa is thought to be Gab1, which binds EGFR (24). The AG1478 treatment abrogated both signals (Fig. 4BGo). IGF-IR was thus found to form a complex with EGFR. Indeed, EGFR protein was detected in the immunoprecipitated lysate from untreated cells, indicating that IGF-IR can form a complex with EGFR regardless of IGF-IR or EGFR activation, confirming the previous report (20). After IGF-I stimulation, a clear band at approximately 95 kDa was detected by an antiphosphotyrosine antibody, which was thought to be the tyrosine-phosphorylated IGF-IR ß-subunit. No signal was detected at approximately 170 kDa, and an antibody raised against phosphorylated EGFR also did not detect any clear bands at approximately 170 kDa, indicating that IGF-IR-bound EGFR is not transactivated by IGF-I stimulation. We conclude that the ability of IGF-IR to precipitate EGFR shows that IGF-IR and EGFR always form a complex, but that the transactivation of EGFR is not induced by IGF-I stimulation.



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  		FIG. 4. Properties of the proteins that coprecipitated with IGF-IR. A, Effect of IGF-I or EGF treatment on the tyrosine phosphorylation of IGF-IR-coprecipitated proteins. Serum-deprived cells were stimulated, or not, with 50 ng/ml IGF-I or EGF for 10 min, and the cell lysates were immunoprecipitated with an antibody against IGF-IR. Immunoprecipitates were subjected to SDS-PAGE and processed for Western blotting using PY20 (top panel). The same blot was reprobed with antibodies against phosphorylated (middle panel) or pan-EGFR (bottom panel). B, Effect of AG1478 treatment on the tyrosine phosphorylation of IGF-IR-coprecipitated proteins. Cells were treated with 1 µM AG1478 30 min before 50 ng/ml EGF treatment. Cells were lysed and prepared for Western blotting as described above.

 
Shc- or Grb2-associated EGFR does not show EGFR transactivation after IGF-I stimulation
To confirm that the transactivation of EGFR does not occur after IGF-I stimulation, we next coprecipitated EGFR with one of its docking proteins, Shc (Fig. 5AGo) or Grb2 (Fig. 5BGo), and determined whether EGFR transactivation could be identified. Western analysis using an antiphosphotyrosine antibody exhibited distinct bands representing tyrosine-phosphorylated proteins, including one at approximately 170 kDa (Fig. 5AGo, top panel). The band with the highest intensity was observed when cells were stimulated with EGF. The antibody recognizing phosphorylated EGFR revealed that phosphorylated EGFR was present in the cell lysates, even in unstimulated cells (third panel). IGF-I did not activate Shc-bound EGFR, and interestingly, the intensity appeared to have somewhat decreased after IGF-I stimulation. Background EGFR autophosphorylation was almost eliminated in IGF-I-treated cells, and a similar level of reduction was observed in EGF-treated cells by pretreatment with AG1478. Tyrosine phosphorylation of a smaller protein of approximately 50 kDa, thought to be Shc p52, was also detectable in the immunoprecipitated cell lysates upon stimulation with EGF (Fig. 5AGo, top panel). Its phosphorylation was not increased by IGF-I treatment, but decreased, as shown by the overexposed panel (second panel). The Western blot of the whole cell lysates before immunoprecipitation confirmed our findings regarding ERK activation (bottom panel). Notably, IGF-I induced ERK activation, whereas AG1478-pre-treated cells did not induce ERK activation even after EGF stimulation despite similar levels of EGFR autophosphorylation. A second docking protein, Grb2, also immunoprecipitated EGFR; again, there was no evidence that IGF-I transactivated EGFR (Fig. 5BGo).



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  		FIG. 5. Autophosphorylation of docking protein-associated EGFR in Ca9–22 cells. A, Autophosphorylation of Shc-associated EGFR. Serum-derived AG1478-treated or untreated (1 µM, 30 min) cells were stimulated with 50 ng/ml IGF-I or EGF for 10 min, and the cell lysates were immunoprecipitated with an antibody against Shc. Immunoprecipitates were subjected to SDS-PAGE and processed for Western blotting using PY20 (top panel). The second panel shows an overexposed image of the top panel in the 50-kDa region. The same blot was reprobed with an antibody against phosphorylated EGFR (third panel) or Shc (fourth panel). The bottom panel represents the Western blot for phosphorylated ERK, performed using part of the whole cell lysates before immunoprecipitation. B, Autophosphorylation of Grb2-associated EGFR. Serum-deprived AG1478-treated or untreated (1 µM, 30 min) cells were stimulated with 10 or 50 ng/ml IGF-I and 50 ng/ml EGF for 10 min. Cell lysates were processed as described for Shc, but instead using an antibody against Grb2. Top panel, PY20; middle panel, phosphorylated EGFR; bottom panel, Western blot for phosphorylated ERK, performed using part of the whole cell lysates before immunoprecipitation.

