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EAST, an Epidermal Growth Factor Receptor- and Eps15-associated Protein with Src Homology 3 and Tyrosine-based Activation Motif Domains

1998; Elsevier BV; Volume: 273; Issue: 33 Linguagem: Inglês

10.1074/jbc.273.33.21408

1083-351X

Olli Lohi, Anssi Poussu, Jari Meriläinen, Sakari Kellokumpu, Veli-Matti Wasenius, Veli‐Pekka Lehto,

Cellular transport and secretion

We describe the cloning and characterization of a new cytoplasmic protein designated epidermal growth factor receptor-associated protein with SH3- andTAM domains (EAST). It contains an Src homology 3 domain in its midregion and a tyrosine-based activation motif in its COOH terminus. Antibodies to EAST recognize a 68-kDa protein that is present in most chicken tissues. An epidermal growth factor (EGF)-dependent association between the EGF receptor (EGFR) and EAST was shown by reciprocal immunoprecipitation/immunoblotting studies with specific antibodies. Activated EGFR catalyzed the tyrosine phosphorylation of EAST, as judged by an in vitro kinase assay with both immunoprecipitated and purified EGFR. Immunoprecipitation/immunoblotting experiments also demonstrated an association between EAST and eps15, an EGFR substrate associated with clathrin-coated pits and vesicles, which is essential in the endocytotic pathway. The association between EAST and eps15 was not affected by EGF treatment. In immunofluorescence microscopy, EAST was shown to partially colocalize with clathrin. The sequence of the NH2-terminal portion of EAST shows a high degree of similarity with a group of proteins involved in endocytosis or vesicle trafficking. Thus, EAST is a novel signal transduction component probably involved in EGF signaling and in the endocytotic machinery. We describe the cloning and characterization of a new cytoplasmic protein designated epidermal growth factor receptor-associated protein with SH3- andTAM domains (EAST). It contains an Src homology 3 domain in its midregion and a tyrosine-based activation motif in its COOH terminus. Antibodies to EAST recognize a 68-kDa protein that is present in most chicken tissues. An epidermal growth factor (EGF)-dependent association between the EGF receptor (EGFR) and EAST was shown by reciprocal immunoprecipitation/immunoblotting studies with specific antibodies. Activated EGFR catalyzed the tyrosine phosphorylation of EAST, as judged by an in vitro kinase assay with both immunoprecipitated and purified EGFR. Immunoprecipitation/immunoblotting experiments also demonstrated an association between EAST and eps15, an EGFR substrate associated with clathrin-coated pits and vesicles, which is essential in the endocytotic pathway. The association between EAST and eps15 was not affected by EGF treatment. In immunofluorescence microscopy, EAST was shown to partially colocalize with clathrin. The sequence of the NH2-terminal portion of EAST shows a high degree of similarity with a group of proteins involved in endocytosis or vesicle trafficking. Thus, EAST is a novel signal transduction component probably involved in EGF signaling and in the endocytotic machinery. Signal transduction proteins are characterized by their capacity to specifically associate with other proteins to form multimolecular assemblies (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar). In the case of receptor tyrosine kinases and receptor tyrosine kinase-induced intracellular signaling, such protein interactions are mediated by distinct protein domains. Among these, the Src homology 2 (SH2) 1The abbreviations used are: SH2Src homology 2SH3Src homology 3EGFepidermal growth factorEGFREGF receptorTAMtyrosine-based activation motifaaamino acid(s)CEHFchicken embryo heart fibroblastPCRpolymerase chain reactionGSTglutathione S-transferasePBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisHAhemagglutininPipes1,4-piperazinediethanesulfonic acidSTAMsignal transducing adaptor molecule. domain, which binds Tyr(P) residues in a specific context, and the Src homology 3 (SH3) domain, which binds sequences characterized by polyproline tracts, are the best known (2Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1021) Google Scholar). In the epidermal growth factor receptor (EGFR), for instance, binding of the epidermal growth factor (EGF) leads to the phosphorylation of multiple tyrosine residues by the kinase activity of the receptor. These Tyr(P) residues serve as docking sites for various downstream, SH2-containing, signaling elements (3Pawson T. Schlessinger J. Curr. Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (577) Google Scholar). These, in turn, can associate with other signaling proteins or substrates via other binding modules, such as SH3 domains. Src homology 2 Src homology 3 epidermal growth factor EGF receptor tyrosine-based activation motif amino acid(s) chicken embryo heart fibroblast polymerase chain reaction glutathione S-transferase phosphate-buffered saline polyacrylamide gel electrophoresis hemagglutinin 1,4-piperazinediethanesulfonic acid signal transducing adaptor molecule. As new proteins and interactions are being discovered, new motifs are also disclosed. Thus, the phosphotyrosine-binding domain present in Shc and IRS-1, for example, recognizes Tyr(P) in a manner different from SH2 domains (4Zhou M.M Ravichandran K.S. Olejniczak E.F. Petros A.M. Meadows R.P. Sattler M. Harlan J.E. Wade W.S. Burakoff S.J. Fesik S.W. Nature. 1995; 378: 584-592Crossref PubMed Scopus (324) Google Scholar). Similarly, a distinct new type of tyrosine-containing, SH2-binding domain, termed tyrosine-based activation motif (TAM), has been found in antigen receptor molecules (5Keegan A.D. Paul W.E. Immunol. Today. 1992; 13: 63-68Abstract Full Text PDF PubMed Scopus (173) Google Scholar). TAM-containing receptors lack an intrinsic kinase activity and use TAM motifs to recruit and activate nonreceptor protein tyrosine kinases, such as members of the Src and Syk families (6Songyuan Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Rafnofsky S. Feldman R.A. Cantley L.C. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (833) Google Scholar), as their effectors. Many of the known signaling pathways are still only partially characterized, and their regulation is poorly understood, rendering the search for new interacting proteins a topic of high priority. For instance, elimination of critical SH2-binding sites of the platelet-derived growth factor and fibroblast growth factor receptors does not abrogate mitogenic signaling, suggesting that as yet unidentified proteins interact with these receptors (7Yu J.C. Heidaran M.A. Pierce J.H. Gutkind J.S. Lombardi D. Ruggiero M. Aaronson S.A. Mol. Cell. Biol. 1991; 11: 3780-3785Crossref PubMed Scopus (65) Google Scholar, 8Mohammadi M. Dikis I. Sorokin A. Burgess W.H. Jaye M. Schlessinger J. Mol. Cell. Biol. 1996; 16: 977-989Crossref PubMed Scopus (342) Google Scholar). The identification of novel receptor-associated components is also required to fill the gaps in our knowledge of the internalization of receptor tyrosine kinases (9Riezman H. Woodman P.G. van Meer G. Marsh M. Cell. 1997; 91: 731-738Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The internalization of these receptors contributes to their down-regulation and thus is an important part of signal transduction (10Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4611) Google Scholar, 11Seaman M.N.J. Burd C.G. Emr S.D. Curr. Opin. Cell Biol. 1996; 8: 549-556Crossref PubMed Scopus (59) Google Scholar). Fazioli et al. (12Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar) recently found a new EGFR substrate that they designated eps15, for EGFR pathwaysubstrate clone 15. Eps15 is phosphorylated by EGFR in response to the activation of the latter by EGF. The unique domain structure of eps15 makes this protein a novel type of EGFR substrate. However, no direct interaction between the EGFR and eps15 could be shown. More recently, a specific interaction between the SH3 domain of Crk and the conserved proline-rich motif of eps15 was demonstrated, suggesting that Crk could mediate the binding of eps15 to the EGFR (13Schumacher C. Knudsen B.S. Ohuchi T. Di Fiore P.P. Glassman R.H. Hanafusa H. J. Biol. Chem. 1995; 270: 15341-15347Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Intriguingly, eps15 binds to proteins operating in endocytosis, such as α-adaptin (14Benmerah A. Gagnon J. Begue B. Megarbane B. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1995; 131: 1831-1838Crossref PubMed Scopus (151) Google Scholar, 15Benmerah A. Begue B. Dautry-Varsat A. Cerf-Bensussan N. J. Biol. Chem. 1996; 271: 12111-12116Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and clathrin (16van Delft S. Schumacher C. Hage W. Verkleij A.J. Henegouwen P.M.P. J. Cell Biol. 1997; 136: 811-821Crossref PubMed Scopus (112) Google Scholar), and associates with clathrin-coated pits (17Tebar F. Sorkina T. Sorkin A. Ericsson M. Kirchhausen T. J. Biol. Chem. 1996; 271: 28727-28730Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). More recently, eps15 was shown to be essential in receptor-mediated endocytosis (18Carbone R. Fre S. Iannolo G. Belleudi F. Mancini P. Pelicci P.G. Torrisi M. Di Fiore P.P. Cancer Res. 1997; 57: 5498-5504PubMed Google Scholar). In this paper, we describe the cloning, the primary structure and functional properties of a new signal transduction protein that associates with the EGFR and eps15. Because of its binding properties and domain structure, we have named it EGF receptor-associated protein with SH3- andTAM domains (EAST). We suggest that EAST, together with eps15, is involved in EGFR-mediated signaling and in the regulation of the endocytotic machinery. Standard solutions, buffers, and procedures for the purification and precipitation of DNA, restriction enzyme digestion, and ligation reactions were as described in Sambrooket al. (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Sequencing was done by the dideoxynucleotide chain termination method of Sanger (32Sanger F. Nicklen S. Coulsen A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52668) Google Scholar), using the T7 Sequencing kit (Pharmacia Biotech Inc.) or using an automated ABI PRISM 377XL DNA Sequencer (Perkin-Elmer). Synthetic oligonucleotides were obtained from the Oligonucleotide Core Facility of Biocenter Oulu or from Pharmacia. For sequence analysis and alignments, the GCG, CLUSTAL W, and COILS programs were used. Fragments of mRNAs encoding for SH3 domain-containing proteins expressed in chicken brain were amplified by reverse transcription and polymerase chain reaction (PCR) using degenerate oligonucleotide primers (20Meriläinen J. Lehto V.-P. Wasenius V.-M. J. Biol. Chem. 1997; 272: 23278-23284Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The original clone of EAST was extended by PCR cloning from different chicken libraries and from RNA and by library screening with cDNA probes. The libraries used included a chicken brain cDNA library (5′Stretch) cloned in λgt10 and λgt11 vectors (CLONTECH) and a chicken genomic library in the EMBL SP6/T7 vector (CLONTECH). RNAs were prepared as described earlier (20Meriläinen J. Lehto V.-P. Wasenius V.-M. J. Biol. Chem. 1997; 272: 23278-23284Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). 5′ rapid amplification of cDNA ends was done using the 5′AmpliFINDER rapid amplification of cDNA ends kit (CLONTECH). Library screening was performed as follows. Filters containing ∼1 × 104 plaques/filter were incubated overnight with an EAST probe in hybridization buffer (50% formamide, 5× SET-buffer, 5× Denhardt's solution, 1% SDS, 50 μg of yeast tRNA). The probe was labeled with [α-32P]dCTP using an Oligolabeling Kit (Pharmacia). After washing several times with 2× SSC (0.3 m NaCl, 30 mm sodium citrate, pH 7.0) and 1% SDS at 42 °C, autoradiography was performed overnight on x-ray film (Kodak). Positive plaques were then collected, plated, and rescreened. The cDNA corresponding to the nucleotides 60–231 of the coding region of EAST was amplified by PCR and used as a probe. Preparation of total RNAs and mRNAs were as described previously (20Meriläinen J. Lehto V.-P. Wasenius V.-M. J. Biol. Chem. 1997; 272: 23278-23284Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), and the hybridization was performed as above. Cultures of chicken embryo heart fibroblasts (CEHF) were established, and the cells were grown as described previously (20Meriläinen J. Lehto V.-P. Wasenius V.