©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Requirements of the Epidermal Growth Factor Receptor for Tyrosine Phosphorylation of eps8 and eps15, Substrates Lacking Src SH2 Homology Domains (*)

Clara V. Alvarez (1)(§), Kjoung-Jin Shon (1)(¶), Mariarosaria Miloso (1)(**), Laura Beguinot (1) (2)(§§)

From the (1)Laboratory of Molecular Oncology, Dipartimento di Ricerca Biologica e Tecnologica, and the (2)Istituto di Neuroscienze e Bioimmagini del CNR, HS Raffaele, Via Olgettina 60, 20132 Milano, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phosphorylation of two newly identified epidermal growth factor (EGF) receptor substrates, eps8 and eps15, which do not possess Src homology (SH2) domains, was investigated using EGF receptor mutants of the autophosphorylation sites and deletion mutants of the carboxyl-terminal region. Two mutants, F5, in which all five tyrosine autophosphorylation sites substituted by phenylalanine, and Dc 123F, in which four tyrosines were removed by deletion and the fifth (Tyr-992) was mutated into phenylalanine, phosphorylated eps8 and eps15 as efficiently as the wild-type receptor. In contrast, SH2-containing substrates, phospholipase C, the GTPase-activating protein of Ras, the p85 subunit of phosphatidylinositol 3 kinase, and the Src and collagen homology protein, are not phosphorylated by the F5 and Dc 123F mutants. A longer EGF receptor deletion mutant, Dc 214, lacking all five autophosphorylation sites, was unable to phosphorylate eps15 but phosphorylated eps8 13-fold more than the wild-type receptor. To determine the EGF receptor region important for phosphorylation of eps8 and eps15, progressive deletion mutants lacking the final 123, 165, 196, and 214 COOH-terminal residues were used. eps8 phosphorylation was progressively increased in Dc 165, Dc 196, and Dc 214 EGF receptor mutants, indicating that removal of the final 214 COOH-terminal residues increases the phosphorylation of this substrate by the EGF receptor. In contrast, eps15 was phosphorylated by Dc 123 and Dc 165 EGF receptor mutants but not by Dc 196 and Dc 214 mutants. This indicates that a region of 30 residues located between Dc 165 and Dc 196 is essential for eps15 phosphorylation. This is the first demonstration of structural requirements in the EGF receptor COOH terminus for efficient phosphorylation of non-SH2-containing substrates. In addition, enhanced eps8 phosphorylation correlates well with the increased transforming potential of EGF receptor deletion mutants Dc 196 and Dc 214, suggesting that this substrate may be involved in mitogenic signaling.


INTRODUCTION

The receptor for epidermal growth factor (EGF-R),()a 175-kDa transmembrane glycoprotein, is a member of the protein-tyrosine kinase family(1, 2) . EGF binding triggers the activation of receptor tyrosine kinase activity, which is essential to induce all responses to EGF(3, 4) . Receptor tyrosine kinase activity leads to autophosphorylation and to tyrosine phosphorylation of specific cellular substrates(1, 2) . Autophosphorylation regulates the biological activity of the EGF-R by influencing receptor kinase activity (5, 6) and, by creating binding sites for substrates. A number of these substrates, such as phospholipase C (PLC), GTPase-activating protein of Ras (GAP), the regulatory subunit of phosphatidylinositol 3 kinase (p85), Src and collagen homology protein (SHC) (for review see Refs. 1 and 7), phosphotyrosine phosphatase Syp (9) and D1(10) , contain sequence motifs called Src homology (SH2) domains.

In some instances, association of an SH2-containing substrate (e.g. PLC) with activated growth factor receptors is considered essential for tyrosine phosphorylation and activation(7) . Mutation of single autophosphorylation sites in the platelet-derived growth factor(11, 12, 13, 14) , the colony-stimulating factor(15) , the fibroblast growth factor(16, 17) , and the nerve growth factor(18, 19) receptors prevents association and tyrosine phosphorylation of the various substrates. In these examples, specificity depends on the sequence motifs surrounding each autophosphorylation site and on the structure of individual SH2 domain. Data with the EGF receptor, however, indicate that autophosphorylation sites can compensate for each other and do not stringently define association motifs for SH2 substrates(20, 21, 22, 23, 24) . The COOH terminus of the EGF-R contains all five autophosphorylation sites (Tyr-1173, Tyr-1148, Tyr-1086, Tyr-1068, and Tyr-992), and substitution or removal of all five is required to completely abolish association and/or phosphorylation of PLC, GAP, and the p85 subunit of phosphatidylinositol 3 kinase(20, 21, 22) . The requirement for SHC and Grb-2 interaction is more stringent because mutation of both Tyr-1173 and Tyr-1148 reduces the binding of SHC to very low levels(22, 23) . Mutation of Tyr-1068, Tyr-1086, and Tyr-1148 abolishes interaction with Grb-2(24) .()

