©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The SH3 Domain of Crk Binds Specifically to a Conserved Proline-rich Motif in Eps15 and Eps15R (*)

Christoph Schumacher (1), Beatrice S. Knudsen (1), Tohru Ohuchi (1), Pier Paolo Di Fiore (2), Robert H. Glassman (1) (3), Hidesaburo Hanafusa (1)(§)

From the (1)Laboratory of Molecular Oncology, The Rockefeller University, New York, New York 10021, (2)Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, and (3)Division of Hematology-Oncology, the New York Hospital-Cornell University Medical Center, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Crk protein belongs to the family of proteins consisting of mainly Src homology 2 and 3 (SH2 and SH3) domains. These proteins are thought to transduce signals from tyrosine kinases to downstream effectors. In order to understand the specificity and effector function of the SH3 domain of Crk, we screened an expression library for binding proteins. We isolated Eps15, a substrate of the epidermal growth factor receptor (EGFR) tyrosine kinase, and Eps15R, a novel protein with high sequence homology to the carboxyl-terminal domain of Eps15. Antibodies raised against a fragment of the Eps15R gene product immunoprecipitated a protein of 145 kDa. Eps15 and Eps15R bound specifically to the amino-terminal SH3 domain of Crk and coprecipitated equivalently with both c-Crk and v-Crk from cell lysates. The amino acid sequences of Eps15 and Eps15R featured several proline-rich regions as putative binding motifs for SH3 domains. In both Eps15 and Eps15R, we identified one proline-rich motif which accounts for their interaction with the Crk SH3 domain. Each binding motif contains the sequence P-X-L-P-X-K, an amino acid stretch that is highly conserved in all proteins known to interact specifically with the first SH3 domain of Crk. Furthermore, we found that immunoprecipitates of activated EGFR-kinase stably bound in vitro-translated Eps15 only in the presence of in vitro-translated v-Crk. Crk might therefore be involved in Eps15-mediated signal transduction through the EGFR.


INTRODUCTION

The protooncogene products of v-crk, c-Crk-II and c-Crk-I, belong to a new family of proteins consisting primarily of Src homology 2 and 3 (SH2 and SH3)()domains while lacking any catalytic domain(1, 2) . Nck and Grb2 are two other members of this emerging family of so-called ``adaptor proteins''(3, 4) . The widely expressed c-Crk-II protein contains an amino-terminal SH2 domain followed by two SH3 domains. The c-Crk-I protein, which is found in embryonic lung cells, and v-Crk do not contain the second SH3 domain. In addition, v-Crk has an amino-terminal Gag region.

Src homology domains have been found in a wide range of proteins involved in cell signaling and are suggested to act as molecular adaptors linking and regulating the subcellular localization and enzymatic activity of functionally diverse molecules(5, 6) . Binding of SH2 domains to tyrosine-phosphorylated regions of growth factor receptors is thought to provide a common mechanism by which regulatory proteins interact specifically with growth factor receptors and thereby couple growth factor stimulation to intracellular signaling pathways (7). SH3 domain interactions have been implicated in the targeting of proteins and in the regulation of small GTP-binding proteins(8, 9) . Thus, SH2 and SH3 domains within one adaptor protein may collaborate to assemble a signaling cascade by recruiting upstream and downstream enzymatic activities into a ternary complex. Grb2 was shown to be complexed via its SH3 domain with Sos, a guanine-nucleotide releasing protein for Ras. Upon growth factor stimulation, the Grb2-Sos complex is recruited from the cytosol to the plasma membrane to activate Ras (10, 11). v-Crk which has been shown to interact via its SH2 domain with tyrosine-phosphorylated epidermal growth factor receptors (EGFR) might have a similar function as Grb2 (12).()Sos and C3G, a cytosolic protein with homology to Ras guanine-nucleotide releasing protein, were recently shown to bind to the first SH3 domain of c-Crk(14, 15) . The demonstration that binding of the Grb2 SH2 domain to phosphotyrosine motifs did not change the binding affinity of the SH3 domain to Sos (16, 17) leads to the suggestion that SH2 and SH3 domains are not allosterically coupled within one adaptor molecule. The SH2 and SH3 domains of Crk, however, may be interdependent, the binding of one domain to its target eventuating in a new interaction involving the adjacent domain. The amino-terminal SH3 domain of c-Crk was shown to target the Abl family tyrosine kinases c-Abl and Arg. The subsequent phosphorylation of the c-Crk protein generates a binding motif for the SH2 domain of Crk(18, 19, 20) .