-M. J. Biol. Chem. 1997; 272: 23278-23284Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). HER-14 cells (a gift from Dr. J. Schlessinger, New York University Medical Center), expressing ∼1 × 104 EGFR/cell (21Rotin D. Margolis B. Mohammadi M. Daly R.J. Daum G. Li N. Fischer E.H. Burgess W.H.M. Ullrich A. Schlessinger J. EMBO J. 1992; 11: 559-567Crossref PubMed Scopus (251) Google Scholar), were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum (Hyclone) and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin sulfate, 0.25 μg/ml amphotericin B). Cells were serum-starved for 12–16 h in 0.2% fetal bovine serum and then stimulated with EGF (100 ng/ml). HeLa cells were maintained in Eagle's minimal essential medium with Earle's salts (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum, 2 mm glutamine, 1% nonessential amino acids, and antibiotics (see above). The anti-EAST antibody was prepared as follows. A GST-EAST fusion protein was produced by cloning the cDNA corresponding to the amino acids 4–188 of the EAST coding region into the pGEX-2-TK vector (Pharmacia) and having it expressed inEscherichia coli (DH5α). The fusion protein was purified according to the manufacturer's instructions (Pharmacia) and was used as an antigen to immunize rabbits. The antisera were collected and affinity-purified on CNBr-activated Sepharose 4B beads (Pharmacia) coated with the corresponding fusion protein. Anti-GST activity was preadsorbed by incubating the affinity-purified antibodies with a recombinant GST coupled to glutathione-Sepharose 4B (Pharmacia). Monoclonal and polyclonal anti-EGFR antibodies were generous gifts from Dr. J. Schlessinger. Monoclonal anti-eps15 antibody was a kind gift from Dr. Pier Paolo Di Fiore (European Institute of Oncology, Milan, Italy). Polyclonal anti-eps15 (sc-534) and anti-HA (sc-805) antibodies and the monoclonal anti-phosphotyrosine antibody (PY99) were purchased from Santa Cruz Biotechnology. Monoclonal anti-HA antibody (12CA5) was from Boehringer Mannheim. Monoclonal anti-clathrin antibody was from Transduction Laboratories. Anti-rabbit and anti-mouse IgG-agaroses were from Sigma. Immunoblotting of tissues and cells was carried out essentially as described previously (20Meriläinen J. Lehto V.-P. Wasenius V.-M. J. Biol. Chem. 1997; 272: 23278-23284Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Cells, grown to 70–90% confluency, were washed with ice-cold PBS and then scraped in a lysis buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mmEGTA, 10% glycerol, 25 mm NaF, 10 μmZnCl2, 1% Triton X-100, 1 mm sodium orthovanadate, 10 mm β-glycerophosphate, 5 μg/ml leupeptin, 5 μg/ml aprotinin, and 1 mmphenylmethylsulfonyl fluoride). Lysates were incubated on ice for 10 min and cleared by centrifugation for 10 min at 14,000 ×g, and the supernatants were collected and used in the assays. For immunoprecipitations, 1–4 mg of proteins were used. Antibodies were added to the cleared lysates and incubated on a nutator for 2 h at 4 °C. Anti-rabbit or anti-mouse IgG-agarose beads were then added, and the incubation was continued for another 2 h at 4 °C. In some cases, cells were pretreated by incubating them with a secondary conjugate for 2 h at 4 °C. Beads were washed 3–5 times with the lysis buffer and then boiled in 2× Laemmli's sample buffer for 5 min. The solubilized immunoprecipitates were separated by 10% SDS-PAGE, transferred onto nitrocellulose filters (Schleicher & Schuell) using a Semi-Dry blotter (KemTech), and subjected to immunoblot analysis. The blots were developed by the ECL method. For repetitive probing, the filters were stripped for 40 min at 62 °C in a stripping buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 100 mm 2-mercaptoethanol), washed extensively, and reprobed. The in vitro immunocomplex kinase assay was performed as follows. HER-14 cells were lysed (as above), and the lysates were used for immunoprecipitation using a monoconal anti-EGFR antibody and anti-mouse IgG-agarose beads. The beads were washed three times with the lysis buffer and once with kinase buffer (0.5% Nonidet P-40, 140 mm NaCl, 25 mm Hepes, pH 7.2, 1 mm MgCl2, 1 mmMnCl2, 2 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 