Recently, novel non-SH2-containing substrates of the EGF receptor have been identified (25) and termed EGF receptor pathway substrates (eps) clone 8(26) , clone 15(27) , and ezrin(28) . Overexpression of eps15 by itself is sufficient to induce transformation of NIH 3T3(27) , and overexpression of eps8 potentiates the mitogenic activity of the EGF receptor(26) , suggesting that both proteins are involved in receptor mitogenic pathway. These substrates do not possess typical SH2 motifs, and only eps8 has been shown to be a direct substrate of activated EGF receptor by in vitro analysis with purified receptor (26). However, eps15 seems to be specific to the EGF receptor pathway, because its phosphorylation is not induced by the ErbB-2 kinase(27) , whereas eps8 and ezrin are phosphorylated by several growth factor receptors(26, 27, 34) .

Because eps8 and eps15 do not possess SH2 domains, it is important to establish how they are phosphorylated by the EGF receptor and what region in the receptor is important to allow their phosphorylation. In this study we have examined the requirement of the receptor COOH terminus and autophosphorylation sites for eps8 and eps15 phosphorylation.


EXPERIMENTAL PROCEDURES

Materials

EGF was from Promega, and I-EGF and I-protein A were from Amersham Corp. Aprotinin, phenylmethylsulfonyl fluoride, and polylysine were from Sigma. Protein G-Sepharose was from Pharmacia. Nitrocellulose membranes were from Schleicher & Schuell. G418, transferrin, cell culture medium, and serum were from Life Technologies, Inc. Monoclonal antibodies against phosphotyrosine and the extracellular domain of the EGF-R were from Upstate Biotechnology Incorporated. Polyclonal antibodies against eps8, eps15, and PLC were kindly provided by Dr. Francesca Fazioli (Dipartimento di Ricerca Biologica e Tecnologica, Milano) and Graham Carpenter (Vanderbilt University, Nashville, TN), respectively, and were previously described(21, 26, 27) .

EGF Receptor Mutants and Cell Culture

The human EGF-R mutant F5 was obtained by site-directed mutagenesis of single tyrosine residues with phenylalanine as described previously(21) . Dc 123F was obtained by deletion of the last 123 amino acids and site-directed mutagenesis of Tyr-992 with phenylalanine(20) , and Dc 214 was obtained by deletion of the final 214 residues(20) . Mutants Dc 196 and Dc 165 were obtained by deletion of the last 196 and 165 carboxyl-terminal residues by site-directed mutagenesis of residue 992 or 1021, respectively, into stop codons using the following oligonucleotides: 5`-GATGCCGACGAGTAGTACTTCCCACAGCAG-3` and 5`-CACCCCAGCAACAATATAGACCGTGTCCTTGCAT-3`. Mutagenesis was performed in M13mp18 encoding the AccII-HincI EGF-R cDNA fragment(3113-3625). A single-stranded template was prepared, and mutagenesis was performed as described by Taylor et al.(32) and confirmed by dideoxy sequencing(33) . The mutated fragments were cloned back into the pMMTV-EGF-R vector, and the mutated EGF-R cDNAs (SacII-XhoI) were subcloned into the pCO11 vector (5) to produce pDc 165 and pDc 196.

NIH 3T3 cells, which contain about 3,000 receptors/cell, were used for transfections. Transfections, G418 selection, foci formations, and growth in agar were performed as described previously(5) . As determined by I-EGF binding and Scatchard analysis, most mutant receptors were expressed at 3-5 10 receptors/cell, whereas F5 mutant had about 2 10 receptor/cell(5) . As control, a NIH 3T3 line (Cl 17) expressing 4 10 human wild-type EGF-R/cell was used(29) . Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% newborn calf serum with penicillin, streptomycin, and glutamine.

