From the Department of Pharmacology, the University of Iowa College of Medicine, Iowa City, Iowa 52242-1109
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ABSTRACT |
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The ErbB2 and ErbB3 proteins together constitute a functional coreceptor for heregulin (neuregulin). Heregulin stimulates the phosphorylation of both coreceptor constituents and initiates a variety of other signaling events, which include phosphorylation of the Shc protein. The role of Shc in heregulin-stimulated signal transduction through the ErbB2·ErbB3 coreceptor was investigated here. Heregulin was found to promote ErbB3/Shc association in NIH-3T3 cells expressing endogenous ErbB2 and recombinant ErbB3. A mutant ErbB3 protein was generated in which Tyr-1325 in a consensus Shc phosphotyrosine-binding domain recognition site was mutated to Phe (ErbB3-Y/F). This mutation abolished the association of Shc with ErbB3 and blocked the activation of mitogen-activated protein kinase by heregulin. Whereas heregulin induced mitogenesis in NIH-3T3 cells transfected with wild-type ErbB3 cDNA, this mitogenic response was markedly attenuated in NIH-3T3 cells transfected with the ErbB3-Y/F cDNA. These results showed a specific interaction of Shc with the ErbB3 receptor protein and demonstrated the importance of this interaction in the activation of mitogenic responses by the ErbB2·ErbB3 heregulin coreceptor complex.
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INTRODUCTION |
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The ErbB3/HER3 receptor protein is a member of the ErbB/HER family of growth factor receptors (1), the prototype of which is the epidermal growth factor (EGF)1 receptor (ErbB1/HER1). Like other members of this family, the ErbB3 protein consists of an extracellular ligand binding domain, a transmembrane domain, an intracellular protein tyrosine kinase domain, and a C-terminal phosphorylation domain. Human heregulins (2) or their rat counterparts, the Neu differentiation factors (3), have been identified as a family of ligands for this receptor. ErbB3 is unique among ErbB/HER family members in that it has an impaired protein tyrosine kinase activity, which has been attributed to the substitution of amino acid residues invariantly conserved in protein tyrosine kinases (4, 5). However, ErbB3 tyrosine residue phosphorylation is observed when ErbB3 is coexpressed with other ErbB family members, apparently through the formation of heterodimeric receptor complexes (6, 7). Cells coexpressing EGF receptor and ErbB3 show an EGF-dependent ErbB3 phosphorylation (8, 9). Heregulin-stimulated phosphorylation of both ErbB2 and ErbB3 occurs in cells coexpressing these proteins (10-12), and although ErbB2 itself does not bind heregulin, ErbB2 and ErbB3 cooperate in the formation of a high affinity heregulin coreceptor complex (10). In addition, heregulin-dependent phosphorylation of EGF receptor and ErbB2 has been attributed to cross-phosphorylation by the kinase-intact heregulin receptor ErbB4 (13, 14).
Among the heterodimers formed within the ErbB family, the ErbB2·ErbB3 coreceptor complex is believed to elicit the most potent mitogenic signal (7, 11, 15). The contribution of ErbB3 to the mitogenic potential of ErbB family coreceptors might be enhanced by its unique C-terminal phosphorylation domain, which possesses several consensus sequences for the binding of signal-transducing proteins, including phosphoinositide (PI) 3-kinase, Grb2, Shc, SH-PTP2, and Src family protein tyrosine kinases (16). Notably, this domain contains six repeats of the consensus motif, Tyr-Xaa-Xaa-Met (YXXM), for binding to the p85 subunit of PI 3-kinase (17, 18). The role of PI 3-kinase in signal transduction by ErbB family coreceptors has begun to be clarified. The EGF-dependent association of PI 3-kinase with the ErbB3 protein has been observed in cancer cells expressing high levels of both EGF receptor and ErbB3 (8, 9). Also, a heregulin-dependent association of PI 3-kinase with ErbB3 has been seen in the context of the ErbB2·ErbB3 coreceptor, and the resulting activation of PI 3-kinase has been shown to be important for heregulin-stimulated mitogenesis (11).