mm β-glycerophosphate). They were then resuspended in 40 μl of kinase buffer containing unlabeled ATP at a final concentration of 50 μm. Incubation was carried out for 20 min at 4 °C. The complexes were then washed twice with kinase buffer to remove the excess ATP. 5 μg of normal or mutated recombinant EAST protein and 6 μCi of [γ-32P]dATP were added, and the mixtures were incubated for 15 min at 37 °C. EAST was then collected by incubating with either glutathione-Sepharose 4B beads or anti-EAST antibody-conjugated IgG-agarose. The beads were then washed twice with kinase buffer, boiled in 2× Laemmli's sample buffer, and subjected to SDS-PAGE (as above) and autoradiography. The direct phosphorylation assay using a purified EGFR kinase domain (Stratagene) was done according to the manufacturer's instructions. The cDNAs used for transfection experiments were produced by PCR using Pfu polymerase (Stratagene). The HA epitope tag was added to the COOH terminus of the constructs by primer design. The authenticity of the constructs was confirmed by DNA sequencing. The NH2-terminal (aa 1–205) and COOH-terminal (aa 260–469) constructs were subcloned into theEcoRI/HindIII cloning site of the pRK5 transfection vector (a gift from Dr. J. Schlessinger). Transient transfections were done using FUGENE 6-reagent (Boehringer Mannheim) according to the manufacturer's instructions. Mutations were produced using the QuikChange site-directed mutagenesis kit (Stratagene). Mutations were confirmed by DNA sequencing. For immunofluorescence microscopy, the cells were grown on glass coverslips. Cells were washed in Hanks' salt solution and then fixed for 10 min with 4% paraformaldehyde in PEM buffer (100 mm Pipes, pH 6.8, 5 mm EGTA, 2 mm MgCl2, and 0.2% Triton X-100). After washing in PBS, the cells were incubated with 10% fetal bovine serum in PBS-glycine for 30 min. They were then overlaid with primary antibody for 30 min, washed, and incubated with either Texas Red-conjugated (Jackson ImmunoResearch) or fluorescein isothiocyanate-conjugated secondary antibody (Caltag Laboratories) for another 30 min. Double-staining experiments were performed as described previously (20Meriläinen J. Lehto V.-P. Wasenius V.-M. J. Biol. Chem. 1997; 272: 23278-23284Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The cells were viewed under an Olympus BH2 fluorescence microscope equipped with appropriate filters. The full-length, NH2-terminal (aa 1–205) and COOH-terminal (aa 260–469) sequences were amplified by PCR using Pfu polymerase (Stratagene). The products were subcloned into theBamHI/EcoRI cloning site of the pGEX-2-TK expression vector (Pharmacia). Mutations were introduced as described above, and the sequences were confirmed by DNA sequencing. Expression of the fusion proteins was induced with isopropyl-1-thio-β-d-galactopyranoside (0.1–0.5 mm) at room temperature for 4–8 h. Cells were spun down, resuspended in PBS containing 1% Triton X-100, sonicated, and incubated on ice for 10 min. After centrifugation, the supernatants were incubated with glutathione-Sepharose 4B beads (Pharmacia). The beads were washed several times in PBS, and the fusion protein was eluted with 20 mm reduced glutathione in 0.1 mTris-HCl, pH 8.0. The amino acid sequence of EAST, deduced from the full-length cDNA isolated, and sequenced as described under “Experimental Procedures,” is shown in Fig.1 A. The protein consists of 468 amino acids with a calculated molecular mass of 52,406 Da. The methionine start codon is in partial agreement with the Kozak consensus sequence. The open reading frame ends with two sequential in-frame stop codons, followed by a putative polyadenylation signal about 300 nucleotides downstream (not shown). An SH3 domain is located in the middle of the sequence (aa 206–259). A sequence similar to the immunoreceptor tyrosine-based activation motif or TAM domains of several immunoreceptors, integrin β4, and STAM, is present in the COOH-terminal half of the sequence (aa 356–375) (Fig.2 A). Together with the high degree of overall similarity, the presence of the canonical pair of closely spaced tyrosines, followed by a leucine three residues downstream, suggests