Mutation of a Shc Binding Site Tyrosine Residue in ErbB3/HER3 Blocks Heregulin-dependent Activation of Mitogen-activated Protein Kinase*

Ulka Vijapurkar, Kunrong ChengDagger , and John G. Koland§

From the Department of Pharmacology, the University of Iowa College of Medicine, Iowa City, Iowa 52242-1109

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- NIH-3T3 cells were purchased from American Type Culture Collection. LipofectAMINE transfection reagent was obtained from Life Technologies, Inc. Recombinant heregulin-beta 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. [gamma -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 right-arrow 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-beta 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 [gamma -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-beta 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Fig. 1.   Expression and heregulin-mediated phosphorylation of ErbB2 and ErbB3 in stably transfected NIH-3T3 cell lines. NIH-3T3 cells were transfected with either the parent pcDNA3 expression vector (-) or pcDNA3 incorporating wild-type (WT) and Tyr-1325 right-arrow Phe mutant (Y/F) ErbB3 cDNAs. Mock-transfected cells and cells expressing ErbB3-Y/F and ErbB3-WT were serum-starved and stimulated with vehicle or 1 nM heregulin-beta 1. A, cell lysates containing 1 mg of protein were immunoprecipitated with ErbB3-specific antibody, and the immunoprecipitates were subjected to SDS-PAGE and immunoblotting with either ErbB3-specific antibody (alpha -B3) or anti-phosphotyrosine antibody (alpha -P-tyr). B, alternatively, cell lysates were blotted with ErbB2-specific antibody (alpha -B2) or immunoprecipitated (IP) with a phosphotyrosine-specific antibody (alpha -P-Tyr) and then immunoblotted (IB) with ErbB2-specific antibody.

As we have previously observed (16), ErbB3-WT showed a constitutive phosphorylation on tyrosine residues that was enhanced by stimulation with heregulin-beta 1 (Fig. 1A). Phosphorylation of the ErbB3-Y/F mutant protein was enhanced to a similar extent as the wild-type protein. Treatment of the mock-transfected cells with heregulin induced no phosphorylation response. The heregulin-dependent phosphorylation of tyrosine residues in ErbB3 was presumably mediated by the ErbB2 protein tyrosine kinase, endogenously present in the NIH-3T3 fibroblasts (Fig. 1B). The endogenous ErbB2 protein in NIH-3T3 cells expressing either ErbB3-WT or ErbB3-Y/F was phosphorylated on tyrosine residues, and this phosphorylation was augmented in response to heregulin (Fig. 1B), which was consistent with the observation that ErbB2 and ErbB3 function as heregulin coreceptors (10, 43).

The Tyr-1325 right-arrow 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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Fig. 2.   Heregulin-stimulated ErbB3/Shc association, Shc phosphorylation, and Shc/Grb2 association. NIH-3T3 cells transfected with vector, ErbB3-WT, or ErbB3-Y/F cDNAs were treated as described in Fig. 1. A, lysates from cells stimulated with heregulin or control vehicle were probed with an Shc-specific antibody (alpha -Shc). All three isoforms of Shc (p46, p52, and p66) were evident. B, Shc was immunoprecipitated from the lysates with a Shc-specific antibody, and the immunoprecipitates were immunoblotted with either ErbB2-specific (alpha -B2) or ErbB3-specific (alpha -B3) antibody. C, Shc immunoprecipitates were also immunoblotted with an anti-phosphotyrosine-horseradish peroxidase conjugate (alpha -P-tyr) or a Grb2-specific antibody (alpha -Grb2). D, in vitro association of the phosphorylated ErbB3 C terminus with Shc proteins. GST-B3 and GST (65 pmol each) were incubated under phosphorylating (+ATP) or nonphosphorylating (-ATP) conditions, allowed to interact with Shc proteins from NIH-3T3 cell lysates, and then precipitated with glutathione-agarose. Association of Shc proteins with precipitated GST-B3 or GST was detected by immunoblotting with a Shc-specific antibody (alpha -Shc). An aliquot of the original cell lysates corresponding to ~1/40 of that in the binding assay was also analyzed and shown for comparison. Phosphorylation of the ErbB3 C-terminal peptide was detected by immunoblotting with anti-phosphotyrosine (alpha -P-tyr).

Heregulin-stimulated Shc Phosphorylation and Shc/Grb2 Association Is Significantly Attenuated in NIH-3T3 Cells Expressing ErbB3-Y/F-- Since the Tyr right-arrow 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.