Growth Factor Treatment and Cell Lysate Preparation

Transfected cells were grown to about 90% confluence in 150-mm polylysine-coated dishes in DMEM containing 10% newborn calf serum. Subsequently cells were incubated for 16 h in DMEM containing 0.5% newborn calf serum, 5 µg/ml transferrin, and 10M sodium selenite. Experiments were initiated with or without 100 ng/ml EGF for 60 min at 4 °C in DMEM supplemented with 20 mM Hepes, pH. 7.4, and 0.1% bovine serum albumin. The capacity of the EGF-R to autophosphorylate, phosphorylate, and associate with cellular substrates is not altered at 4 °C(21, 22) , and preliminary experiments showed that the maximal level of phosphorylation was achieved under these conditions. Identical results were obtained when lysates were prepared after cell incubation with EGF at 37 °C for 10 min. Lysates were prepared by washing the monolayers with ice-cold phosphate-buffered saline and scraping the cells in ice-cold lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 20 µM Na pyrophosphate, 10 mM orthovanadate, 4 µM phenylmethylsulfonyl fluoride, and 1 mg/ml aprotinin). Lysates were centrifuged at 14,000 g for 15 min at 4° C. Total proteins were measured by the Bio-Rad method.

Immunoprecipitation and Western Blot Analysis

Total cellular lysates (3.5 mg of proteins) were immunoprecipitated with agarose-conjugated anti-Tyr(P) or with protein G-Sepharose coupled with eps8 and eps15 antibodies. The immunoprecipitates were washed five times with HNTG buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100) and boiled in Laemmli buffer. Samples were run on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were then incubated for 2 h with primary antibodies (anti-eps8, anti-eps15, or anti-Tyr(P) Ab), extensively washed and incubated with 0.2 µCi/ml I-protein A, washed extensively, and exposed for autoradiography. Specific bands were quantitated using a PhosphorImager (Molecular Dynamics). Quantitation of phosphotyrosine recovered from eps8 and eps15 was performed as described previously(21) . To examine tyrosine phosphorylation of total cellular proteins in response to EGF, an aliquot of cell lysates (50-100 µg) was mixed with 2 Laemmli buffer, loaded on a 7.5% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and blotted with anti-Tyr(P) Ab.

In Vitro Kinase Activity

The EGF receptor was immunoprecipitated from cellular extracts, as described above. Immunoprecipitated receptors were incubated with 100 ng/ml EGF for 10 min at room temperature and then incubated for 2 min at 4° C with 15 mM MgCl, 20 mM MnCl, 10 µCi of [-P]ATP and various concentrations of a dodecapeptide corresponding to the sequence surrounding Tyr-1173, ranging from 0.0065 to 0.65 µM or various concentrations of ATP from 0.003 to 100 µM. The peptide NH-AENAEYLRVAPQ-COOH was kindly provided by Dr. Ettore Appella (Laboratory of Cellular Biology, NIH). Reactions were terminated by the addition of 7.5 µl of 5 SDS Laemmli buffer. A 4-µl aliquot of the samples was then loaded on high density SDS-polyacrylamide gels, run with the Phast system, dried, and exposed at -80° C for autoradiogaphy. Quantitation was performed using a PhosphorImager.


RESULTS AND DISCUSSION

To determine whether tyrosine phosphorylation of the EGF receptor is necessary for phosphorylation of eps8 and eps15 proteins, NIH 3T3 cells expressing wild-type or mutant receptors were used. Fig. 1A schematically represents the EGF receptor mutants used in this study. In the F5 mutant, all five autophosphorylation sites were mutated into phenylalanine, whereas in the Dc 123F, four autophosphorylation sites were removed by deletion, and the fifth site, Tyr-992, was mutated to phenylalanine(20, 21) . In addition, we used mutants with progressive deletions of the COOH terminus, creating receptors lacking the final 123, 165, 196, and 214 COOH-terminal amino acids(20, 21) . Clones of NIH 3T3 cells expressing EGF receptor mutants were chosen to express comparable levels of receptor, as shown in Fig. 1B and confirmed by I-EGF binding (3-5 10 receptors/cell) (data not shown). The F5 mutant receptor was expressed at a slightly lower level (about 50%) than wild-type receptor.