Like other ErbB family members, the ErbB3 protein incorporates a consensus motif, Asn-Pro-Xaa-Tyr (NPXY), for binding to the Shc protein. Shc is an adapter protein that contains a C-terminal SH2 domain and an N-terminal phosphotyrosine-binding domain. The phosphotyrosine-binding domain of Shc specifically binds to phosphotyrosine in the NPXY sequence context (19-22) and mediates the binding of Shc to the EGF (23-25) and insulin (25, 26) receptors. Receptor-associated Shc is rapidly phosphorylated (27-29) and subsequently binds a Grb2·Sos complex, which results in the translocation of the complex to the plasma membrane (30, 31). Sos, a guanine nucleotide exchange protein, then activates Ras (32, 33), which in turn stimulates the mitogen-activated protein kinase (MAPK) cascade (34, 35). Shc has been implicated in mitogenic signaling by epidermal growth factor (36), platelet-derived growth factor (37), nerve growth factor (38), and insulin (39) receptors.
The Shc protein has been shown to associate with phosphorylated ErbB3 (28, 40), and heregulin has been found to stimulate the phosphorylation of Shc (40). These findings suggest a possible contribution of the Shc signaling pathway to heregulin-stimulated mitogenesis. Synthetic phosphopeptide competition experiments have indicated that Tyr-1309 in human ErbB3 is the binding site of Shc (28). By mutating the corresponding tyrosine residue in the putative Shc binding site of the rat ErbB3 receptor protein, we have in the present study examined the heregulin-stimulated interaction of Shc with the ErbB3 receptor, and we have investigated the role of Shc in mitogenesis mediated by the ErbB2·ErbB3 coreceptor complex.
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EXPERIMENTAL PROCEDURES |
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Materials--
NIH-3T3 cells were purchased from American Type
Culture Collection. LipofectAMINE transfection reagent was obtained
from Life Technologies, Inc. Recombinant heregulin-1 and antibodies
recognizing ErbB2 (Ab-1) and ErbB3 (2C3, 2F12) were purchased from
NeoMarkers. Anti-phosphotyrosine (PY20), recombinant PY20 conjugated to
horseradish peroxidase, anti-Shc, and anti-Grb2 were purchased from
Transduction Laboratories. Anti-p85 was purchased from Upstate
Biotechnology. A mitogen-activated protein kinase-specific antibody
recognizing both Erk1 and Erk2 isoforms (Zymed Laboratories
Inc.) and distinct Erk1-specific and Erk2-specific antibodies
(Santa Cruz) were also procured. Recombinant platelet-derived growth
factor-BB and wortmannin were purchased from Sigma. Horseradish
peroxidase-conjugated secondary antibodies and enhanced
chemiluminescence (ECL) reagents were purchased from Amersham Pharmacia
Biotech. [
-32P]ATP (3000 Ci/mmol) and
[methyl-3H]thymidine (90 Ci/mmol) were
acquired from NEN Life Science Products. The recombinant EGF receptor
protein tyrosine domain, consisting of amino acid residues 645-972 of
the parent receptor, was expressed with the baculovirus/insect system
and purified as described previously (5).
Generation of an ErbB3 Tyr-1325 Phe Mutant Protein--
The
rat ErbB3 cDNA (16) was mutated by use of the Ex-Site Mutagenesis
kit from Stratagene. A tyrosine codon corresponding to amino acid 1325 was replaced with a phenylalanine codon with a 33-base pair
reverse mutagenic primer 5'-GGGAAAAGCCGGCTGTGCCAGAAATCGGGGTTG-3' and the ErbB3 expression plasmid pcDNA3-B3 (16) as the
template for the polymerase chain reaction mutagenesis. The altered
region of the cDNA was subcloned into the parent expression
vector to yield the mutant ErbB3 receptor cDNA expression
vector (pcDNA3-B3-Y/F). The affected region was sequenced to verify
the accuracy of polymerase chain reaction amplification.
Cell Culture-- NIH-3T3 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere. After transfection with the pcDNA3-B3-Y/F mutant expression plasmid using LipofectAMINE reagent, stable NIH-3T3 clones were selected with Geneticin (G418) and screened for the expression of the mutant receptor protein by Western blotting. A stable NIH-3T3 cell line expressing ErbB3-WT was isolated as described previously (16). For some [3H]thymidine incorporation assays nonclonal pools of NIH-3T3 cells transfected with pcDNA3-B3-WT and pcDNA3-B3-Y/F were grown under Geneticin selection. Equivalent expression of wild-type and mutant receptors was verified by immunoblotting.
