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

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 (1). 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) 1 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 (2). 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 (3). These, in turn, can associate with other signaling proteins or substrates via other binding modules, such as SH3 domains.
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 (4). Similarly, a distinct new type of tyrosine-containing, SH2-binding domain, termed tyrosine-based activation motif (TAM), has been found in antigen receptor molecules (5). 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 (6), 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 (7,8). The identification of novel receptor-associated components is also required to fill the gaps in our knowledge of the internalization of receptor tyrosine kinases (9). The internalization of these receptors contributes to their down-regulation and thus is an important part of signal transduction (10,11).
Fazioli et al. (12) recently found a new EGFR substrate that they designated eps15, for EGFR pathway substrate 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 (13). Intriguingly, eps15 binds to proteins operating in endocytosis, such as ␣-adaptin (14,15) and clathrin (16), and associates with clathrin-coated pits (17). More recently, eps15 was shown to be essential in receptormediated endocytosis (18).
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-and TAM domains (EAST). We suggest that EAST, together with eps15, is involved in EGFR-mediated signaling and in the regulation of the endocytotic machinery.

EXPERIMENTAL PROCEDURES
General Procedures-Standard solutions, buffers, and procedures for the purification and precipitation of DNA, restriction enzyme digestion, and ligation reactions were as described in Sambrook et al. (19). Sequencing was done by the dideoxynucleotide chain termination method of Sanger (32), 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.
cDNA Cloning of EAST-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 (20). 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 (5ЈStretch) cloned in gt10 and gt11 vectors (CLONTECH) and a chicken genomic library in the EMBL SP6/T7 vector (CLONTECH). RNAs were prepared as described earlier (20). 5Ј rapid amplification of cDNA ends was done using the 5ЈAmpliFINDER rapid amplification of cDNA ends kit (CLONTECH). Library screening was performed as follows. Filters containing ϳ1 ϫ 10 4 plaques/filter were incubated overnight with an EAST probe in hybridization buffer (50% formamide, 5ϫ SET-buffer, 5ϫ Denhardt's solution, 1% SDS, 50 g of yeast tRNA). The probe was labeled with [␣-32 P]dCTP using an Oligolabeling Kit (Pharmacia). After washing several times with 2ϫ 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.
Northern Blot Analysis-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 (20), and the hybridization was performed as above.
Antibodies-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 in Escherichia coli (DH5␣). 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 CNBractivated 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 antimouse IgG-agaroses were from Sigma.
Immunoblotting and Immunoprecipitation-Immunoblotting of tissues and cells was carried out essentially as described previously (20). 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 mM EGTA, 10% glycerol, 25 mM NaF, 10 M ZnCl 2 , 1% Triton X-100, 1 mM sodium orthovanadate, 10 mM ␤-glycerophosphate, 5 g/ml leupeptin, 5 g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Lysates were incubated on ice for 10 min and cleared by centrifugation for 10 min at 14,000 ϫ 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 2ϫ 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.
Kinase Assays-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 MgCl 2 , 1 mM MnCl 2 , 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, and 10 mM ␤-glycerophosphate). They were then resuspended in 40 l of kinase buffer containing unlabeled ATP at a final concentration of 50 M. 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 g of normal or mutated recombinant EAST protein and 6 Ci of [␥-32 P]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 2ϫ 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.
Transient Transfection-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 NH 2 -terminal (aa 1-205) and COOH-terminal (aa 260 -469) constructs were subcloned into the EcoRI/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.
Immunofluorescence Microscopy-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 MgCl 2 , 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 (20). The cells were viewed under an Olympus BH2 fluorescence microscope equipped with appropriate filters.
GST Fusion Proteins-The full-length, NH 2 -terminal (aa 1-205) and COOH-terminal (aa 260 -469) sequences were amplified by PCR using Pfu polymerase (Stratagene). The products were subcloned into the BamHI/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-␤-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 M Tris-HCl, pH 8.0.

