©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tyrosine Phosphorylation of the c-cbl Proto-oncogene Protein Product and Association with Epidermal Growth Factor (EGF) Receptor upon EGF Stimulation (*)

(Received for publication, May 25, 1995; and in revised form, July 7, 1995)

Maria L. Galisteo (1)(§) Ivan Dikic (1) Andreas G. Batzer (1) Wallace Y. Langdon (2) Joseph Schlessinger (1)(¶)

From the  (1)Department of Pharmacology, New York University Medical Center, New York, New York 10016 and the (2)Department of Biochemistry, University of Western Australia, Nedlands, Western Australia 6009, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p120 becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120 homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120 is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.


INTRODUCTION

The cbl oncogene was identified as the transforming gene of the Cas NS-1 murine leukemia virus(1) . The transforming product of this virus is a Gag-v-Cbl fusion protein in which 40 kDa are encoded by v-cbl. Cloning of the mouse and human c-cbl proto-oncogenes (2) revealed that v-cbl is a truncated form of c-cbl, encoding only 355 NH(2)-terminal amino acids. The primary sequence of p120 contains an NH(2)-terminal domain with a putative nuclear localization signal, followed by a RING zinc finger motif. The COOH-terminal half of the protein contains multiple proline-rich stretches that may function as ligands for SH3 domains. Both the RING zinc finger and the proline-rich domain are absent in v-Cbl. Whereas v-Cbl could be found both in the cytoplasm and nucleus of cells infected with the Cas NS-1 retrovirus, p120is exclusively cytosolic(3) .

Recently, p120 was found to be tyrosine-phosphorylated upon activation of the T cell receptor, and an in vitro interaction with the adaptor protein Grb2, independent of T cell receptor activation, has been reported(4) . In addition, an interaction between the SH3 domains of the adaptor protein Nck and the proline-rich domain of p120 has been reported(5) . Detailed analysis of the primary structure of p120 revealed the presence of the motif PPVPPR, which is identical to the Grb2 binding site in the Ras guanine nucleotide releasing factor ``Sos''(6) . This observation prompted us to examine in detail a possible interaction between p120 and Grb2 in the context of the Ras signaling pathway initiated by the epidermal growth factor receptor (EGFR). (^1)In this report we demonstrate that p120 becomes strongly tyrosine-phosphorylated upon stimulation of EGFR, platelet-derived growth factor (PDGF), nerve growth factor (NGF), and fibroblast growth factor (FGFR1) receptors. We also demonstrate that p120 associates with activated EGFR both in vivo and in vitro. These results suggest that p120 plays a role in an early step of the EGFR signaling pathway.


MATERIALS AND METHODS

Cell Lines

HER14 (expressing wild-type EGFR), Y5F (expressing an EGFR mutant with the five main tyrosine autophosphorylation sites mutated to phenylalanine), CD196 (expressing an EGFR deletion mutant lacking the COOH-terminal 196 amino acids), kinase-negative (K721A) cell lines, all derived from NIH 3T3, have been described previously(7, 8) . They were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. For starvation, these cells were incubated in DMEM alone (for the CD196 cell line) or DMEM supplemented with 0.1% calf serum for 20-24 h before lysis. PC12-derived cells (PC12-NGFR1 and PC12-FGFR1 cells) were grown in DMEM supplemented with 10% fetal bovine serum and 5% horse serum. These cells were starved for 40-48 h in DMEM supplemented with 0.1% fetal bovine serum and 0.1% horse serum.

Antibodies

Affinity-purified polyclonal C-15 antibodies (Santa Cruz Biotechnology) were used for p120immunoprecipitation, and monoclonal 108 (9) for EGFR immunoprecipitation. NGF receptor was immunoprecipitated using affinity purified anti-NGFR (TrkA) antibodies (Oncogene Science). FGFR1 was blotted with antibodies described in (10) . For immunoblotting of p120, R2 antibodies (3) were used. For Shc immunoblotting, polyclonal 410 antibodies (11) were used. For EGFR immunoblotting, RK2, alpha-C, or alpha-F (9) antibodies were used. Polyclonal antibodies number 72 were used to detect phosphotyrosine-containing proteins(12) . Antibody number 86 (13) was used for Grb2 immunoblotting.

Cell Lysis, Immunoprecipitation, and Western Blotting

Cells were serum-starved and then left untreated or stimulated for 5 min at 37 °C with EGF (Intergen, 100 ng/ml), NGF (Harlan Bioproduct for Science, 50 ng/ml), aFGF (100 ng/ml), and heparin (50 µg/ml)(14) , PDGF (Intergen, 50 ng/ml), or insulin (100 nM, Sigma). Cells were washed twice with ice-cold phosphate base saline and lysed in buffer containing phosphatase inhibitors as described(12) . For immunoprecipitation, pre-cleared lysates (3 mg of total protein) were incubated with antibodies for at least 90 min. Immunocomplexes were recovered on protein A-Sepharose 4B beads (Zymed), washed three times with lysis buffer and once with phosphate base saline. 2 Laemmli buffer was added, and the samples were boiled for 4 min, subjected to SDS-PAGE (9% in most cases), transferred to nitrocellulose membranes (Micron Separations Inc.), and immunoblotted according to previously published procedures(15) . Bound proteins were visualized by enhanced chemiluminescence (DuPont NEN).