Cell Stimulation, Immunoprecipitation, and
Immunoblotting--
Prior to stimulation with growth factor, cells
were starved for 18 h in low serum medium (DMEM containing 0.1%
fetal bovine serum). Starved cells were washed once with low serum
medium and incubated with heregulin-1 (1 nM final
concentration) diluted in culture medium containing 0.1% bovine serum
albumin, or the dilution vehicle, for 5-7 min at 37 °C. Cells were
washed immediately with ice-cold phosphate-buffered saline and lysed
with Nonidet P-40 lysis buffer (1% Nonidet P-40, 50 mM
Hepes/Na, 150 mM sodium chloride, 2 mM EDTA, 3 mM EGTA, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 50 mM sodium fluoride,
2 µg/ml pepstatin A, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, pH 7.4). The whole cell
lysates were centrifuged for 10 min at 13,000 × g.
After protein concentration was assayed, the supernatants were
immunoprecipitated with appropriate antibodies (8). The
immunoprecipitates and cell lysate samples were resolved by SDS-PAGE,
transferred to a polyvinylidene difluoride membrane, and detected with
the indicated antibodies by ECL luminography.
In Vitro Binding Assays-- A C-terminal peptide fragment of ErbB3 (residues 1311-1339) containing Tyr-1325 was generated as a GST fusion protein (GST-B3) and purified as described previously (41). GST-B3 or GST (65 pmol each) was incubated in buffer A (20 mM Hepes/ Na, 50 mM sodium chloride, 10% (v/v) glycerol, pH 7.4) supplemented with 10 mM MnCl2, 0.1% Triton X-100, and 0.1 µg of EGF receptor protein tyrosine kinase domain (5) in the absence or presence of 50 µM ATP for 30 min at 22 °C (total volume 10 µl). The mixtures were diluted into 375 µl of lysate from NIH-3T3 cells (2 mg/ml protein), incubated for 30 min on ice, and then allowed to bind glutathione-agarose (100 µl of a 1:1 suspension in buffer A) for 1 h at 4 °C. The agarose suspensions were centrifuged for 1 min at 600 × g. The pellets were washed twice in 500 µl of ice-cold Nonidet P-40 lysis buffer and then suspended in gel sample buffer. Pellets and cell lysate samples (20 µg of protein) were resolved by SDS-PAGE and immunoblotted with anti-Shc and anti-phosphotyrosine.
Mitogen-activated Protein Kinase Assay--
Mitogen-activated
protein kinase (MAPK) from cells stimulated with heregulin or control
vehicle was immunoprecipitated with a combination of Erk1 and Erk2
antibodies as described above. The washed immunoprecipitates were
suspended in 30 µl of reaction buffer containing 10 mM
Hepes/ Na, 10 mM MgCl2, pH 7.4, and 8 µg of
myelin basic protein (MBP). The reaction was initiated by adding 3 µl
of 100 µM ATP containing 5 µCi of
[-32P]ATP and incubated for 15 min at 30 °C. The
reaction was quenched with sample buffer, and the proteins were
subjected to SDS-PAGE. The gel was subsequently dried, exposed to
autoradiographic film, and MBP phosphorylation quantified by
scintillation counting of excised gel bands. In the MAPK gel shift
assay, cell lysate supernatants from heregulin-, platelet-derived
growth factor-, or vehicle-stimulated cells were subjected to Western
blotting with an antibody recognizing both the Erk1 and Erk2 isoforms
of MAPK. Here in SDS-PAGE the amount of bisacrylamide in the gel was
reduced (acrylamide:bisacrylamide, 30:0.04). and the electrode buffer
was twice-concentrated (42).