Figure 1: Schematic representation and EGF receptor levels of EGF COOH-terminal receptor mutants. A, the COOH-terminal domain of the EGF receptor is shown with the five autophosphorylation sites, Tyr-992, Tyr-1068, Tyr-1086, Tyr-1148, and Tyr-1173. F5 has all five tyrosines mutated to phenylalanines (F). Dc 123F has a deletion of the 123 COOH-terminal amino acids and substitution of Tyr-992 with phenylalanine. Dc 165, Dc 196, and Dc 214 have a deletion of the COOH-terminal 165, 196, and 214 amino acids, respectively. F5, Dc 123F, and Dc 214 were previously published (20, 21). The numbers of EGF binding sites were calculated from Scatchard plot analysis of I-EGF binding data (5). Y, Tyr. B, equal amounts of total lysates (150 µg of proteins) from NIH 3T3 expressing wild-type (WT) EGF-R (Cl 17, 4 10 EGF-R/cell, lane 5) and mutant cells, F5 (lane 1), Dc 123F (lane 2), Dc 165 (lane 3), Dc 196 (lane 4), and Dc 214 (lane 6) were run on a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose, and anti-EGF-R mAb was used to detect the EGF-R. Exposure of the autoradiogram was for 15 h.



Phosphorylation of eps15 and eps15 by EGF Receptors Lacking the Autophosphorylation Sites

To study eps8 and eps15 phosphorylation, lysates of EGF-stimulated cells were immunoprecipitated with antiphosphotyrosine antibodies and analyzed for the presence of eps8 and eps15 by Western blotting. As previously observed, eps8 and eps15 were phosphorylated only in EGF-stimulated receptor transfected cells, and no phosphorylation was detected in EGF-treated, untransfected NIH 3T3 cells (data not shown; Refs. 26 and 27). A long EGF-R deletion mutant lacking almost the entire COOH terminus was investigated in order to address the role of the COOH terminus in eps8 and eps15 phosphorylation. As shown in Fig. 2, eps8 phosphorylation was dramatically increased in cells expressing the Dc 214 mutant, whereas eps15 phosphorylation was almost abolished (Fig. 2A, lane 6). Quantitation indicates that phosphorylation by Dc 214 was increased 13-fold for eps8 and decreased by 80% for eps15 (). eps8 appeared as two bands of 92 and 68 kDa, the latter representing a degradation product(26) . To test the role of the EGF receptor autophosphorylation sites in tyrosine phosphorylation of eps8 and eps15, the F5 receptor mutant was used. Fig. 2shows that phosphorylation of eps8 and eps15 was equally effective by the wild-type and F5 mutant receptors (Fig. 2A, lane 2). Quantitation of eps8 and eps15 phosphorylation by the F5 mutant and wild-type receptor normalized to the amount of receptor expressed indicated that eps8 was equally phosphorylated, whereas eps15 was slightly more phosphorylated (about 2-fold) by the F5 than by wild-type receptors. Control blots indicated that the levels of eps8 and eps15 were identical in all unstimulated and EGF-treated cells (Fig. 2B). Phosphorylation of eps8 (Fig. 3, lane 2) and eps15 (see Fig. 5, lane 2) by the Dc 123F mutant was similar to that of the wild-type receptor. eps15 appeared mainly as a single band of 142 kDa in unstimulated cells and as two bands of 142 and 155 kDa in EGF-treated cells, both of which contained phosphotyrosine. The appearance of the 155-kDa band as a predominant form in EGF-treated cells suggests that the more slowly migrating form is due to phosphorylation. eps15 has been reported to be phosphorylated on Tyr and Ser/Thr residues(25) . However, it is not known whether this slower migration is due to tyrosine phosphorylation or also to serine/threonine phosphorylation induced by EGF. In addition, identical results were obtained when eps8 and eps15 were first immunoprecipitated with their specific antibodies and then blotted with phosphotyrosine antibodies (data not shown). These data, therefore, indicate that the five EGF-R autophosphorylation sites are not necessary for efficient tyrosine phosphorylation of these two substrates that lack SH2 domains. These results contrast with the inability of the F5 and Dc 123F mutants to phosphorylate and/or associate with SH2-containing substrates, such as PLC, GAP, the p85 subunit of phosphatidylinositol 3 kinase, SHC, and Grb-2(20, 21, 22) . Indeed, control experiments showed that phosphorylation of PLC was undetectable with the F5 and Dc 123F mutants (data not shown), as described previously(21, 22) .