Preparation of GST Fusion Proteins

A DNA fragment encoding amino acids 1-486, of human c-cbl was amplified by polymerase chain reaction and subcloned into the BamHI site in pGSTag (gift of D. Ron), in-frame with GSTag (``N-Cbl''). This protein contains a serine residue that can be phosphorylated using [P]ATP and protein kinase A(16) . C-Cbl, comprising amino acids 487-906 of p120, was constructed from a NcoI/PstI fragment (1278 base pairs) of human p120 cDNA which was filled in with Klenow and subcloned into the SmaI site of pGSTag. Escherichia coli cells were transformed, and fusion proteins were produced and affinity-purified on glutathione-Sepharose beads (17) . 15 µg of fusion protein were radiolabeled with 1 mCi of [-P]ATP (DuPont NEN) as described previously(16) .

Filter Binding Assay with P-Labeled Proteins

Upon SDS-PAGE and transfer to nitrocellulose, filters were blocked in block buffer (20 mM Hepes, pH 7.5, 5 mM MgCl(2), 1 mM KCl, 5 mM dithiothreitol, 5 mM NaF, 0.02% sodium azide, 5% non-fat dry milk) for 3 h at room temperature and then incubated overnight at 4 °C with a [P]-N-Cbl probe (specific activity of 9 10^6 cpm/ml in block buffer). The filter was extensively washed in Tris-buffered saline containing 0.1% Triton X-100 and exposed to film.


RESULTS AND DISCUSSION

We examined the possibility that p120 was involved in the EGFR-mediated signaling pathway. Untreated and EGF-stimulated HER14 cell lysates were immunoprecipitated with anti-p120antibodies, and tyrosine-phosphorylated proteins were detected with antibodies against phosphotyrosine (Fig. 1A). Two strongly tyrosine-phosphorylated proteins with apparent molecular masses of approximately 120 and 170 kDa, as well as a weak band of approximately 55 kDa, were detected in lysates from EGF-stimulated cells. Immunoblotting with anti-p120 antibodies revealed that the 120-kDa protein corresponds to p120 (Fig. 1B). Immunoblotting with anti-EGFR antibodies or with anti-Shc antibodies demonstrated that the 170- and 55-kDa polypeptides represent the EGFR and Shc protein, respectively (Fig. 1B). Immunoblotting with anti-Grb2 antibodies demonstrated that this protein was present in this complex (Fig. 1B). p120 could also be detected in activated EGFR immunoprecipitates (Fig. 1C). These results show that p120 associates in vivo with activated EGFR.


Figure 1: EGF-induced tyrosine phosphorylation and association of p120 with activated EGFR. A, lysates from untreated and EGF-stimulated HER14 cells were incubated with affinity-purified anti-p120 antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE on a 9% gel and analyzed by immunoblotting with anti-Tyr(P) antibodies. B, the blot in A was stripped and re-probed with anti-EGFR (alpha-C), anti-p120, anti-Shc, and anti-Grb2 antibodies. C, EGFR complexes from untreated and EGF-stimulated HER14 cell lysates were immunoprecipitated with anti-EGFR antibodies (RK2) and analyzed by immunoblotting with anti-EGFR and anti-p120antibodies. 70 µg of whole cell lysates were also loaded in C.



We next investigated whether p120 is phosphorylated upon stimulation with other growth factors. Addition of NGF or aFGF to PC12 cells induced tyrosine phosphorylation of p120 (Fig. 2, A and B). However, we could not detect association between p120 and the corresponding activated receptors. Immunoblotting with Grb2 or Shc antibodies failed to reveal the presence of these proteins in p120immunoprecipitates (not shown). PDGF also induced tyrosine phosphorylation of p120 (Fig. 2C), whereas insulin stimulation of CHO cells overexpressing the insulin receptor did not have any effect (Fig. 2D).