[3H]Thymidine Incorporation Assay--
Cells were
plated at a density of 5 × 105/well in 6-well dishes,
grown for 24 h, and then serum-deprived for 18 h in DMEM
containing 0.1% fetal bovine serum. Cells were then stimulated with
varying concentrations of heregulin-1 for 18 h, after which 0.5 µCi/ml of [methyl-3H]thymidine was added to
each well, and the cells were further incubated for 4 h. For
experiments with wortmannin, either Me2SO or wortmannin
(100 nM) in Me2SO was added 30 min prior to
stimulation of cells with either vehicle or heregulin (10 nM). Cells were then washed twice with cold
phosphate-buffered saline, extracted with 5% trichloroacetic acid, and
then solubilized in 0.1 M sodium hydroxide. The
radioactivity incorporated into DNA was measured by scintillation
counting.
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RESULTS |
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Heregulin-dependent Phosphorylation of Wild-type and Mutant ErbB3 Proteins in Stably Transfected NIH-3T3 Cells-- By using site-directed mutagenesis, we created a mutant ErbB3 protein in which the candidate Shc binding site residue, Tyr-1325 (28), was substituted with phenylalanine (ErbB3-Y/F). NIH-3T3 fibroblast cell lines that stably expressed high levels of the wild-type (ErbB3-WT) and mutant (ErbB3-Y/F) receptor proteins were isolated. To confirm the expression of the receptor proteins, cell lysates were analyzed by immunoprecipitation followed by Western blotting with an ErbB3-specific antibody. The transfected fibroblasts expressed comparable levels of wild-type and mutant ErbB3 proteins (Fig. 1A). Cells transfected with the parent expression vector did not express detectable ErbB3.
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The Tyr-1325 Phe Point Mutation in ErbB3 Abolishes
Heregulin-dependent ErbB3/Shc Association--
To assay
the association of Shc with the wild-type and mutant ErbB3 receptor
proteins, lysates of stably transfected NIH-3T3 cells were
immunoprecipitated with an Shc-specific antibody and subsequently
immunoblotted with an ErbB3-specific antibody. Lysates from
mock-transfected cells and cells expressing either the mutant or
wild-type receptor protein showed the presence of each isoform of Shc,
p46, p52, and p66, in similar amounts across the cell lines (Fig.
2A). From cells expressing the
wild-type receptor, the ErbB3 protein coimmunoprecipitated with Shc,
which suggested that Shc constitutively associated with ErbB3. However,
this ErbB3/Shc association was significantly enhanced following
stimulation with heregulin. In contrast, Shc immunoprecipitates from
cells expressing ErbB3-Y/F showed no presence of the ErbB3-Y/F protein
(Fig. 2B). Thus, the mutation of a single tyrosine in the
NPXY sequence motif in the ErbB3 receptor abolished
association of Shc with ErbB3. Mock-transfected cells showed no
heregulin-dependent ErbB3/Shc association. Interestingly,
no association of Shc with ErbB2 was evident in any of the cells (Fig.
2B) (see "Discussion").
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Heregulin-stimulated Shc Phosphorylation and Shc/Grb2 Association
Is Significantly Attenuated in NIH-3T3 Cells Expressing
ErbB3-Y/F--
Since the Tyr Phe mutation blocked the interaction
of Shc with the ErbB3 receptor, we examined the effect of this mutation on heregulin signaling by the ErbB2·ErbB3 coreceptor. To investigate potential heregulin-stimulated Shc phosphorylation, Shc
immunoprecipitates were probed with a phosphotyrosine-specific
antibody. An increase in the phosphorylation of the Shc proteins was
seen in response to heregulin in cells expressing the wild-type
receptor. Among the three isoforms of Shc, p52 seemed to be
preferentially phosphorylated in response to heregulin. In cells
expressing ErbB3-Y/F the heregulin-induced phosphorylation of Shc was
significantly reduced as compared with cells expressing the wild-type
receptor (Fig. 2C). Heregulin did not stimulate Shc
phosphorylation in the mock-transfected cells.