that this region is a genuine TAM. Secondary structure prediction analysis of EAST using the COILS program predicts a high propensity for α-helicity in the NH2 terminus (aa 160–225) and in the region partially overlapping the TAM (aa 330–365). A schematic representation of the structural features of EAST is given in Fig. 1 B.Figure 2Sequence alignments. A, sequence alignment of the TAM of EAST with a representative set of TAMs. B, alignment of the amino acid sequence of EAST with STAM, VPS27, Hrs, and Tom-1b. A single fully conserved residue is indicated by an asterisk, and a strong (>60%) conservation is indicated by a dot. The residues are shadedaccording to the following scheme: white letter on ablack background and black letter on a gray background when an identical residue or similarity, respectively, has an occurrence of more than 60% in a position. The accession numbers of the sequences are as follows: EAST, AJ224514; STAM, U43900; Tom-1b, Y08741; Hrs, D84064; VPS27, P40343.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A sequence homology search of the protein data banks revealed a high degree of overall similarity of EAST with the STAM (22Takeshita T. Arita T. Asao H. Tanaka N. Higuchi M. Kuroda H. Kaneko K. Munakata H. Endo Y. Fujita T. Sugamura K. Biochem. Biophys. Res. Commun. 1996; 225: 1035-1039Crossref PubMed Scopus (69) Google Scholar). In the NH2-terminal half, the proteins are 68% identical (74% homologous). The SH3 domains are 84% identical (84% homologous). Lower degrees of identity (52%) and homology (59%) were seen in the COOH-terminal portion including the TAM. The COOH termini of EAST and STAM appear to be unique. Partial similarities were also found with some other proteins, including VPS27, a yeast protein involved in endocytotic trafficking (23Piper R.C. Cooper A.A. Yang H. Stevens T.H. J. Cell Biol. 1995; 131: 603-617Crossref PubMed Scopus (342) Google Scholar), Hrs, a receptor tyrosine kinase substrate (24Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (145) Google Scholar), and Tom-1, a v-Myb target gene product (25Burk O. Worpenberg S. Haenig B. Klempnauer K.-H. EMBO J. 1997; 16: 1371-1380Crossref PubMed Scopus (60) Google Scholar) (Fig. 2 B). The homologies were found within the first 200 amino acids of these proteins and were found not to reflect the presence of any known protein motif. Northern blot analysis of chicken tissues revealed a major RNA transcript of ∼1.9 kilobases that was present in about equal amounts in chicken brain, heart, liver, lung, gizzard, gut, skin, eye, skeletal muscle, and kidney (Fig.3 A). A weaker signal of 5 kilobases was also detected (data not shown), suggesting the possible existence of two closely related genes or of differential splicing events giving rise to two isoforms of EAST. The widespread expression of EAST was verified by immunoblot analysis using an affinity-purified anti-EAST antibody raised against the NH2 terminus of EAST (see “Experimental Procedures”). A single polypeptide band of ∼68 kDa was detected in almost all of the tissues and cells (Fig.3 B). The high relative electrophoretic mobility of EAST deviates considerably from its calculated molecular mass, suggesting a secondary structure that resists denaturation by boiling in 2-mercaptoethanol and SDS. The presence of a TAM domain in EAST prompted us to study the phosphorylation state of EAST in response to treatment with growth factors. HER-14 cells, stably transfected with EGFR cDNA (21Rotin D. Margolis B. Mohammadi M. Daly R.J. Daum G. Li N. Fischer E.H. Burgess W.H.M. Ullrich A. Schlessinger J. EMBO J. 1992; 11: 559-567Crossref PubMed Scopus (251) Google Scholar), were stimulated with EGF after overnight serum starvation. EAST was then immunoprecipitated with anti-EAST antibodies, and the precipitate was analyzed for phosphorylation by immunoblotting with anti-phosphotyrosine antibody. As seen in Fig. 4, EAST was tyrosine-phosphorylated within 1 min of the EGF treatment and maintained a high level of phosphorylation for over 30 min. To investigate whether the phosphorylation of EAST was specific to EGF, we also tested the effect of platelet-derived growth factor, lysophosphatidic