Figure 2: Phosphorylation of eps8 and eps15 substrates by F5 and Dc 214 EGF receptor mutants. A, cells were treated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) 100 ng/ml EGF for 60 min at 4 °C, and the lysates were prepared. Equal amounts of lysates (3 mg of proteins) from F5 (lanes 1 and 2), wild-type (lanes 3 and 4), and Dc 214 (lanes 5 and 6) expressing cells were immunoprecipitated with anti-Ptyr mAb, run on 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose filters, and then reacted with anti-eps8 Ab (left) or anti-eps15 Ab (right). Exposure of the blots was for 7 and 13 h, respectively. B, to compare levels of expression of eps8 and eps15, equal amounts of lysates (150 µg of proteins) were directly blotted with anti-eps8 Ab (left) or anti-eps15 Ab (right). Quantitation of the autoradiograms was performed with the PhosphorImager. Exposure of the blots was for 5 and 12 h, respectively. w.t., wild type.




Figure 3: Phosphorylation of eps8 by EGF receptor deletion mutants. A, cells were treated with (lanes 2, 4, 6, 8, and 10) or without (lanes 1, 3, 5, 7, and 9) 100 ng/ml EGF for 60 min at 4 °C, and the lysates were prepared. Equal amounts of lysates (3 mg of proteins) from Dc 123F (lanes 1 and 2), Dc 165 (lanes3 and 4), Dc 196 (lanes5 and 6), wild-type (lanes 7 and 8), and Dc 214 (lanes 9 and 10) expressing cells were immunoprecipitated with anti-Ptyr mAb, run on 10% SDS-polyacrylamide gels, transferred to nitrocellulose filters, and then reacted with anti-eps8 Ab. Exposure of the autoradiogram was for 18 h. B, to compare levels of expression of eps8, equal amounts of lysates (150 µg of proteins) were directly blotted with anti-eps8 Ab (lanes 1-10). Exposure of the autoradiogram was for 20 h. w.t., wild type.




Figure 5: Phosphorylation of eps15 by EGF receptor deletion mutants. A, cells were treated with (lanes 2, 4, 6, 8, and 10) or without (lanes 1, 3, 5, 7, and 9) 100 ng/ml EGF for 60 min at 4 °C, and the lysates were prepared. Equal amounts of lysates (3 mg of proteins) from Dc 123F (lanes 1 and 2), Dc 165 (lanes 3 and 4), Dc 196 (lanes 5 and 6), wild-type (lanes 7 and 8), and Dc 214 (lanes 9 and 10) expressing cells were immunoprecipitated with anti-Ptyr mAb, run on 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose filters, and reacted with anti-eps15 Ab. Exposure of the autoradiogram was for 24 h. B, to compare levels of expression of eps15, equal amounts of lysates (150 µg of proteins) were directly blotted with anti-eps15 Ab (lanes 1-10). Exposure of the autoradiogram was for 22 h.



Phosphorylation of eps8 by EGF Receptor Deletion Mutants

Shorter EGF receptor deletion mutants were used to better define the region of the EGF-R carboxyl terminus responsible for modulating eps8 and eps15 phosphorylation. As shown in Fig. 3, eps8 was similarly phosphorylated by the wild-type, Dc 123F, and Dc 165 receptors. However, its phosphorylation was greatly increased by the Dc 196 and Dc 214 mutants. Quantitation relative to wild-type phosphorylation indicated that the Dc 196 and Dc 214 mutants had 9- and 13-fold increases, respectively, in eps8 phosphorylation (Tables I and II).

The behavior of eps8 phosphorylation resembles that of two other EGF receptor SH2 substrates, GAP (21, 22) and SCH(22, 23) , which can be efficiently phosphorylated by the Dc 214 mutant, although they are unable to form an association complex with the mutant receptor. In contrast to GAP and SHC, however, Dc 214 and Dc 196 phosphorylated eps8 to a much greater extent than wild-type receptor, possibly because of the reduced interaction and phosphorylation of some of the other SH2-containing substrates, like PLC, p85, and Grb-2.

Co-immunoprecipitation experiments, performed by immunoprecipitation with EGF-R antibodies and Western blotting with eps8 antibodies, indicated that, although very little association could be detected with the wild-type receptor, the Dc 196 and Dc 214 receptor mutants were able to associate directly with eps8, mostly with the 68-kDa form, (Fig. 4). In addition, eps8 associated constitutively with the Dc 196 and Dc 214 mutant, whereas, under the same conditions, association with the wild-type receptor could be detected only in the presence of EGF.