A Phosphorylated ErbB3 C-terminal Peptide Interacts with Shc Proteins in Vitro-- To determine whether Tyr-1325 in the ErbB3 C terminus could when phosphorylated serve as a binding site for the Shc protein, we expressed a short C-terminal peptide fragment of ErbB3 (residues 1311-1339) containing only one tyrosine residue, Tyr-1325, as a GST fusion protein (GST-B3), and we used this protein in in vitro binding assays. Here the GST-B3 fusion protein was first phosphorylated with a recombinant EGF receptor protein tyrosine kinase domain (5) and then incubated with lysates of NIH-3T3 cells containing the Shc proteins. After precipitation of the phosphorylated GST-B3 protein with glutathione-agarose, associated Shc proteins were detected by Western blotting (Fig. 2D). Control experiments showed that the GST domain was not phosphorylated under these conditions and did not significantly interact with the Shc proteins. Also, the interaction of Shc with GST-B3 was dependent upon prior phosphorylation of the fusion protein. These results indicated that the C-terminal NPXY motif in ErbB3 could serve when phosphorylated as an Shc-binding site.
Heregulin-stimulated Activation of MAPK Is Impaired in NIH-3T3 Cells Expressing the ErbB3-Y/F Mutant Protein-- Receptor-mediated Shc phosphorylation and Shc/Grb2 association would be predicted to result in the activation of the Ras/MAPK signaling pathway. Potential heregulin-stimulated activation of MAPK was characterized in NIH-3T3 cells expressing ErbB2·ErbB3 coreceptors (Fig. 3). The ErbB3-Y/F receptor protein, which failed to interact with Shc, was used to examine the involvement of Shc in the activation of MAPK via the ErbB2·ErbB3 coreceptor. The activation of MAPK in the NIH-3T3 transfectants was detected by gel mobility shift assays (Fig. 3A) and in vitro phosphorylation assays employing the exogenous substrate myelin basic protein (MBP) (Fig. 3B). In NIH-3T3 cells expressing ErbB3-WT, MAPK was clearly activated in response to heregulin. This was evident by the retarded migration of both the p42 (Erk2) and p44 (Erk1) isoforms of MAPK in gel shift assays (Fig. 3A). Also, MAPK immunoprecipitates from heregulin-stimulated cells expressing ErbB3-WT showed strong MBP phosphorylation in immune complex kinase assays (Fig. 3B). In contrast, NIH-3T3 cells expressing ErbB3-Y/F showed no MAPK activation in response to heregulin. Since NIH-3T3 fibroblasts endogenously express receptors for platelet-derived growth factor, it was of interest to see whether this factor stimulated the activation of MAPK in the various transfected cell lines. MAPK was clearly activated in response to platelet-derived growth factor in the mock-transfected cells and in cells expressing either ErbB3-Y/F or ErbB3-WT, as was evident by the retarded migration of both the p42 and p44 isoforms of MAPK in the gel shift assay (Fig. 3A). Possibly, the failure of the ErbB3-Y/F protein to activate MAPK in response to heregulin resulted from its inability to interact with Shc and mediate an Shc/Grb2 association.
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Association of PI 3-Kinase with Wild-type and Mutant ErbB3 Proteins in Transfected NIH-3T3 Fibroblasts-- Previous studies have reported an association of PI 3-kinase with the ErbB3 protein in both the EGF receptor·ErbB3 (8, 9) and ErbB2·ErbB3 (11) coreceptor contexts. Given that ErbB3-Y/F failed to both interact with Shc and activate MAPK, we sought to determine if the mutant receptor could still associate with PI 3-kinase and therefore potentially signal through the PI 3-kinase pathway. Immunoblotting analyses of ErbB3 immunoprecipitates from control cells and cells expressing either ErbB3-WT or ErbB3-Y/F showed the presence of the p85 regulatory subunit of PI 3-kinase (Fig. 4), which indicated that the mutant ErbB3 protein retained its ability to interact with PI 3-kinase. Heregulin-stimulated association of p85 with ErbB3 was variable, which could have been due to the high basal association seen in the transfected NIH-3T3 cells.