The molecular nature of the interactions between the SH2 and SH3 modules of Crk and their respective protein ligands is likely to determine signal specificity and therefore effector function. SH2 domain-phosphopeptide interactions have been well characterized, predicting that a phosphotyrosine residue is required for binding and that neighboring residues confer specificity. The Crk SH2 domain was shown to preferentially bind peptides that contain a phosphorylated Y-X-X-P motif(21, 22) . Progress toward the definition of binding motifs for SH3 domains and their functions has been made by screening expression libraries for SH3 domain binding proteins. The first such protein described was 3BP1(23) , which bound to the SH3 domain of Abl. The binding motif for the SH3 domain has been localized to a sequence rich in proline residues(24) . Similar results have been obtained from screens of combinatorial peptide libraries (25). Solution and crystal structures of SH3 domains revealed that the core of the domain consists of two perpendicular, anti-parallel, three-stranded -sheets. The most highly conserved residues form a hydrophobic surface which has been identified as ligand binding site (26, 27). SH3 domains recognize proline-rich motifs possessing the left-handed type II polyproline helix conformation. The pseudosymmetry of the polyproline type II helix explains the observation that proline-rich motifs interact in both axial orientations with SH3 domains(28, 29) . The polyproline helix has three residues per turn. Every third residue, that is residues at positions i and i + 3 lie on the same face of the helix(30, 31) . Two proline residues spaced by two amino acids, the P-X-X-P motif (i, i + 3), directly intercalate between the aromatic residues on the hydrophobic surface of the SH3 domain. Other prolines in the ligand appear to promote the helix formation, whereas neighboring non-proline residues are thought to determine specificity(25, 32) . The binding motif P-X-L-P-X-K(R) is present in all proteins demonstrated to interact with the amino-terminal Crk SH3 domain: Sos, Abl, Arg, and C3G(33) .

Identification of additional Crk SH3 domain binding proteins and determination of their respective binding motifs may help to elucidate the cellular effector function of Crk and further substantiate the P-X-L-P-X-K(R) motif as a specific Crk SH3 domain binding sequence. In the present work, we cloned two proteins which interact specifically with the SH3 domain of Crk by screening an expression library. One protein was identified as Eps15, an EGFR tyrosine kinase substrate which is involved in the control of cell proliferation(34) . The second protein exhibits a novel sequence related to Eps15 which we called Eps15R (R for related). Both proteins interact with the SH3 domain of Crk via a consensus P-A-L-P-P-K binding motif. Furthermore, we present evidence for a prolonged stable association of Eps15 with the stimulated EGFR in the presence of v-Crk.


MATERIALS AND METHODS

SH3 Domains Containing GST-Fusion Proteins

The expression vectors for glutathione S-transferase (GST), GST-Crk[SH3], GST-Crk[SH3][SH3], and GST-Nck have been described(18) . The expression vector for GST-Grb2 was a gift from Tadaomi Takenawa(35) , and the GST-Src[SH3] construct was kindly provided by David Baltimore (23). Expression and purification of the GST-fusion proteins was done as described(36) . S-Labeling of GST-Crk[SH3][SH3] was performed as detailed(37) .