Figure 2: Tyrosine-phosphorylation of p120is induced upon stimulation with aFGF, NGF, and PDGF. A, PC12 cells were left untreated or treated with acidic FGF and heparin and lysates immunoprecipitated with anti-p120 (C-15) antibodies or anti-FGFR1 antibodies. B, PC12 cells were left untreated or stimulated with NGF and lysates immunoprecipitated with C-15 or anti-NGFR antibodies. C, HER14 cells were left untreated or stimulated with EGF or PDGF and lysates immunoprecipitated with C-15 antibodies. D, CHO-IR cells were left untreated or stimulated with insulin and lysates immunoprecipitated with anti-p120 antibodies. All immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with R2 and anti-phosphotyrosine antibodies. PC12 NGFR1 and PC12 FGFR1 express approximately 2.5 10^5 NGF receptors (TrkA)/cell and 2 10^5 FGF receptor/cell, respectively. CHO-IR cells express 1.2 10^6 insulin receptors/cell.



Several previously characterized EGFR mutants were used to dissect the interaction between p120 and the EGFR. Tyrosine phosphorylation of p120 was examined in NIH 3T3 cells expressing an EGFR mutant lacking the carboxyl-terminal tail (CD196) (7) , a kinase-negative receptor mutant (K721A)(8) , or a receptor lacking five tyrosine autophosphorylation sites (Y5F)(7) . Background levels of tyrosine-phosphorylated p120 were detected with the EGF-stimulated kinase negative mutant (Fig. 3). This indicates that EGFR tyrosine kinase activity is essential for p120 phosphorylation, either by directly phosphorylating p120 or by activating other protein tyrosine kinases. In the CD196 and Y5F mutants, p120 was still phosphorylated upon growth factor stimulation, although to a lesser extent as compared with wild-type EGFR (Fig. 3). Thus, the tyrosine-phosphorylated EGFR tail facilitates p120 phosphorylation, although association with the EGFR tail is not essential for EGF-induced tyrosine phosphorylation of p120. Immunoblotting with anti-Grb2 antibodies showed the absence of this protein in p120immunoprecipitates from any of the mutant cell lines (not shown).


Figure 3: Effect of EGFR mutants on tyrosine phosphorylation of p120. Lysates derived from the indicated cell lines, either unstimulated or EGF-stimulated, were incubated with affinity-purified anti-p120 (C-15) antibodies. Immunocomplexes were analyzed by SDS-PAGE and immunoblotted with anti-p120 (R2) or anti-Tyr(P) antibodies.



Interaction between p120 and activated EGFR must occur either directly or through an adaptor protein. We investigated the possibility of a direct interaction between p120 and EGFR using filter binding assays with GST-p120 fusion proteins. -P-Radiolabeled N-Cbl and C-Cbl, encompassing amino acids 1-486 and 487-906 of human p120, respectively, were used to probe filters containing immunoprecipitates of EGFR from untreated or EGF-stimulated HER14, CD196, or Y5F cell lysates. In this assay, N-Cbl showed strong binding to tyrosine-autophosphorylated wild-type EGFR (Fig. 4A). The probe also bound, although more weakly, to activated Y5F mutant receptor, which becomes phosphorylated on several new tyrosine residues upon activation (7) . No binding to CD196 EGFR deletion mutant was detected. We also examined the binding of N-Cbl to a purified cytoplasmic domain of the receptor (EGFR-1C)(18) , which had been incubated with or without ATP, rendering EGFR-1C heavily phosphorylated or unphosphorylated, respectively. As expected, N-Cbl bound to the phosphorylated form of EGFR-1C (Fig. 4A). These data show that p120 is able to bind directly to activated EGFR in vitro. N-Cbl was not tyrosine-phosphorylated, thereby demonstrating that tyrosine-phosphorylation of p120 is not essential for receptor binding. In contrast, a -[P]fusion protein of the same specific activity containing amino acid residues 487-906 (C-Cbl) did not bind to activated EGFR or any other proteins in the same assay (not shown).


Figure 4: A p120 deletion mutant (N-Cbl) binds to tyrosine-autophosphorylated EGFR in vitro. Untreated and EGF-stimulated cell lysates (HER14, CD196, and Y5F) were incubated with monoclonal anti-EGFR antibodies. EGFR-1C is a purified protein that contains the entire cytoplasmic domain of EGFR. 1 µg of EGFR-1C was incubated with or without 1 mM ATP in 50 µl of Hepes, pH 7.5, for 10 min at room temperature, and the autophosphorylation reaction was stopped by addition of 2 Laemmli buffer. 70 ng of each EGFR-1C sample, half of each HER14 immunoprecipitate, half of each CD196 immunoprecipitate, and one-fourth of each Y5F immunoprecipitate were electrophoresed through two equivalent 9% acrylamide gels and transferred to nitrocellulose. One filter was probed with -[P]N-Cbl (A), stripped, and re-probed with anti-EGFR (alpha-F) antibodies (C). The second filter was immunoblotted with anti-Tyr(P) antibodies (B).