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Heregulin-stimulated DNA Synthesis in NIH-3T3 Cells Expressing ErbB3-WT and ErbB3-Y/F-- In order to determine whether the wild-type and mutant ErbB3 proteins mediated a mitogenic response to heregulin, cellular DNA synthesis was analyzed with a [3H]thymidine incorporation assay. The results of a representative experiment are shown in Fig. 5A. Mock-transfected NIH-3T3 cells showed no enhanced [3H]thymidine uptake in response to heregulin. Cells expressing ErbB3-WT showed a dose-dependent uptake of [3H]thymidine with a significant stimulation seen at a 0.1 nM concentration of heregulin. Cells expressing ErbB3-Y/F showed an attenuated mitogenic response relative to those expressing the wild-type receptor protein. Interestingly, the high basal activity seen in the cells expressing ErbB3-WT was absent in cells expressing ErbB3-Y/F. Heregulin-stimulated [3H]thymidine incorporation, defined as the difference between basal incorporation and that stimulated by 10 nM heregulin, was compared between cells expressing either ErbB3-Y/F or ErbB3-WT (see Fig. 6). In five separate experiments, heregulin-stimulated DNA synthesis mediated by ErbB3-Y/F was found to range between 15 and 60% that mediated by ErbB3-WT. Heregulin-stimulated DNA synthesis was also studied with nonclonal pools of cells transfected with ErbB3-WT and ErbB3-Y/F cDNAs to ensure that the attenuated mitogenic response seen with clonal cells expressing ErbB3-Y/F was not an effect of clonal variation. Fig. 5B shows the results of a representative experiment with nonclonal cells expressing moderate levels of the wild-type and mutant receptor. Nonclonal cells expressing ErbB3-Y/F showed a significantly attenuated mitogenic response when compared with cells expressing ErbB3-WT. These results indicated that the association of Shc with the ErbB3 protein and the ensuing activation of the Ras/MAPK signaling pathway contributed to the mitogenic potential of the ErbB2·ErbB3 heregulin coreceptor.
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Effect of Wortmannin on Heregulin-stimulated [3H]Thymidine Incorporation-- Given that the ErbB3-Y/F protein retained the ability to associate with the p85 regulatory subunit of PI 3-kinase (Fig. 4), it was considered that activation of the PI 3-kinase pathway might account for the residual mitogenic activity seen in cells expressing ErbB3-Y/F. To determine the contribution of PI 3-kinase to the stimulation of DNA synthesis by heregulin, we examined the effect of wortmannin, a PI 3-kinase inhibitor, on [3H]thymidine uptake in cells expressing either ErbB3-WT or ErbB3-Y/F (Fig. 6). Cells were treated with or without wortmannin for 30 min prior to stimulation with either vehicle or 10 nM heregulin. In the representative experiment shown in Fig. 6, heregulin-stimulated DNA synthesis mediated by ErbB3-Y/F was found to be 39% that mediated by ErbB3-WT. Wortmannin decreased heregulin-stimulated [3H]thymidine incorporation in cells expressing ErbB3-Y/F by almost 45% and to a lesser extent (20%) in cells expressing ErbB3-WT. These results implicated PI 3-kinase as another contributor in mitogenic signaling by ErbB2·ErbB3 heregulin coreceptors. A similar effect of wortmannin on heregulin-stimulated DNA synthesis was previously observed in a study of fibroblasts expressing ectopic ErbB2 and ErbB3 proteins (11).
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DISCUSSION |
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The ErbB2 and ErbB3 proteins together constitute a functional heregulin coreceptor (10). Whereas heregulin is a ligand for the ErbB3 receptor protein, ErbB2 does not independently bind heregulin with significant affinity (10, 43), although it may in the context of an ErbB2·ErbB3 heterodimer cooperate in the high affinity binding of heregulin (10). The ErbB3 protein appears to be devoid of intrinsic kinase activity (4, 5). However, C-terminal tyrosine residues of both ErbB2 and ErbB3 are phosphorylated upon stimulation of the ErbB2·ErbB3 coreceptor (10), which is apparently mediated by the protein tyrosine kinase activity of ErbB2 (12). Hence, both ErbB2 and ErbB3, by complementing the functions of one another, can play important roles in heregulin signaling.
Heterodimerization might also increase the diversity of signaling through the activated ErbB2·ErbB3 coreceptor. However, the signal transduction pathways activated by the ErbB2·ErbB3 coreceptor have not been thoroughly characterized. The coupling of PI 3-kinase to ErbB3 in response to heregulin in the ErbB2·ErbB3 coreceptor context (11) and in response to EGF in cells overexpressing EGF receptor and ErbB3 (8, 9) has been documented. The Shc adapter protein has been shown to be phosphorylated in response to heregulin in cells overexpressing both ErbB3 and ErbB4 (40) and in cells overexpressing ErbB4 alone (44). The identification of the potential binding site of Shc on the ErbB3 C terminus by use of peptide competition assays (28) and the heregulin-stimulated ErbB3/Shc association demonstrated in cells expressing the ErbB2 and ErbB3 proteins (7, 40) have implicated Shc in heregulin signal transduction.