Library Screening

A 16-day stage mouse embryo cDNA expression library constructed in phage EXlox vector (Novagen) was screened for Crk SH3 domain binding proteins. Binding to expressed proteins was detected with S-labeled GST-Crk[SH3][SH3] for the first screen and unlabeled GST-Crk[SH3][SH3] followed by anti-GST antibody and I-labeled Protein A (Amersham) for subsequent screens. 13 clones were purified from approximately 8 10 plaques. Automatic conversion of phage recombinants to plox plasmids was generated by infection of a bacterial host expressing the P1 cre recombinase (BM25.8 bacterial strain from Novagen), which in turn recognizes the loxP sites and forms the plasmid by site-specific recombination. Terminal sequencing of the plasmid preparations by the dideoxy-chain termination method using a commercial kit (UBI) revealed 6 independent clones. 4 clones were identified by a GenBank search using the BLAST program(38) , while 1 clone was determined by Southern hybridization. The final clone revealed a novel nucleotide sequence.

Eps15 and Eps15R GST-Fusion Constructs

The 1.6-kb partial cDNA clone of eps15 was excised from plox with EcoRI and HindIII and subcloned into pBluescript II SK (Stratagene). A fragment of 0.8 kb was cut out from the insert with PstI, and the purified vector was religated. The shortened insert of 0.8 kb was excised from pBluescript with BamHI and MscI and directionally subcloned into pGEX-3X to generate pGEX-Eps15aa713-884. The 0.8-kb partial clone of eps15R was excised from plox with EcoRI and HindIII and directly subcloned into pGEX-1N. The constructs were sequenced through the junctions to verify sequence fidelity and orientation. All regions of the eps15 and eps15R nucleotide sequences which encode proline-rich motifs (prm) were oligo-synthesized and cloned into pGEX-1N digested with EcoRI to generate pGEX-Eps15prm1, pGEX-Eps15prm2, pGEX-Eps15prm3, pGEX-Eps15Rprm2R, and pGEX-Eps15Rprm3R. The proline-rich peptide constructs were sequenced entirely. The full-length construct of eps15, pGEX-Eps15aa1-897, has been described(34) . Expression and purification of all GST-fusion proteins was done as detailed(36) . Purity and integrity of the fusion proteins was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (39) and Coomassie Blue staining.

Antisera

The Eps15 antiserum (RF99) was generated by immunizing rabbits with the GST-Eps15aa713-884 protein. The Eps15R antiserum (RF148) was produced in rabbits with the cloned GST-Eps15R fusion protein as immunogen. Polyclonal antibodies against GST were produced by immunizing rabbits with GST and purifying the sera through an affinity column. Anti-c-Crk (265) and anti-Gag (3C2) have been described(40) . A monoclonal anti-EGFR antibody directed against the extracellular receptor domain was purchased from Amersham.

Antibody Binding to Eps15 and Eps15R

Antibody binding to Eps15 and Eps15R was tested by immunoprecipitation and immunoblotting. HeLa cells were harvested in RIPA buffer containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2.5 mM EDTA, 1 mM dithiothreitol, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, 100 kallikrein inactivating units/ml aprotinin, 1.0 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 µM sodium molybdate, and 10 mM sodium fluoride. Lysates were cleared from particulate material by centrifugation for 10 min at 10,000 g. Protein concentration of cell lysates was determined by the Bradford method (Bio-Rad). Precipitations were performed with 0.5 mg of total lysate proteins and 5 µl of either anti-Eps15 or anti-Eps15R antiserum in 0.5 ml of RIPA buffer. The antibody complexes were immunopurified with Protein G-Sepharose beads (Pharmacia Biotech Inc.) and washed three times with RIPA buffer. Proteins were then separated by SDS-PAGE, transferred onto nitrocellulose, and probed with either anti-Eps15 or anti-Eps15R serum, each diluted 1:300 in binding buffer. Binding buffer contained Tris-buffered saline, 1 mM EDTA, 0.1% (v/v) Tween 20, 0.02% (w/v) sodium azide, 2% (w/v) bovine serum albumin, 1 mM dithiothreitol, and the aforementioned protease and phosphatase inhibitors. Bound antibodies were detected with I-labeled Protein A and autoradiography.