 
Collectively, our results indicate that IGF-I induces ERK activation without the transactivation of EGFR in Ca9–22 cells; however, the process is dependent on basal EGFR-TK activity.

IGF-I induces both Ras and c-Raf activation, but only the latter is significantly inhibited by AG1478 treatment
To understand the basal EGFR-TK activity-dependent mechanism occurring in Ca9–22 cells, we next examined the activation of downstream factors after IGF-I stimulation, such as Ras and c-Raf. To investigate Ras activation, cell lysates were pulled down by the GST-RBD of c-Raf at the indicated times after stimulation, and Ras activity was determined by the level of RBD-trapped Ras demonstrated by Western blotting (Fig. 6AGo). Upon application of EGF, Ras activation immediately increased and reached a peak about 2–5 min after stimulation. Activation disappeared at 10 min after stimulation. Although Shc was not activated by IGF-I stimulation (Fig. 5AGo), significant Ras activation was detected upon IGF-I stimulation and exhibited a time course similar to that observed upon EGF stimulation. AG1478 treatment abrogated EGF-induced Ras activation, but IGF-I-induced activation was not significantly affected by the treatment (Fig. 6BGo). These results clearly demonstrate that IGF-I-induced Ras activation is independent of EGFR-TK activity and that Ras activation alone, without the basal EGFR-TK activity, is insufficient to activate ERK after IGF-I stimulation in Ca9–22 cells.



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  		FIG. 6. Ras and c-Raf activation after IGF-I stimulation in Ca9–22 cells. A, Time course of Ras activation after IGF-I or EGF stimulation. Serum-deprived cells were stimulated, or not, with 50 ng/ml IGF-I or EGF at the indicated times, and the resulting cell lysates were incubated with the GST-tagged RBD. The amount of trapped Ras was determined by Western blotting, as described in Materials and Methods. The whole cell lysates remaining after the procedure were used for the detection of pan-Ras as a control. c, unstimulated control. B, Effect of AG1478 treatment on IGF-I- or EGF-induced Ras activation. Serum-deprived AG1478-treated or untreated (1 µM, 30 min) cells were stimulated with 50 ng/ml IGF-I or EGF for 2 min, and Ras activation was determined as described above (top panel). The activation of Ras was quantitated using NIH Image (bottom panel). C, c-Raf kinase assay after IGF-I stimulation in Ca9–22 cells. Serum-deprived AG 1478-treated or untreated (1 µM, 30 min) cells were stimulated with 50 ng/ml IGF-I or EGF for 5 min, and c-Raf activity was determined as described in Materials and Methods. Each data point represents the mean ± SD of three separate experiments. *, P < 0.01; **, P < 0.001 (vs. unstimulated cells, by ANOVA). c, unstimulated control. D, Effect of a specific MEK inhibitor on IGF-I-induced ERK activation. Serum-deprived PD98059-treated or untreated (1, 5, 25, and 50 µM, 30 min) cells were stimulated with 50 ng/ml IGF-I or EGF for 10 min. Cell lysates were subjected to SDS-PAGE and processed for Western blotting.

 
In general, ERK is believed to be activated through the sequential activation of the Ras/c-Raf/MEK/ERK pathway (11, 12, 13). However, the c-Raf activation mechanism is still unresolved. We therefore performed a c-Raf kinase assay using immunoprecipitation with an anti-c-Raf antibody. The immunoprecipitates were then combined with recombinant MEK and ERK2, and the uptake of [{gamma}-32P]ATP was quantitated using myelin basic protein as a substrate (Fig. 6CGo). IGF-I significantly induced c-Raf activation, and it was of great interest that AG1478 treatment reduced the activation below the level observed in untreated controls. EGF induction enhanced levels of c-Raf activity compared with IGF-I induction, and AG1478 treatment reduced the EGF-induced activity to the level observed in IGF-I-treated cells in the presence of AG1478. These results indicate that c-Raf activity reflects ERK activity induced by either EGF or IGF-I even in combination with AG1478 treatment. Therefore, c-Raf may be the key kinase involved in the regulation of the IGF-I-induced ERK activation. In the context of the present assay system, activated c-Raf should activate ERK through MEK activation. In concurrence with this idea, IGF-I-induced ERK activation was abrogated in vivo by a specific inhibitor of MEK, PD98059, confirming that the c-Raf/MEK/ERK pathway is involved in IGF-I-induced ERK activation in Ca9–22 cells (Fig. 6DGo).