Figure 4: Co-immunoprecipitation of EGF receptor Dc 196 and Dc 214 mutants with eps8.A, cells were treated with (lane 2, 4, and 6) or without (lanes 1, 3, and 5) 100 ng/ml EGF for 60 min at 4 °C, and the lysates were prepared. Equal amounts of lysates (5 mg of proteins) from wild-type (lanes 1 and 2), Dc 196 (lanes 3 and 4), and Dc 214 (lanes 5 and 6) expressing cells were immunoprecipitated with anti-EGF-R mAb, run on 10% SDS-polyacrylamide gels, transferred to nitrocellulose filters, and then reacted with anti-eps8 Ab. Exposure of the autoradiogram was for 72 h. eps8 indicates the 96- and 68-kDa forms of eps8. B, to compare levels of expression of eps8, equal amounts of lysates (150 µg of proteins) were directly blotted with anti-eps8 Ab (lanes 1-6). Exposure of the autoradiogram was for 20 h. eps8 indicates the 96- and 68-kDa forms of eps8. C, to compare levels of EGF receptor immunoprecipitated, 1/40 of the immunoprecipitation (lanes 3-6) and 1/20 (lane 1 and 2) were directly blotted with anti-EGF-R Ab (lanes 1-6). Exposure of the autoradiogram was for 12 h.



Phosphorylation of eps15 by EGF Receptor Deletion Mutants

The same EGF receptor deletion mutants were also tested for their ability to phosphorylate eps15. As shown in Fig. 5, Dc 123F and Dc 165 phosphorylated eps15 similarly to the wild-type EGF receptor, whereas Dc 196 and Dc 214 were essentially unable to phosphorylate eps15. Quantitation indicated that phosphorylation of eps15 by Dc 196 and Dc 214 was decreased to 10 and 20%, respectively, of the wild-type receptor level ().

The phosphorylation properties of eps15 were similar to those of PLC, which could not be phosphorylated by either the Dc 196 or the Dc 214 deletion mutants. In the case of PLC, the mechanism is fairly evident, because these two mutants lack all five autophosphorylation sites. On the contrary, the reason for the decreased capacity of phosphorylation of eps15 is not evident. Our results delimit a region of the EGF receptor, encompassing residues 991-1021 (between Dc 165 and Dc 196), which seems critical for eps15 phosphorylation. The slightly higher phosphorylation of eps15 by the F5 and Dc 123F mutants compared with wild-type receptor (less than 2-fold) may be due to the lack of binding and activation of a tyrosine phosphatase. In particular, the major EGF receptor binding site for Tyr PTPase 1B is Tyr-992(31) , which is absent in the F5 and Dc 123F mutants. Because it is not known whether eps15 is a direct EGF receptor substrate because its association with the receptor cannot be detected or whether it is phosphorylated by another tyrosine kinase activated by EGF receptor, it is difficult at the moment to interpret the role of these 30 amino acids in the EGF receptor tail. One possibility is that both eps15 and the EGF receptor bind an adaptor protein independently of the autophosphorylation sites, which would bring eps15 in contact with the EGF receptor. Alternatively, this region may be important in allowing EGF receptor activation of another tyrosine kinase(35) . Finally, it is also possible that the lack of eps15 phosphorylation is due to a change in conformation of the two EGF receptor mutants that does not allow eps15 phosphorylation.

In Vitro Kinase Activity of EGF Receptors Lacking the Autophosphorylation Sites

To test the kinase activity of the the F5 and Dc 123F mutant receptors, in vitro kinase activity toward a synthetic peptide corresponding to the amino acids surrounding Tyr-1173 was measured. As shown in , F5 and Dc 123F mutant receptors showed a 3-fold higher K for the peptide than the wild-type EGF receptor. Therefore, in vitro kinase activity of an EGF receptor lacking the autophosphorylation sites was lower than that of the wild-type but not totally abolished. Also, tyrosine phosphorylation of total cellular proteins in vivo is known to be drastically decreased but not completely absent in the F5 and Dc 123F mutant receptor (Ref. 21; data not shown), possibly due to the presence of a compensatory autophosphorylation site.