Far Western Blot

Expression of EXlox phage cDNA inserts was induced with isopropyl-1-thio--D-galactopyranoside for 3 h after infection of a bacterial host strain carrying the gene for T7 RNA polymerase (BL21[DE3]pLysE bacterial strain from Novagen). 5 ml of bacterial culture were pelleted by centrifugation (3000 rpm for 10 min) and lysed in 0.5 ml of RIPA buffer. Insoluble material was pelleted by microcentrifugation at 11,000 g for 10 min and analyzed by SDS-PAGE. Separated proteins were transferred onto nitrocellulose membranes (Immobilon) and blocked in binding buffer. Membranes were probed with GST-fusion proteins at 1 µg/ml in binding buffer for 3 h. Bound GST-fusion proteins were detected with a polyclonal antiserum to GST followed by I-labeled Protein A (Amersham).

Precipitation of Crk from Cell Lysates

Parental human carcinoma A431 cells and lines overexpressing c-Crk or v-Crk were cultured as described(41) . v-Crk-transformed rat fibroblasts and v-Crk expressing 3Y1 cells were also utilized(42) . Cell lysis was performed in 1% Nonidet P-40 buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and the inhibitors used for RIPA buffer. 10 µg of GST-fusion protein was incubated for 3 h at 4 °C with either 1 mg of protein from total A431 cell lysate or 300 µg from total v-Crk/3Y1 cell lysate in 0.5 ml of Nonidet P-40 lysis buffer. 15 µl of glutathione-Sepharose beads (Pharmacia Biotech Inc.) were subsequently added for 40 min to collect the protein complexes. All samples were washed four times with ice-cold Nonidet P-40 lysis buffer, boiled in electrophoresis buffer, and analyzed by SDS-PAGE. 50 µg of total cell lysate proteins were subjected to SDS-PAGE as a control. Western blots were blocked and probed with appropriate antisera to the Crk proteins. For detection of the primary antibodies, I-labeled Protein A or I-labeled antisera specific for mouse immunoglobulins (Amersham) were used.

In Vitro Transcription and Translation

The pCEV27-Eps15 plasmid (34) was linearized with SfiI and blunt-ended with the Klenow DNA polymerase, whereas the pBluescript-v-Crk plasmid (41) was linearized with XbaI. This allowed for selective transcription of the respective inserts and not of the remainder of the plasmid. After phenol extraction and ethanol precipitation of the linearized plasmids, 1 µg of plasmid DNA was used in 50 µl of transcription reactions using riboprobe reagents (Promega). Transcription reactions were performed with SP6 RNA polymerase for eps15 and T7 RNA polymerase for v-crk at 38 °C for 1 h. After transcription, RNase-free DNase (RQ1; Promega) was added to the reaction samples to digest plasmid DNA. Following phenol extraction, the RNAs were precipitated with ethanol and resuspended in 30 µl of RNase-free sterile water. For the in vitro translation, 10 µl of the in vitro-transcribed RNAs were incubated with rabbit reticulocyte lysate (Promega) and [S]methionine (Amersham) in a final volume of 60 µl under the conditions suggested by the manufacturer. Reactions were analyzed by SDS-PAGE and fluorography using an autoradiographic image enhancer (National Diagnostics).

Analysis of Eps15 Binding to the EGF Receptor in Presence of v-Crk

For each binding reaction, 1 µl of anti-EGFR antibody was incubated with 100 µg of total A431 lysate proteins for 90 min at 4 °C. Following an additional incubation for 40 min at 4 °C in the presence of 15 µl of Protein G-Sepharose beads (Pharmacia), the EGFR immunocomplexes were washed four times with ice-cold HNTG buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol and all previously mentioned protease and phosphatase inhibitors. The EGFR immunocomplexes were thereupon incubated with 5 µl of the in vitro-translated proteins (v-Crk and/or Eps15) in a final volume of 100 µl for 90 min at 4 °C. As indicated in the figure legends, EGFR tyrosine kinase was either activated by addition of 7 mM MnCl and 60 µM ATP followed by an incubation for 30 min at 30 °C or inactivated by the addition of 10 mM EDTA. Bead-coupled EGFR complexes were finally regenerated by centrifugation and washed four times with HNTG buffer. All binding reactions were analyzed by SDS-PAGE and fluorography.