The purposes of this study were to demonstrate the binding of Shc to a
specific residue in the ErbB3 C terminus in response to heregulin and
to determine if this heregulin-induced binding event contributed to the
mitogenic response elicited by the ErbB2·ErbB3 coreceptor. We
addressed these questions by site-directed mutagenesis of Tyr-1325 in
the putative Shc binding site (NPXY motif) (21-24) in the
ErbB3 C terminus. Expression of the ErbB3-Y/F mutant protein in NIH-3T3
fibroblasts expressing endogenous ErbB2 resulted in the formation of
functional heregulin coreceptors (Fig. 1). Heregulin stimulated the
phosphorylation of the mutant ErbB3 protein to a similar extent as the
wild-type protein (Fig. 1A). However, the Tyr-1325 Phe
substitution abolished interaction of ErbB3 with Shc (Fig.
2B), which suggested that Shc specifically bound to
phosphorylated Tyr-1325 in the ErbB3 C terminus. The potential of
phosphorylated Tyr-1325 of ErbB3 to interact with Shc proteins was
subsequently demonstrated by in vitro binding experiments (Fig. 2D). The observations that heregulin did not (i)
stimulate the phosphorylation of Shc (Fig. 2C), (ii)
stimulate association of Shc with Grb2 (Fig. 2C), or (iii)
activate MAPK (Fig. 3) in NIH-3T3 cells expressing the ErbB3-Y/F
receptor suggested that heregulin-stimulated ErbB3/Shc association was
necessary for the activation of these downstream signaling events.
Also, it was apparent that any possible interaction of Grb2 with the
activated ErbB2 or ErbB3 protein could not effectively activate the
Ras/MAPK signaling pathway in the absence of Shc involvement.
Previous studies of NIH-3T3 fibroblasts (29) and T47D mammary carcinoma cells (40) have reported an ErbB2/Shc interaction. In the former study, a chimeric EGF receptor/ErbB2 protein was expressed in NIH-3T3 fibroblasts, and an EGF-dependent association of Shc with the ErbB2 cytoplasmic domain was seen. The latter study of T47D cells documented a heregulin-stimulated ErbB2/Shc association in addition to ErbB3/Shc association. Also, the catalytically activated rat ErbB2/Neu oncogene product was found to interact with Shc via an Asn-Leu-Tyr-Tyr (NLYY) sequence motif in the receptor C terminus (20, 45). In contrast, we failed to see any ErbB2/Shc interaction in the NIH-3T3 transfectants in response to heregulin (Fig. 2B). One possible explanation for these apparent discrepancies is that in each of these cases phosphorylation of the ErbB2 cytosolic domain occurred in the context of a coreceptor complex with different constituents, which could have resulted in the phosphorylation of distinct subsets of tyrosine residues in the ErbB2 C terminus. In the cases of the chimeric EGF receptor/ErbB2 protein and the ErbB2/Neu oncogene product, ErbB2 phosphorylation presumably was mediated by the ErbB2 catalytic domain. In the case of T47D cells, which express all four members of the ErbB family, the ErbB2 protein may have been phosphorylated within a heterodimeric complex with the kinase-active heregulin receptor ErbB4. In the present case, ErbB2 phosphorylation likely occurred in the context of a dimeric complex with the kinase-deficient ErbB3 protein. ErbB2 phosphorylation in this context would require either an intramolecular mechanism (46) or a mechanism involving higher order receptor oligomers (47, 48). Alternatively, our failure to detect ErbB2/Shc association in NIH-3T3 cells overexpressing recombinant ErbB3 in the presence of endogenous ErbB2 might have reflected a relatively low ratio of ErbB2 and ErbB3 protein levels. Indeed, Pinkas-Kramarski et al. (7) have previously demonstrated an ErbB2/Shc association in cells overexpressing both ErbB2 and ErbB3.