Anti-Shc immunoprecipitates were also probed with a Grb2-specific antibody. Grb2 was found to be constitutively associated with Shc, but this association was increased in response to heregulin in cells expressing ErbB3-WT. No increase in heregulin-stimulated Shc/Grb2 association was seen in cells expressing the ErbB2-Y/F mutant receptor. Vector-transfected cells also showed no heregulin-dependent Shc/Grb2 association (Fig. 2C). These results indicated that Shc phosphorylation and Shc/Grb2 association were potentiated by the binding of Shc to ErbB3, which was apparently mediated by Tyr-1325 in the ErbB3 C terminus.

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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Fig. 3.   Heregulin-stimulated activation of mitogen-activated protein kinase. A, cells stimulated with heregulin (1 nM), platelet-derived growth factor (50 ng/ml), or vehicle were lysed as described in Fig. 1. Cell lysates containing 70 µg of total protein were subjected to a gel mobility shift assay of MAPK activation as described under "Experimental Procedures." Both the p42 and p44 isoforms of MAPK are indicated. The appearance of more slowly migrating bands in cells transfected with wild-type ErbB3 cDNA in response to heregulin and in all three cell lines in response to platelet-derived growth factor indicated the activation of the MAPK isoforms. B, alternatively, MAPK was immunoprecipitated from heregulin-stimulated and control cells, and the immunoprecipitates were subjected to in vitro MAPK assays with MBP and [gamma -32P]ATP as substrates. Error bars represent the standard error of three independent experiments.

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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Fig. 4.   Association of PI 3-kinase with ErbB3-WT and ErbB3-Y/F in stably transfected NIH-3T3 fibroblasts. Transfected cells were treated as described in Fig. 1. Lysates from vehicle- or heregulin-stimulated cells were probed with an antibody recognizing the p85 regulatory subunit of PI 3-kinase (alpha -p85). Cell lysates containing 1 mg of protein were also immunoprecipitated (IP) with ErbB3-specific antibody (alpha -B3), and the immunoprecipitates were subjected to SDS-PAGE and immunoblotting (IB) with p85-specific antibody.

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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Fig. 5.   Heregulin-stimulated [3H]thymidine uptake by NIH-3T3 cells expressing ErbB3-WT and ErbB3-Y/F. A, NIH-3T3 clones stably transfected with pcDNA3 (triangle ), pcDNA3-B3-WT (open circle ), or pcDNA3-B3-Y/F () were serum-starved overnight followed by treatment with varying concentrations of heregulin for 18 h. [methyl-3H]thymidine was added to the stimulation medium, and its incorporation into DNA was determined after 4 h. B, NIH-3T3 cells transfected with pcDNA3-B3-WT (open circle ), or pcDNA3-B3-Y/F () were grown under Geneticin selection for 4 weeks to generate nonclonal pools expressing wild-type and mutant receptors. [3H]Thymidine incorporation was analyzed as in A. The inset shows expression levels of ErbB3-WT and ErbB3-Y/F in the nonclonal transfected cells as determined by immunoblotting of cell lysates with ErbB3 antibody (alpha -B3). Error bars represent the standard deviation of triplicate assays.


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Fig. 6.   Effect of inhibition of PI 3-kinase activity on heregulin-stimulated [3H]thymidine uptake. Serum-starved cells transfected with pcDNA3, pcDNA3-B3-Y/F, or pcDNA3-B3-WT expression vectors were incubated with wortmannin (Wort; 100 nM) or vehicle for 30 min prior to stimulation with either vehicle or heregulin (10 nM) for 18 h. [3H]Thymidine incorporation assays were performed as indicated in Fig. 5. Heregulin-stimulated [3H]thymidine incorporation (Delta [3H]thymidine incorporation) was determined in triplicate assays by subtracting basal incorporation from that stimulated by 10 nM heregulin.

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).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 right-arrow 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 right-arrow 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 (Delta [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.

    FOOTNOTES

* 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.