RESULTS

Isolation of cDNA Clones Encoding Binding Proteins for the SH3 Domain of Crk

A commercial 16-day-old mouse embryo cDNA expression library was screened for Crk SH3 domain binding proteins. Six cDNA clones were purified and terminally sequenced, four of which were identified by computer-assisted sequence homology search with the GenBank data base. One cDNA clone of 1.6 kb corresponded to the newly discovered EGF receptor pathway substrate, Eps15, a 145-kDa protein phosphorylated by the activated EGFR tyrosine kinase (34), whereas three clones were translational artifacts of prothymosin (43), skeletal muscle actin(44) , and -1-globin(45) , respectively. Translational frame shifts of these clones led to artificial synthesis of Crk SH3 domain binding sequences. One cDNA clone was identified by Southern hybridization as the recently cloned Crk SH3 domain binding protein, C3G(15, 33) . The sixth cDNA clone of 0.8 kb revealed a unique nucleotide sequence which was entirely determined.

Identification of an Eps15-related Gene Product

The sequence of the 0.8-kb cDNA clone predicted an open reading frame of approximately 0.4 kb encoding 139 amino acids. The novel protein sequence revealed a 45% identity (61% similarity) to the carboxyl-terminal end of Eps15 and was therefore called Eps15-related, Eps15R (Fig. 1). To analyze the full-length gene product of this novel sequence and to compare it with Eps15, we generated polyclonal antisera against both proteins. The cDNA encoding amino acids 713-884 of Eps15 and the entire clone of Eps15R were expressed as GST-fusion proteins for use as immunogens. When added to HeLa cell lysate, the anti-Eps15 serum immunoprecipitated two proteins, a predominant one of 145 kDa and another of 155 kDa; the serum against Eps15R reacted with a single protein of 145 kDa (Fig. 2). On immunoblots, both antisera reacted with the immunoprecipitated 145-kDa protein species. To further show that these antisera recognize different epitopes, we immunoblotted the HeLa cell lysates and supernatants after immunoprecipitation with each antiserum. Anti-Eps15 antisera partly depleted both the 145-kDa and the 155-kDa bands, while anti-Eps15R antisera partly depleted only the 145-kDa band. The 145-kDa band might represent comigrating Eps15 and Eps15R proteins. The nature of the 155-kDa protein is unknown.


Figure 1: Amino acid sequence comparison between Eps15 and the partial gene product encoded by the eps15-related clone. The identity (colons) between the two amino acid sequences is 45%, while the similarity (colons and dots) is 61%. The boxed proline-rich domains are candidate binding motifs for the SH3 domain of Crk.




Figure 2: Identification of the Eps15R gene product. Western analysis using specific antisera noted on the bottom of figure were performed on (i) total HeLa cell lysates, (ii) HeLa cell lysates immunoprecipitated (IP) with anti-Eps15 or anti-Eps15R, and (iii) supernatants (SN) of the lysates after immunoprecipitation. Bound antibodies were detected with I-labeled Protein A. Molecular mass markers in kilodaltons are shown on the left. The 97-kDa band is nonspecific.



Specificity of the Isolated Crk-binding Proteins for Various SH3 Domains

Binding specificity of the expressed phage cDNA inserts of eps15 and eps15R was assessed by far Western blots probed with a panel of different SH3 domains fused to GST (Fig. 3). Partial eps15 (1.6 kb) and eps15R (0.8 kb) clones expressed as T7 gene10 carboxyl-terminal fusions bound strongly to the first SH3 domain of Crk, weakly to full-length Grb2, and negligibly to the SH3 domains of Nck and Src.


Figure 3: Binding specificity of cloned Eps15 and Eps15R gene products to various SH3 domains. Cloned phage inserts of eps15R and eps15 were expressed as fusions with 260-amino-acid T7 gene10 protein and analyzed by SDS-PAGE. The Western blots were probed with GST-Crk[SH3], GST-Grb2, GST-Nck, GST-Src[SH3], and GST as shown in the panels from left to right. Binding was detected with a polyclonal antibody to GST followed by I-labeled Protein A. Molecular mass standards are indicated in kilodaltons.