In each of the EGFR mutants, the extent of binding of N-Cbl to EGFR correlated with tyrosine autophosphorylation of the receptor (Fig. 4). This suggests that N-Cbl binds to the phosphotyrosine-containing region of EGFR, similar to binding of SH2 domains(19) . However, p120 has no SH2 domains. It has been recently reported that a domain in the NH(2) terminus of Shc is able to bind directly to phosphotyrosine containing sequences in EGFR (20, 21) and NGFR(22) , but a similar sequence does not exist in p120. It is noteworthy that the amino terminus half of p120, which is the region responsible for binding directly to activated EGFR, is highly conserved between p120 and its Caenorhabditis elegans homologue, Sli-1(23) . Since most of this region is present in the oncogenic v-Cbl protein, our findings raise the possibility that v-Cbl might interact with EGFR in vivo. On the other hand, the carboxyl terminus half of p120, which contains a number of proline-rich sequences that may serve as SH3 binding sites(24) , is mostly absent in Sli-1(23) .

Both the amino-terminal region (1-486) and the proline-rich region (487-906) of p120 were able to form a complex with activated EGFR when incubated with EGF-stimulated HER14 lysates (not shown). This raises the possibility that, in living cells, p120 can bind to activated EGFR directly and indirectly. We have previously shown that Grb2 can bind both directly and indirectly (via Shc) to the EGFR (12) and directly (25) and indirectly (via PTP1D) to the PDGF receptor(26) . By analogy with other cases, it is possible that complex formation between the proline-rich region of p120 and activated EGFR is mediated by a protein that contains both SH2 and SH3 domains. The SH2 domain could mediate interaction with activated EGFR, and the SH3 domain may bind to proline-rich sequences of p120. It was recently reported that p120 can interact with the adaptor proteins Grb2 (4) and Nck(5) . We analyzed the sequence of p120 for possible Grb2 SH2 binding sites, but no sequences corresponding to the consensus for this binding were found. Structural and functional studies on SH3 domains have allowed the identification of proline-rich sequences as SH3 domain ligands(24) . p120 contains a number of proline-rich sequences that fit this consensus. One sequence in particular, amino acids 494-499 (PPVPPR), is identical to a known Grb2 binding site in the Sos protein(6) , although flanking residues, which also play a role in binding to the SH3 domain, are different. We have shown that Grb2 and Shc were present in p120immunoprecipitates from EGF-stimulated HER14 cells. However, little or no Grb2 and Shc was present in p120 immunoprecipitates of lysates from NIH 3T3 cells overexpressing CD196, Y5F, and K721A EGFR mutants or from PC12 cells overexpressing NGF or aFGF receptors (not shown). Morever, co-immunoprecipitation of p120 with either Grb2 or Shc was detected only upon EGFR activation and autophosphorylation. These data argue against a direct interaction in vivo between p120 and Grb2 or Shc. Their presence in p120 immunoprecipitates is probably due to tertiary complex formation with activated EGFR molecules, rather than with p120. Attempts to identify a putative adaptor protein which would interact with EGFR and p120in HER14 cells, by means of co-immunoprecipitation experiments, were unsuccesful for Nck, the p85 subunit of PI3-kinase, Crk, p120, and PLC.

Recent genetic experiments (23) implicate the protein product of sli-1, a C. elegans homologue of the mammalian p120, as a negative regulator of the vulval induction pathway initiated by Let-23, the C. elegans homologue of the mammalian EGFR(27) . Genetic studies have shown that mutations in sli-1 can rescue weak loss-of-function alleles of let-23, sem-5, and, partially, let-60 and suggest that Sli-1 may act as a negative regulator of the vulval induction pathway at the Let-23/Sem-5 step (23) . The studies presented here show that p120 is associated with activated EGFR and becomes phosphorylated on tyrosine residues in response to EGF, FGF, PDGF, and NGF stimulation. By analogy with Sli-1 modulating Let-23 function, p120 may negatively regulate EGFR signaling, for example by directly blocking binding sites of adaptor proteins in the receptor. Elucidation of the exact role of p120 in signaling via receptor tyrosine kinases will provide further information about the network of interactions that control this important signal transduction pathway.


FOOTNOTES

*
This work was supported by grants from Sugen Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
On leave from the Department of Physical Chemistry, University of Granada (Spain) and was supported in part by a grant from the Direccion General de Investigacion Cientifica y Tecnica (DGICYT), Spanish Ministry of Education and Science.

To whom correspondence should be addressed. Tel.: 212-263-7111; Fax: 212-263-7133.

(^1)
The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; aFGF, acidic fibroblast growth factor; FGFR, fibroblast growth factor receptor; NGF, nerve growth factor; NGFR, nerve growth factor receptor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; SH3, Src homology 3; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We thank P. Sternberg for sharing unpublished results, N. Li for providing the CD196 and Y5F cell lines, M. Mohammadi for EGFR-1C, T. Spivak for recombinant aFGF, and K. Nelson for oligonucleotide synthesis. We thank Drs. M. Lemmon and C. Gordon for useful comments on the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.