Sequence and Domain Structure of EAST-
The amino acid sequence of EAST, deduced from the full-length cDNA isolated, and sequenced as described under "Experimental Procedures," is shown in Fig. 1A. 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 ␤4, and STAM, is present in the COOH-terminal half of the sequence (aa 356 -375) ( Fig. 2A). 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 ␣-helicity in the NH 2 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. 1B.
A sequence homology search of the protein data banks revealed a high degree of overall similarity of EAST with the STAM (22). In the NH 2 -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 (23), Hrs, a receptor tyrosine kinase substrate (24), and Tom-1, a v-Myb target gene product (25) (Fig. 2B). 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.
Expression of EAST in Chicken Tissues-Northern blot analysis of chicken tissues revealed a major RNA transcript of ϳ1.9 kilobases that was present in about equal amounts in chicken brain, heart, liver, lung, gizzard, gut, skin, eye, skeletal muscle, and kidney (Fig. 3A). 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 affinitypurified anti-EAST antibody raised against the NH 2 terminus of EAST (see "Experimental Procedures"). A single polypeptide band of ϳ68 kDa was detected in almost all of the tissues and cells (Fig. 3B). 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.
Tyrosine Phosphorylation of EAST-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 (21), 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.
Association of EAST with 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. 5A). The co-immunoprecipitation of EGFR with EAST, seen even in serum-starved, nonstimulated cells, was clearly enhanced after EGF treatment (Fig. 5A). 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.  5B). 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.  6A). 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. 6B 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. 6B). We also made a preliminary attempt to characterize the sites of phosphorylation in EAST. For that purpose, NH 2 -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 NH 2 -terminal half (Fig. 6B). 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, Tyr 359 and Tyr 372 , in the TAM domain. By analogy with other TAMcontaining 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. 6B), suggesting that tyrosines other than Tyr 359 and Tyr 372 of the TAM are the phosphorylation targets of the EGFR.
Association of EAST with Eps15-Eps15 is an EGFR substrate (12) 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 (26,27) and that the interaction can be abolished by a point mutation of any of these three amino acids. An NPF motif is present in the NH 2 terminus of EAST (aa 7-9). To test whether this motif mediates a direct interaction between EAST and eps15, we overexpressed an HA-tagged, mutated NH 2 -terminal EAST (aa 1-205) in HER-14 cells and performed immunoprecipitations with anti-HA antibody. The substitution of Asn by Asp could not abolish the association between EAST and eps15 (data not shown).
Immunolocalization of EAST-A single band of 68 kDa was recognized by affinity-purified anti-EAST antibodies in immunoblot analyses of CEHF lysates (Fig. 3B), whereas no bands were seen with the preimmune serum (data not shown), demonstrating the specificity of the antibody.
In immunofluorescence microscopy of fixed and permeabilized CEHFs with anti-EAST antibodies, a distinct membraneassociated staining was observed (Fig. 8A). This reaction was blocked by the preincubation of the antibody with the bacterially produced EAST (Fig. 8B) but not with GST alone (data not shown). A closer examination of the staining revealed a dotted or vesicular membrane-associated staining. Often, it was also seen in cell protuberances, which probably represent membrane ruffles. In some cells, the staining pattern partially coincided with actin fibers and focal adhesions (data not shown).
In view of the association between EAST and the endocytosis-associated eps15, we investigated the subcellular distribution of EAST in relation to proteins involved in receptor-mediated endocytosis. This was studied in HeLa cells using antibodies to clathrin, a marker for clathrin-coated pits and vesicles formed during the early stages of endocytosis; EAST displayed a membrane-associated distribution, with spot-like condensations (Fig. 9A), and a partial colocalization with clathrin (Fig. 9B). This strongly suggests that EAST is localized in structures associated with endocytosis.