Heregulin is mitogenic to a variety of cell types (49) including human
mammary cancer cells (2) in which ErbB3 and other members of the ErbB
family are often overexpressed. The ErbB2·ErbB3 heterodimeric complex
has been shown to be capable of mediating mitogenic and proliferative
responses to heregulin, and PI 3-kinase has been shown to be involved
in these responses (11). Because the binding of Shc to the
ErbB2·ErbB3 coreceptor expressed in our transfected fibroblast cell
lines appeared to be directly mediated by the phosphorylation of
Tyr-1325 of ErbB3, the Tyr-1325 Phe mutant ErbB3 protein could be
exploited in the investigation of the role of Shc in mitogenic
signaling by the ErbB2·ErbB3 heregulin coreceptor.
Whereas heregulin stimulated a dose-dependent increase in
DNA synthesis in cells expressing ErbB3-WT, this response was
significantly attenuated in cells expressing ErbB3-Y/F (Fig. 5). The
high basal mitogenic activity displayed by cells expressing ErbB3-WT
was not shown by cells expressing ErbB3-Y/F. Qualitatively similar results were observed when either clonal cells expressing high levels
of ErbB3-WT and ErbB3-Y/F or nonclonal pools of cells expressing moderate levels of the ErbB3 proteins were examined, although the
residual mitogenic activity of the ErbB3-Y/F protein was enhanced in
the clonal cells. The heregulin-stimulated component of DNA synthesis
([3H]thymidine incorporation) in clonal cells
expressing ErbB3-Y/F was found to be significantly lower than in clonal
cells expressing ErbB3-WT (Fig. 6). The residual mitogenic response to
heregulin seen in the cells expressing ErbB3-Y/F could have reflected
the activation of the PI 3-kinase pathway (11), which would presumably not be blocked by the Shc binding site mutation. Indeed, ErbB3-Y/F was
able to associate with the p85 regulatory subunit of PI 3-kinase to a
similar extent as was ErbB3-WT (Fig. 4). Also, pretreatment with the PI
3-kinase inhibitor wortmannin decreased heregulin-stimulated [3H]thymidine uptake in cells expressing ErbB3-Y/F as
well as in cells expressing ErbB3-WT (Fig. 6). Whereas the residual
mitogenic activity seen in cells expressing ErbB3-Y/F might therefore
be attributed in part to the activation of the PI 3-kinase pathway, we
conclude that Shc-mediated signaling events contributed significantly to mitogenic signaling by the ErbB2·ErbB3 heregulin coreceptor.
In summary, the results presented in this study indicated that Tyr-1325 in the ErbB3 C terminus is a primary site for the interaction of Shc with the ErbB2·ErbB3 coreceptor complex. Mutation of this tyrosine to phenylalanine abolished association of Shc with ErbB3, blocked activation of the MAPK signaling pathway, and attenuated the mitogenic response to heregulin. Our studies have thus demonstrated that heregulin-induced association of Shc with ErbB3 can initiate signaling events that contribute significantly to the mitogenic effect of heregulin on cells expressing ErbB2·ErbB3 coreceptors.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK44684 and U. S. Army Research and Development Command Grant DAMD17-94-J-4185. Services were provided by the University of Iowa Diabetes and Endocrinology Research Center supported by National Institutes of Health Grant DK25295.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Cell Biology, Neurobiology and Anatomy,
Loyola University Medical Center, Maywood, IL 60153.
§ To whom correspondence should be addressed. Tel.: 319-335-6508; Fax: 319-335-8930.
The abbreviations used are:
EGF, epidermal
growth factor; Erk, extracellularly regulated kinase; DMEM, Dulbecco's
modified Eagle's medium; Grb2, growth factor receptor-bound protein 2; GST, glutathione S-transferaseGST-B3, GST
fusion protein incorporating rat ErbB3 residues 1311-1339MAPK, mitogen-activated protein kinaseMBP, myelin basic proteinNPXY, Asn-Pro-Xaa-Tyr sequence motifPAGE, polyacrylamide
gel electrophoresisPI, phosphoinositideWT, wild-typeY/F, Tyr Phe amino acid substitutionYXXM, Tyr-Xaa-Xaa-Met sequence
motif.
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