Dagger 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 right-arrow Phe amino acid substitutionYXXM, Tyr-Xaa-Xaa-Met sequence motif.
    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Fantl, W. J., Johnson, D. E., and Williams, L. T. (1993) Annu. Rev. Biochem. 62, 453-481[CrossRef][Medline] [Order article via Infotrieve]
  2. Holmes, W. E., Sliwkowski, M. X., Akita, R. W., Henzel, W. J., Lee, J., Park, J. W., Yansura, D., Abadi, N., Raab, H., Lewis, G. D., Shepard, H. M., Kuang, W. J., Wood, W. I., Goeddel, D. V., and Vandlen, R. L. (1992) Science 256, 1205-1210[Medline] [Order article via Infotrieve]
  3. Wen, D., Peles, E., Cupples, R., Suggs, S. V., Bacus, S. S., Luo, Y., Trail, G., Hu, S., Silbiger, S. M., Levy, R. B., Koski, R. A., Lu, H. S., and Yarden, Y. (1992) Cell 69, 559-572[Medline] [Order article via Infotrieve]
  4. Guy, P. M., Platko, J. V., Cantley, L. C., Cerione, R. A., and Carraway, K. L., III (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8132-8136[Abstract]
  5. Sierke, S. L., Cheng, K., Kim, H.-H., and Koland, J. G. (1997) Biochem. J. 322, 757-763[Medline] [Order article via Infotrieve]
  6. Carraway, K. L., III, and Cantley, L. C. (1994) Cell 78, 5-8[Medline] [Order article via Infotrieve]
  7. Pinkas-Kramarski, R., Soussan, L., Waterman, H., Levkowitz, G., Alroy, I., Klapper, L., Lavi, S., Seger, R., Ratzkin, B. J., Sela, M., and Yarden, Y. (1996) EMBO J. 15, 2452-2467[Abstract]
  8. Kim, H.-H., Sierke, S. L., and Koland, J. G. (1994) J. Biol. Chem. 269, 24747-24755[Abstract/Free Full Text]
  9. Soltoff, S. P., Carraway, K. L., III, Prigent, S. A., Gullick, W. G., and Cantley, L. C. (1994) Mol. Cell. Biol. 14, 3550-3558[Abstract]
  10. Sliwkowski, M. X., Schaefer, G., Akita, R. W., Lofgren, J. A., Fitzpatrick, V. D., Nuijens, A., Fendly, B. M., Cerione, R. A., Vandlen, R. L., and Carraway, K. L., III (1994) J. Biol. Chem. 269, 14661-14665[Abstract/Free Full Text]
  11. Carraway, K. L., III, Soltoff, S. P., Diamonti, A. J., and Cantley, L. C. (1995) J. Biol. Chem. 270, 7111-7116[Abstract/Free Full Text]
  12. Kim, H.-H., Vijapurkar, U., Hellyer, N. J., Bravo, D., and Koland, J. G. (1998) Biochem. J. 334, 189-195[Medline] [Order article via Infotrieve]
  13. Plowman, G. D., Green, J. M., Culouscou, J. M., Carlton, G. W., Rothwell, V. M., and Buckley, S. (1993) Nature 366, 473-475[CrossRef][Medline] [Order article via Infotrieve]
  14. Tzahar, E., Levkowitz, G., Karunagaran, D., Yi, L., Peles, E., Lavi, S., Chang, D., Liu, N., Yayon, A., Wen, D., and Yarden, Y. (1994) J. Biol. Chem. 269, 25226-25233[Abstract/Free Full Text]
  15. Pinkas-Kramarski, R., Shelly, M., Glathe, S., Ratzkin, B. J., and Yarden, Y. (1996) J. Biol. Chem. 271, 19029-19032[Abstract/Free Full Text]
  16. Hellyer, N. J., Kim, H.-H., Greaves, C. H., Sierke, S. L., and Koland, J. G. (1995) Gene (Amst.) 165, 279-284[CrossRef][Medline] [Order article via Infotrieve]
  17. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778[Medline] [Order article via Infotrieve]
  18. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302[Medline] [Order article via Infotrieve]
  19. Laminet, A. A., Apell, G., Conroy, L., and Kavanaugh, W. M. (1996) J. Biol. Chem. 271, 264-269[Abstract/Free Full Text]
  20. Kavanaugh, W. M., Turck, C. W., and Williams, L. T. (1995) Science 268, 1177-1179[Medline] [Order article via Infotrieve]
  21. Blaikie, P., Immanuel, D., Wu, J., Li, N., Yajnik, V., and Margolis, B. (1994) J. Biol. Chem. 269, 32031-32034[Abstract/Free Full Text]
  22. Zhou, M.-M., Harlan, J. E., Wade, W. S., Crosby, S., Ravichandran, K. S., Burakoff, S. J., and Fesik, S. W. (1995) J. Biol. Chem. 270, 31119-31123[Abstract/Free Full Text]
  23. Batzer, A. G., Blaikie, P., Nelson, K., Schlessinger, J., and Margolis, B. (1995) Mol. Cell. Biol. 15, 4403-4409[Abstract]
  24. Okabayashi, Y., Kido, Y., Okutani, T., Sugimoto, Y., Sakaguchi, K., and Kasuga, M. (1994) J. Biol. Chem. 269, 18674-18678[Abstract/Free Full Text]