Transient Expression of EAST in Cultured Cells-In view of the similarity of the NH 2 terminus of EAST with endocytosisassociated proteins, and the presence of a TAM in its COOHterminal half, we hypothesized that these domains serve as "linker" regions to relevant subcellular targets. Therefore, using HA-tagged constructs and anti-HA antibodies, we studied the distribution of exogenously expressed NH 2 -terminal and COOH-terminal portions of EAST in HeLa cells. As shown in Fig. 9C, the NH 2 -terminal construct located in vesicle-like structures along the plasma membrane. In double-immunostaining experiments, the NH 2 -terminal construct partially colocalized with clathrin (Fig. 9D). The COOH-terminal construct, on the other hand, showed a distinct localization to perinuclear vesicles (Fig. 9E). The results suggest that the NH 2 teminus of EAST may serve as a targeting domain that couples EAST to the endocytotic machinery. The localization of the COOH terminus, although quite distinct, was still associated with vesicular structures. This may indicate that the NH 2 -and COOH-terminal regions of EAST associate with different endocytotic vesicles at different stages of endocytosis. We were unable to study the localization of overexpressed, full-length, EAST protein in cultured cells because of its rapid degradation.

DISCUSSION
In this study we report the cloning and characterization of a new 68-kDa protein, which we have named EAST. It contains an SH3 domain, a TAM domain, and an NH 2 -terminal region similar to the NH 2 termini of proteins involved in endocytosis. EAST associates with eps15 and EGFR. In immunofluorescence microscopy, a membrane-associated and vesicular staining, coinciding with that of clathrin, was seen.
The present results link EAST to both EGFR and eps15, suggesting that EAST could serve as a linker between these two molecules. By virtue of its TAM and SH3 domains, EAST appears to be a bona fide signaling protein that could play a role in pathways involving tyrosine kinases. This was demonstrated by showing that EAST co-immunoprecipitates with the EGFR from lysates of EGF-treated HER-14 cells and that it becomes phosphorylated on tyrosine in response to EGF. A distinct phosphorylation of EAST was seen already within 1 min after stimulation, a time scale that corresponds well to the autophosphorylation of EGFR. The association between EAST and the EGFR was confirmed in reciprocal co-immunoprecipitation experiments using anti-EGFR and anti-EAST antibodies. Significantly more EGFR and EAST were seen complexed after EGF treatment, indicating that the phosphorylation of either one or both of the components enhances the interaction.
Co-immunoprecipitation experiments to determine whether eps15 interacts with EAST were prompted by the presence of a band of about 150 kDa in anti-EAST immunoprecipitates, and an interaction between eps15 and EAST could be confirmed by reciprocal co-immunoprecipitation using anti-eps15 and anti-EAST antibodies. Contrasting with the case of EGFR, there was no difference between naive and EGF-treated cells in the amount of EAST co-immunoprecipitating with eps15, indicating that the eps15-EAST interaction is not dependent on EGFinduced phosphorylation. Whether the interaction is direct or not remains yet to be elucidated, because the association be-tween EAST and eps15 could not be abolished by mutating the NPF motif of EAST.
Originally, eps15 was isolated in an effort to uncover as yet uncharacterized effector pathways emanating from the EGFR and erbB-2/neu receptors (12). Eps15 has a unique domain structure. There are three eps15 homology domains in the amino terminus, and DPF (aspartic acid-proline-phenylalanine) repeats in the carboxyl terminus. In the central region, there is a domain with an ␣-helical conformation and a probable coiled-coil supramolecular organization. From the immunoprecipitation experiments using lysates of unstimulated cells and cells stimulated either with EGF or with other growth factors, it was concluded that the phosphorylation of eps15 is relatively specific to the EGF and the EGFR (12,16). However, attempts to demonstrate a direct interaction between the EGFR and eps15 failed, leaving unanswered the question of whether eps15 is a direct substrate of the EGFR or whether it binds to and is phosphorylated by a kinase(s) downstream of the EGFR (12). More recently, Schumacher et al. (13) isolated eps15 in an expression library search for proteins that bind to the SH3 domain of Crk. They suggested that Crk might be a crucial link between the EGFR and eps15 in the mitogenic signaling emanating from the EGFR. However, the interaction between eps15 and Crk could not be shown by co-immunoprecipitation from cell lysates. The present results clearly show an association between EAST and EGFR on the one hand and between EAST and eps15 on the other hand. Moreover, immunoprecipitation with anti-EAST antibodies also brings down both the EGFR and eps15, strongly suggesting that EAST serves as a "bridge" between the EGFR and eps15. The notion of a direct interaction between EAST and the EGFR has to be reconciled with the fact that in EAST there are no domains, such as SH2 or phosphotyrosine binding, that are usually found in proteins binding to activated receptor tyrosine kinases. The lack of an obvious binding motif is not unique to EAST, however, because c-Cbl, a proto-oncogene protein that becomes strongly phosphorylated and associates with activated EGFR, is also devoid of a distinct receptor-binding motif (28). The possibility of nonphosphotyrosine-mediated binding of the cytoplasmic tail of EGFR should also be considered. In fact, phosphorylation of eps15 occurs even in cells carrying a mutant EGFR in which all five autophosphorylation sites are abolished (29), suggesting that the association of eps15 with the EGFR, whether direct or indirect, is not based on "conventional" phosphotyrosine recognition.