We then sought to determine whether Eps15 and Eps15R interact equivalently with both v-Crk and c-Crk. GST-fusion proteins of Eps15 and Eps15R ligand precipitated both c-Crk and v-Crk from overexpressing A431 cell lysates. GST-Eps15 corresponded to the carboxyl-terminal amino acids 713-884 of the full-length Eps15 protein while GST-Eps15R comprised the carboxyl-terminal 139 amino acids; these represent homologous regions and contain the putative Crk binding motif. Probing of the Western blots with specific antibodies to the Crk species revealed that both of these GST-fusion proteins bind equivalently to c-Crk and v-Crk (Fig. 4). 5 min of stimulation of A431 cells with 50 ng/ml EGF had no impact on Crk binding to the Eps15 and Eps15R fusion proteins.()


Figure 4: Precipitation of c-Crk and v-Crk from cell lysates with GST-Eps15 or GST-Eps15R. A431 cell lysates overexpressing c-Crk or v-Crk were either ligand-precipitated by GST-fusion proteins and analyzed by SDS-PAGE or directly separated by SDS-PAGE. GST was fused to either full-length Eps15 or to the partial Eps15R protein. The Western blot was probed for the presence of c-Crk with polyclonal antiserum followed by I-labeled Protein A; the presence of v-Crk was detected with a monoclonal antibody to Gag followed by I-labeled sheep anti-mouse antibody. Molecular mass standards (in kDa) are indicated on the left.



Amino Acid Sequences in Eps15 and Eps15R Responsible for Binding to the SH3 Domain of Crk

We generated GST-fusion peptides of proline-rich regions identified within Eps15 and Eps15R (boxed amino acid sequences in Fig. 1) as putative binding motifs for the first SH3 domain of Crk. There were three proline-rich motifs identified in the Eps15 protein sequence: (i) proline-rich motif 1 (prm1) for amino acids 206-216; (ii) prm2 for amino acids 770-780, and (iii) prm3 for amino acids 781-792. In Eps15R, we found amino acid stretches homologous to prm2 and prm3 and termed them prm2R and prm3R, respectively. Each GST-proline-rich motif (GST-prm) was analyzed separately for its respective ability to precipitate v-Crk from v-Crk expressing 3Y1 cell lysates (Fig. 5A). Western blots probed with anti-Gag monoclonal antibody showed that the amino-terminal SH3 domain of Crk binds preferentially to particular proline-rich motifs (prm2 and prm2R) within Eps15 and Eps15R, respectively. The amino-terminal proline-rich motif (prm1) in Eps15 lacks a P-X-X-P binding motif and consequently did not bind to the Crk SH3 domain (Fig. 5B). prm2 and prm2R, the regions responsible for the association of Eps15/15R with the SH3 domain of Crk, are highly homologous regions with identical P-A-L-P-P-K motif. A third proline-rich motif, prm3 or prm3R, bound only weakly the SH3 domain of Crk despite the presence of P-X-X-P motifs neighbored by charged lysine and arginine residues.


Figure 5: Identification of the binding motif for the SH3 domain of Crk in Eps15 and Eps15R. A, GST-fusion protein precipitates of v-Crk expressing 3Y1 cell lysates were subjected to SDS-PAGE and transferred to nitrocellulose. The Western blot was analyzed for the presence of v-Crk with a monoclonal antibody to Gag followed by I-labeled sheep anti-mouse immunoglobulin. GST was fused to full-length Eps15 protein, to the partial Eps15R protein, and to proline-rich motifs (prm1, -2, -3, -2R, and -3R) which had been indicated in Fig. 1. The migration of molecular mass markers (in kDa) are shown on the left. B, alignment of the proline-rich motifs (prm) of Eps15 and Eps15R. Indicated are the amino acid boundaries in the Eps15 protein sequence and their relative Crk binding affinities.