Transforming Activity of EGF Receptor Deletion Mutants and eps8 Phosphorylation

Finally, phosphorylation of eps8 correlates well with the high transforming ability of the Dc 196 and Dc 214 mutants. As shown in , EGF-dependent transforming ability of the F5, Dc 123F(5, 21) , and Dc 165 mutants is very low (about 10% of that of the wild-type EGF receptor), whereas in longer deletions the wild-type transformation level is recovered (100 and 120%, respectively, in Dc 196 and Dc 214). In addition, Dc 196 and Dc 214 possessed low transforming activity even in the absence of EGF (15 and 20% of EGF-dependent wild-type EGF-R, respectively). Transformation in the absence of EGF has never been observed with the wild-type or any other EGF receptor mutant(5, 27, 29) . These data were confirmed when the ability to grow in agar was estimated (data not shown). The high transforming activity of Dc 214 and Dc 196 also correlates well with higher tyrosine phosphorylation of endogenous proteins observed using phosphotyrosine blots of total cellular proteins (Ref. 21; data not shown).

In conclusion, the EGF receptor COOH-terminal tail has multiple complex functions that can be mapped to different regions. The extreme COOH terminus with the autophosphorylation sites positively affects receptor biological activity and binding to SH2 substrates; the upstream region contains the EGF-R internalization domains(8, 20, 36) . Finally, a small intermediate region of 30 amino acids negatively influences EGF-R biological and transforming ability and affects phosphorylation of the two newly identified substrates eps8 and eps15.

  
Table: In vitro kinase activity of wild-type and mutant EGF receptors lacking all five autophosphorylation sites

Equal amounts of cell lysates (3 mg of proteins) were immunoprecipitated with EGF-R antibodies. Aliquots of the immunoprecipitates were washed and incubated with 100 nM EGF for 30 min at 20 °C and then incubated with 10 µM [P]ATP, and various concentrations of Tyr-1173 peptide ranging from 0.0065 to 0.65 mM. Reactions were terminated by the addition of 2 Laemmli buffer, and 4-µl aliquots were run on Phast gels, dried, and exposed for autoradiography. The Tyr(P) peptide bands were scanned and quantified with the PhosphorImager. K values are in µM; V values are in µmol/min.


  
Table: Comparison of transforming activity and tyrosine phosphorylation of eps8 and eps15 by different EGF receptor mutants



FOOTNOTES

*
This work was supported by grants from the Associazione Italiana Ricerca sul Cancro and from CNR, PF-ACRO (to L. B.). 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.

§
Supported by a fellowship from Ministro de Educacion y Ciencia, Spain. Present address: Dept. of Physiology, Faculty of Medicine Santiago de Compostela, Spain.

Present address: Ontario Cancer Institute, Toronto, Canada.

**
Supported by a fellowship from Fondazione HS Raffaele, Milano, Italy.

§§
To whom correspondence should be addressed: Laboratory of Molecular Biology, Dipartimento di Ricerca Biologica e Tecnologica, HS Raffaele, Via Olgettina 60, 20132 Milano, Italy. Tel.: 39-2-2643-4747; Fax: 39-2-2643-4844.

The abbreviations used are: EGF-R, epidermal growth factor receptor; EGF, epidermal growth factor; PLC, phospholipase C; GAP, GTPase-activating protein of Ras; SHC, Src and collagen homology proteins; SH2, Src homology; DMEM, Dulbecco's modified Eagle's medium; Ab, antibody; mAb, monoclonal antibody.

L. Beguinot, C. V. Alvarez, and M. Miloso, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Graham Carpenter (Department of Biochemistry, Vanderbilt University) for many helpful suggestions, for careful revision of the manuscript, and for providing anti-PLC Ab; Dr. Francesca Fazioli (Laboratory of Molecular Genetics, Dipartimento di Ricerca Biologica e Tecnologica, Milano) and Dr. PierPaolo Di Fiore (Laboratory of Molecular and Cellular Biology, NIH) for helpful discussions and for anti-eps8 and anti-eps-15 Ab; Dr. Ettore Appella (Laboratory of Cellular Biology, NIH) for the precious gift of the 1173 peptide; Dr. Maria Mazzotti for great help with tissue culture; Dr. Paola Castagnino for help with anti-EGF-R blots (Laboratory of Molecular and Cellular Biology, NIH); and Dr. Claudio de Santis (Dipartimento di Ricerca Biologica e Tecnologica, Milano) for help with the Phast gel system.


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