Recent studies have convincingly shown that eps15 is closely associated with the endocytotic machinery. First, it associates with several proteins known to be components of clathrincoated pits and vesicles, including clathrin and adaptor protein 2 (14 -16). Second, it localizes to the coated pits (17). Third, microinjection of anti-eps15 antibodies to cells inhibits the internalization of the EGFR and of the transferrin receptor (18). In view of the distinct role(s) of eps15 in endocytosis and receptor internalization, it is reasonable to propose that EAST also plays a role in these processes.
In immunofluorescence microscopy, EAST exhibited a general membrane-associated staining and spot-like condensations, which, by their staining with anti-clathrin antibodies, were identified as clathrin-coated pits and vesicles. Thus, both the physical interaction between eps15 and EAST and the codistribution of both proteins with clathrin-associated vesicles are indicative of a role for EAST in endocytosis. Its exact role in coated pit formation or regulation remains, however, to be elucidated. It is tempting to speculate that EAST could serve as a linker between the EGFR and eps15 and thus contribute to the internalization of the EGFR.
Protein data bank sequence comparisons revealed a number of proteins exhibiting sequence similarity with EAST. The highest degree of homology was seen with STAM, a protein originally discovered on the basis of its tyrosine phosphorylation in response to interleukin-2 treatment (22). EAST and STAM share the same SH3/TAM domain organization, which would warrant the claim that they are parent members of a new class of signal transduction proteins with this signature. To our knowledge, EAST and STAM are the first examples of nonreceptor proteins possessing a TAM. Given the role of the TAM in immunoreceptors as a docking site for various nonreceptor kinases, we hypothesize that EAST also has a bridge role, by relaying the linkage between EGFR and the endocytotic machinery. The putative TAM-binding partner remains to be identified.
A local homology was seen between the NH 2 termini of EAST, VPS27, Hrs, and Tom-1. Hrs (hepatocyte growth factorregulated tyrosine kinase substrate) is a phosphotyrosine protein induced by hepatocyte growth factor, EGF, and plateletderived growth factor (24), and it was recently shown to be associated with early endosomes (30). Hrs-2, a homologue of Hrs, was shown to be associated with SNAP-25 and is thought to modulate vesicular transport (31). VPS27 is also implicated in vesicular trafficking in yeast, in which it controls vacuolar and endocytotic traffic through a prevacuolar compartment (23). An association with vesicular/endosomal trafficking appears to be a common denominator between EAST, STAM, Hrs, and VPS27, suggesting a role for the homologous NH 2 -terminal region in the regulation of these processes. More direct evidence supporting this notion was obtained in transfection experiments, in which the NH 2 -terminal portion of EAST colocal- ized distinctly with clathrin. The fourth member of the group, Tom-1 (25), is only partially characterized, and its function is still unknown.
In conclusion, the present data strongly suggest that EAST is involved in the early stages of endocytosis and, more specifically, in the formation of clathrin-coated pits and vesicles. Hence, EAST may play an important role in the down-regulation of receptor tyrosine kinases, especially the EGFR, and may thus regulate the signaling potency of the EGFR. We propose that EAST serves as a scaffolding device in receptor-mediated endocytosis, bringing together the EGFR, eps15, eps15-associated proteins, and other uncharacterized components of the complex machinery of the early stages of endocytosis.