   Discussion
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 Abstract
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 Materials and Methods
 Results
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References
 
Based on evidence from previous reports, we believed that the EGFR-TK-dependent, IGF-I-induced ERK activation was necessarily accompanied by EGFR transactivation (18, 19), which was considered to play an intermediate step in this pathway. It was reported that IGF-I-induced ERK activation, but not Akt activation, was abrogated by specific EGFR-TK inhibitors, and that EGFR autophosphorylation was increased after IGF-I stimulation, indicating that this mechanism of activation is EGFR-TK transactivation dependent. IGF-IR, however, possesses its own means of ERK activation through Shc/Ras/c-Raf and 14-3-3 protein/c-Raf pathways (7, 8), which raised the question of why EGFR-TK transactivation would be required. Furthermore, if IGF-I-induced ERK activation is indeed due to EGFR-TK transactivation, the corresponding level of EGFR activation obtained directly with EGF stimulation should provide a level of ERK activation similar to that obtained with IGF-I stimulation; however, this hypothesis has not been confirmed. In a recent manuscript, Ahmad et al. (20) reported that a low level of IGF-IR-bound EGFR transactivation was detected upon IGF-I stimulation. The researchers then concluded that the significant IGF-I-induced ERK activation could be attributed to the observed subtle EGFR transactivation. We were concerned, however, whether such a low level of EGFR transactivation by IGF-I stimulation would be sufficient to induce ERK activation to a level comparable to that obtained upon EGF stimulation. These considerations led us to construct a hypothesis that there exists an EGFR-TK transactivation-independent, but EGFR-TK inhibitor-sensitive, mechanism that controls IGF-I-induced ERK activation.

In the present study we have used the oral carcinoma cell line Ca9–22 to identify a mechanism for IGF-I-induced ERK activation that is dependent on basal EGFR-TK activity, but does not require EGFR transactivation. The primary findings supporting this hypothesis are as follows: 1) IGF-I-induced ERK activation was abrogated when cells were pretreated with the EGFR-TK specific inhibitor AG1478; 2) neither the pan-MMPs inhibitor nor the HB-EGF scavenger inhibited IGF-I-induced ERK activation; 3) IGF-I did not stimulate global EGFR autophosphorylation; 4) we confirmed that under the present conditions, undetectable levels of EGFR-TK activation by IGF-I stimulation would be unable to induce a significant level of ERK activation; 5) IGF-IR was found in a complex with EGFR, but IGF-I did not stimulate autophosphorylation of IGF-IR-bound EGFR; 6) Shc- or Grb2-bound EGFR did not exhibit an increase in autophosphorylation beyond basal levels upon IGF-I stimulation; and 7) AG1478 treatment abrogated basal EGFR-TK activity.

Our observations thus suggested a novel mechanism for IGF-I-induced ERK activation, which we sought to elucidate. In addition, we attempted to find downstream regulatory factors in the ERK activation cascade, whose activation depends on basal EGFR-TK activity. Unexpectedly, we found that Shc was not activated significantly by IGF-I stimulation; however, IGF-I could still induce significant Ras activation. Although Ras can be efficiently activated through the Shc/Grb2/Sos pathway (25, 26), IRS proteins can also mediate Ras activation (27), which may be what occurred under our experimental conditions. Indeed, IGF-I was able to induce tyrosine phosphorylation of IRS-1, but was not affected by AG1478 treatment (data not shown), a property shared by IGF-I-induced Ras activation. We therefore concluded that basal EGFR-TK activity is unlikely to be involved in the ERK activation cascade before the recruitment of Ras. We reasoned that the factors responsible for the basal EGFR-TK-dependent mechanism probably exist downstream of Ras. c-Raf is a cytosolic serine/threonine kinase that is located downstream of Ras in the ERK activation cascade (13). Although the mechanism of Ras-induced c-Raf activation is not fully understood, in vitro experiments have shown that Ras activation alone is insufficient to activate c-Raf (28), which we have confirmed in the present study. The results of the c-Raf kinase assay clearly demonstrated that IGF-I can induce significant activation of c-Raf in a manner dependent on basal EGFR-TK activity, indicating that the level of ERK activation is determined by the sum of its basal activation level, which originates from basal EGFR-TK activity, with the IGF-I-induced c-Raf activation. These results also imply that c-Raf may be the key kinase involved in the regulation of IGF-I-induced ERK activation. The mechanism by which the basal EGFR-TK activity directly or indirectly regulates c-Raf activation has yet to be determined; however, a specific MEK inhibitor, PD98059, can abrogate IGF-I-induced ERK activation, indicating that MEK activation is involved in this process, potentially linking c-Raf directly to MEK/ERK activation.