acid, phorbol myristic acid, and bradykinin. A low level of tyrosine phosphorylation of EAST could be detected after platelet-derived growth factor treatment of Swiss 3T3 cells but not after treatment with lysophosphatidic acid, phorbol myristic acid, or bradykinin (data not shown). Thus, the phosphorylation of EAST seems to be primarily associated with receptor tyrosine kinases and, in particular, with the EGFR. Several tyrosine-phosphorylated proteins, representing putative EAST-interacting proteins, co-precipitated with EAST (data not shown). The most prominent of these had a molecular mass of about 180 kDa, suggestive of EGFR. This was confirmed by immunoblotting with anti-EGFR antibody (Fig.5 A). The co-immunoprecipitation of EGFR with EAST, seen even in serum-starved, nonstimulated cells, was clearly enhanced after EGF treatment (Fig.5 A). Immunoprecipitation with anti-EGFR antibodies and blotting with anti-EAST antibodies again demonstrated a distinct co-immunoprecipitation of the two proteins, the extent of which was significantly increased upon EGF treatment (Fig. 5 B). The results demonstrate an EGF-dependent association between EGFR and EAST. To determine whether the EGF-induced phosphorylation of EAST is because of a direct action of EGFR or is mediated by some other proteins, we performed an in vitro immunocomplex kinase assay utilizing specific anti-EGFR antibodies to immunoprecipitate EGFR and bacterially produced EAST as a substrate; EAST was phosphorylated under these conditions (Fig. 6 A). Although suggestive of a direct effect of EGFR on EAST, it does not rule out the possibility that the phosphorylation is because of some other kinase(s) present in the immunocomplex. Therefore, the experiment was repeated by using purified, recombinant EGFR kinase domain and bacterially produced EAST. Fig. 6 B shows that EAST was also phosphorylated under these conditions. Then we repeated the experiment by digesting the fusion protein with thrombin, which leads to a cleavage of EAST from its fusion partner GST. A distinct phosphorylation of EAST was seen, verifying that EAST is the target of the kinase activity (Fig.6 B). We also made a preliminary attempt to characterize the sites of phosphorylation in EAST. For that purpose, NH2- and COOH-terminal parts of EAST were expressed as fusion proteins and subjected to phosphorylation assay as above. Phosphorylation of both fusion proteins was seen with a slightly stronger intensity in the NH2-terminal half (Fig. 6 B). These results strongly suggest that EAST is a direct substrate of EGFR. However, there are no typical motifs, such as SH2 and phosphotyrosine-binding domains, that could serve as EGFR-binding sites in EAST. On the other hand, there are two tyrosine residues, Tyr359and Tyr372, in the TAM domain. By analogy with other TAM-containing proteins, these residues are potential phosphorylation sites. To investigate the possibility that they are the targets of the EGF-induced phosphorylation, both residues were mutated to phenylalanines, and the phosphorylation assay was repeated using the mutated EAST as a substrate. The results indicate that the wild-type and mutated EAST are phosphorylated to similar extents (Fig.6 B), suggesting that tyrosines other than Tyr359and Tyr372 of the TAM are the phosphorylation targets of the EGFR. Eps15 is an EGFR substrate (12Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar) with a molecular mass of about 150 kDa. A band of about that size was present on the Tyr(P) immunoblot of the anti-EAST immunoprecipitate (data not shown). Reciprocal immunoprecipitation/immunoblotting experiments using anti-eps15 and anti-EAST antibodies revealed that eps15 and EAST co-immunoprecipitated. This association was independent of EGF treatment, because equal amounts of coprecipitated proteins were present in the immunocomplexes before and after treatment of HER-14 cells with EGF (Fig. 7). In vitro studies with synthetic peptides have shown that eps15 homology domains of eps15 recognize a short core motif, NPF, in interacting proteins (26Wong W.T. Schumacher C. Salcini A.E. Romano A. Castagnino P. Pelicci P.G. Di Fiore P.P. Proc. Natl Ac