The binding motifs identified in Eps15 and Eps15R are remarkably similar to binding sequences in other known Crk SH3 domain interacting proteins (). They share with all listed proteins a P-X-L-P-X-K sequence. However, an arginine three amino acid residues carboxyl-terminal to the P-X-X-P motif is present in several Crk-binding proteins yet absent in both Eps15 and Eps15R.

Binding of v-Crk and Eps15 to the EGF Receptor

We found that the SH3 domain of Crk interacts in vitro with a specific proline-rich motif (prm2) within Eps15. Eps15 is an EGFR substrate which gets phosphorylated on tyrosine upon EGFR-kinase activation. However, a stable association of Eps15 with the EGFR has not been observed(34) . The SH2 domain of v-Crk binds in vitro and in vivo to the tyrosine-phosphorylated EGFR(12, 41) . Therefore, it was of interest to investigate how v-Crk and Eps15 might modulate each other's interactions with the EGFR.

Plasmids containing full-length eps15 and v-crk were transcribed in vitro, and the purified RNAs were translated in rabbit reticulocyte lysates in the presence of [S]methionine. The radiolabeled proteins were then tested for their abilities to bind to Sepharose-coupled EGFR (Fig. 6). In the absence of v-Crk, the phosphorylation of Eps15 by the immunopurified EGFR tyrosine kinase occurred without stable binding of the substrate to the kinase (Fig. 6, lane 1); Eps15 phosphorylation was confirmed by using radiolabeled ATP. In contrast, stable binding of Eps15 to the activated EGFR kinase was observed in the presence of v-Crk (Fig. 6, lane 2). Following binding to the autophosphorylated EGFR, v-Crk was phosphorylated by the activated receptor tyrosine kinase resulting in a doublet (Fig. 6, lanes 2, 4, and 5); the doublet reflects binding of both phosphorylated and unphosphorylated v-Crk to the EGFR. The tyrosine phosphorylation occurs within the Gag domain of v-Crk.()Both v-Crk bands showed similar intensities on the autoradiogram suggesting that roughly 50% of v-Crk bound to the EGFR was phosphorylated under the conditions of our experiment. However, as demonstrated by intensity differences between lanes 3 and 4, the total amount of v-Crk that associated with the EGFR was augmented if v-Crk remained unphosphorylated; phosphorylation of v-Crk appears to accelerate its dissociation from the receptor.


Figure 6: Interaction of Eps15 and v-Crk with the EGF receptor in vitro. In vitro-translated [S]methionine-labeled Eps15 and v-Crk were tested for their abilities to bind to Sepharose bead-coupled EGFR under various conditions. EGFR kinase reaction was initiated by the addition of ATP and MnCl in the presence of v-Crk, Eps15, or both. When indicated, the kinase reaction was terminated through addition of EDTA. The EGFR beads were then centrifuged and analyzed by SDS-PAGE and fluorography. Lane 1, kinase reaction with the EGFR and Eps15. Lane 2, kinase reaction with the EGFR, Eps15, and v-Crk. Lane 3, kinase reaction with the EGFR and Eps15 followed by kinase termination and addition of v-Crk. Lane 4, kinase reaction with the EGFR and v-Crk followed by kinase termination and addition of Eps15. Lane 5, kinase reaction with the EGFR, Eps15, v-Crk, and GST-prm2, the latter being a GST fusion protein of the 11-amino-acid proline-rich binding motif of Eps15.



A stable association of Eps15 with the EGFR occurred only in the presence of v-Crk and an active receptor tyrosine kinase (Fig. 6, lane 2). In lane 3, a kinase reaction was initiated in the presence of Eps15, and v-Crk was added subsequent to kinase inactivation with EDTA. The inactivation of the kinase was complete as shown by lack of a v-Crk band shift. Conversely, in lane 4, a kinase reaction was initiated in the presence of v-Crk, and Eps15 was added after kinase termination. Therefore, EGFR kinase inactivation did not prohibit binding of v-Crk; however, blocking kinase action of the EGFR-v-Crk complex did abolish subsequent binding of Eps15. Furthermore, phosphorylation of v-Crk was not sufficient for Eps15 binding as EGFR kinase inactivation after phosphorylation of v-Crk did not permit association of Eps15 (lane 4). Binding of Eps15 to the EGFR-v-Crk complex was competitively inhibited by excess GST-prm2 (Fig. 6, lane 5), while GST alone had no effect.