  25. Ricketts, W. A., Rose, D. W., Shoelson, S., and Olefsky, J. M. (1996) J. Biol. Chem. 271, 26165-26169[Abstract/Free Full Text]
  26. Gustafson, T. A., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J. (1995) Mol. Cell. Biol. 15, 2500-2508[Abstract]
  27. Batzer, A. G., Rotin, D., Ureña, J. M., Skolnik, E. Y., and Schlessinger, J. (1994) Mol. Cell. Biol. 14, 5192-5201[Abstract]
  28. Prigent, S. A., and Gullick, W. J. (1994) EMBO J. 13, 2831-2841[Abstract]
  29. Segatto, O., Pelicci, G., Giuli, S., Digiesi, G., Di Fiore, P. P., McGlade, J., Pawson, T., and Pelicci, P. G. (1993) Oncogene 8, 2105-2112[Medline] [Order article via Infotrieve]
  30. Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Li, W., Batzer, A., Thomas, S., Brugge, J., Pelicci, P. G., Schlessinger, J., and Pawson, T. (1992) Nature 360, 689-692[CrossRef][Medline] [Order article via Infotrieve]
  31. Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T., and Bowtell, D. (1993) Nature 363, 83-85[CrossRef][Medline] [Order article via Infotrieve]
  32. Chardin, P., Camonis, J. H., Gale, N. W., Van Aelst, L., Schlessinger, J., Wigler, M. H., and Bar-Sagi, D. (1993) Science 260, 1338-1343[Medline] [Order article via Infotrieve]
  33. Egan, S. E., Giddings, B. W., Brooks, M. W., Buday, L., Sizeland, A. M., and Weinberg, R. A. (1993) Nature 363, 45-51[CrossRef][Medline] [Order article via Infotrieve]
  34. de Vries-Smits, A. M. M., Burgering, B. M. T., Leevers, S. J., Marshall, C. J., and Bos, J. L. (1992) Nature 357, 602-604[CrossRef][Medline] [Order article via Infotrieve]
  35. Robbins, D. J., Zhen, E., Cheng, M., Xu, S., Ebert, D., and Cobb, M. H. (1994) Adv. Cancer Res. 63, 93-116[Medline] [Order article via Infotrieve]
  36. Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Grignani, F., Pawson, T., and Pelicci, P. G. (1992) Cell 70, 93-104[Medline] [Order article via Infotrieve]
  37. Yokote, K., Mori, S., Hansen, K., McGlade, J., Pawson, T., Heldin, C.-H., and Claesson-Welsh, L. (1994) J. Biol. Chem. 269, 15337-15343[Abstract/Free Full Text]
  38. Obermeier, A., Lammers, R., Weismuller, K.-H., Jung, G., Schlessinger, J., and Ullrich, A. (1993) J. Biol. Chem. 268, 22963-22966[Abstract/Free Full Text]
  39. Pronk, G. J., McGlade, J., Pelicci, G., Pawson, T., and Bos, J. L. (1993) J. Biol. Chem. 268, 5748-5753[Abstract/Free Full Text]
  40. Graus-Porta, D., Beerli, R. R., and Hynes, N. E. (1995) Mol. Cell. Biol. 15, 1182-1191[Abstract]
  41. Koland, J. G., O'Brien, K. M., and Cerione, R. A. (1990) Biochem. Biophys. Res. Commun. 166, 90-100[Medline] [Order article via Infotrieve]
  42. Posada, J., and Cooper, J. A. (1992) Science 255, 212-215[Medline] [Order article via Infotrieve]
  43. Horan, T., Wen, J., Arakawa, T., Liu, N., Brankow, D., Hu, S., Ratzkin, B., and Philo, J. S. (1995) J. Biol. Chem. 270, 24604-24608[Abstract/Free Full Text]
  44. Culouscou, J.-M., Carlton, G. W., and Aruffo, A. (1995) J. Biol. Chem. 270, 12857-12863[Abstract/Free Full Text]
  45. Dankort, D. L., Wang, Z., Blackmore, V., Moran, M. F., and Muller, W. J. (1997) Mol. Cell. Biol. 17, 5410-25[Abstract]
  46. Koland, J. G., and Cerione, R. A. (1988) J. Biol. Chem. 263, 2230-2237[Abstract/Free Full Text]
  47. Mohammadi, M., Schlessinger, J., and Hubbard, S. R. (1996) Cell 86, 577-587[Medline] [Order article via Infotrieve]
  48. Gamett, D. C., Pearson, G., Cerione, R. A., and Friedberg, I. (1997) J. Biol. Chem. 272, 12052-12056[Abstract/Free Full Text]
  49. Marchionni, M. A., Goodearl, A. D. J., Chen, M. S., Bermingham-McDonogh, O., Kirk, C., Hendricks, M., Danehy, F., Misumi, D., Sudhalter, J., Kobayashi, K., Wroblewski, D., Lynch, C., Baldassare, M., Hiles, I., Davis, J. B., Hsuan, J. J., Totty, N. F., Otsu, M., McBurney, R. N., Waterfield, M. D., Stroobant, P., and Gwynne, D. (1993) Nature 362, 312-318[CrossRef][Medline] [Order article via Infotrieve]


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