Ruiter et al. (29) reported that submicromolar doses of alkyl-lysophospholipids induced ERK activation without EGFR transactivation, but that activation could be abrogated by AG1478 treatment. The researchers also observed that the alkyl-lysophospholipids induced EGFR internalization, and they concluded that the internalization of EGFR with basal TK activity is important for ERK activation (29). These data concur with our observations. IGF-I induces internalization of IGF-IR (30); therefore, EGFR should be cointernalized because these two receptors form a complex in Ca9–22 cells. Because c-Raf activation is likely to occur in the endosomes (31), EGFR molecules with basal activity, when brought to endosomes, may increase c-Raf activity. IGF-IR thus may be a carrier of EGFR to bring it to endosomes, leading to ERK activation. This possibility is being tested in our laboratory by immunofluorescence staining.

The dependence of IGF-I-induced ERK activation on EGFR-TK activity is still puzzling, considering that IGF-IR possesses its own ERK activation pathways. We did demonstrate that the EGFR-TK dependence was not always present, as shown in WT cells overexpressing IGF-IR. In contrast, Ca9–22 cells overexpressing EGFR did exhibit basal EGFR-TK activity dependence. These observations raise the possibility that the relative expression levels of EGFR and IGF-IR determine the level of EGFR-TK dependence. The availability of EGFR appears to facilitate efficient ERK activation, which may overcome any inefficiencies on the part of IGF-IR.

In this study we have demonstrated that EGFR-TK inhibitors can inhibit signals that originate from other receptors even in the absence of EGFR transactivation. This may prove to have clinical implications for antitumor therapies that use EGFR-TK inhibitors; however, extrapolations from the results of only a single cell line must be carefully considered. ZD1839 has already been used in a clinical setting, but side-effects, including rashes and interstitial pneumonia, have been reported as well as a large heterogeneity in the tumor response (32, 33, 34). The use of EGFR-specific agents may contribute to the inhibition of unexpected cross-talk signaling with EGFR, which may be important for normal or tumor tissue functioning. Therefore, the elucidation of mechanisms for cross-talk between EGFR and various heterologous receptors will shed additional light on the construction of a therapeutic strategy that minimizes side-effects and maximizes tumor response.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (13218045) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M.M.).

Abbreviations: ADBI, 20 mM 4-Morpholinepropanesulfonic acid (pH 7.2), 25 mM ß-glycerophosphate, 5 mM EGTA, 1 mM NaVO4, and 1 mM dithiothreitol; DTT, dithiothreitol; EGFR, epidermal growth factor receptor; EGFR-TK, epidermal growth factor receptor tyrosine kinase; ERK, extracellular signal-regulated kinase; GST, glutathione-S-transferase; HB-EGF, heparin-binding epidermal growth factor; HRP, horseradish peroxidase; IGF-IR, IGF-I receptor; IRS-1, insulin receptor substrate-1; MBP, myelin basic protein; MEF, mouse embryo fibroblast; MEK, MAPK kinase; MMP, matrix metalloproteinase; PDGFR, platelet-derived growth factor receptor; PI3-K, phosphatidylinositol 3'-kinase; PMSF, phenylmethylsulfonylfluoride; RBD, Ras binding domain; SDS, sodium dodecyl sulfate; TBST, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20; TK, tyrosine kinase; WT, wild-type.

Received June 3, 2004.

Accepted for publication July 15, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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