Eps15 phosphorylation is not sufficient for binding as demonstrated by the fact that when kinase-inactive EGFR is incubated with phosphorylated Eps15 and v-Crk, no binding was detected. Therefore, Eps15 bound to the EGFR only if (i) v-Crk was complexed and (ii) the EGFR kinase was active.


DISCUSSION

We identified by expression cloning two new targets of the SH3 domain of Crk. Our first clone, eps15, or EGFR pathway substrate 15(34) , was isolated as a protein that was phosphorylated on tyrosine upon stimulation of the EGFR. Overexpression of eps15 led to transformation of NIH3T3 cells suggesting a possible role of Eps15 in a mitogenic EGFR signaling pathway(34) . The human homologue of eps15 bears 89% similarity to murine eps15 and has been mapped to chromosome 1p31-p32.(46) . This region is a hot spot for nonrandom chromosomal abnormalities, exhibiting deletions in neuroblastoma as well as translocations in acute lymphoblastic leukemia(47, 48) . Indeed, two translocations t(1;11) (p32;q11) detected in myeloid leukemias fuse the HRX gene to AF-1p(49) , the latter being identical with human Eps15 by sequence comparison(46) . HRX (also called MLL, ALL-1, HTRX) is a putative transcription factor containing DNA binding domains, AT-hooks, zinc fingers, and methyltransferase regions(50, 51) . The structural features of the Eps15 protein suggested its subdivision into three domains(34) . Domain I contains a candidate tyrosine phosphorylation site and EF-hand helix-loop-helix calcium binding motifs. The amino acid sequence of domain I in Eps15 is 88% similar to the amino-terminal region of End3p, a protein required for the internalization step of endocytosis and for actin cytoskeletal organization in yeast(52) . Domain II includes a coiled-coil region, while domain III has both proline rich regions and repeats of aspartic acid-proline-phenylalanine, the latter suggestive of methyltransferase activity. It is intriguing that both HRX and Eps15 contain putative methyltransferase regions. The oncogenicity of HRX-Eps15 may be related to the fusion protein's ability to methylate DNA and thereby affect transcription. Nonetheless, neither the physiological function of Eps15 nor its role in neoplastic transformation has been elucidated in any detail.

Our second clone contained the partial sequence of a protein that is highly homologous to the carboxyl-terminal end of Eps15. We named this protein Eps15R for Eps15-related. Antibodies generated against this new protein immunoprecipitated a 145-kDa protein which was also detected by serum generated against the Eps15 protein. Anti-Eps15 antibodies recognized a major species migrating at 145 kDa and a minor species at 155 kDa. The molecular nature of those two proteins is not clear. One possibility is that p145 corresponds to Eps15R and p155 to Eps15. It has also been suggested that the 155-kDa species represents a post-translationally modified form of the 145-kDa Eps15 species(34) . This would be consistent with our data if both Eps15 and Eps15R are 145 kDa in the unmodified state and if Eps15R does not undergo significant alterations after translation. A final possibility is that Eps15R does not exist in HeLa cells as a protein and that the antisera generated against the expressed clone in fact recognizes the 145-kDa species of Eps15. Full-length sequencing of Eps15R will be necessary to ascertain whether or not there are functional or structural differences between Eps15 and Eps15R. We will also need to generate antibodies against unique epitopes in Eps15R and Eps15.

We identified in the carboxyl termini of Eps15 and Eps15R proline-rich motifs, prm2 and prm2R, which mediated the association of these proteins to the SH3 domain of Crk. prm2 and prm2R contain a sequence of identical amino acids, P-A-L-P-P-K, which also comprises a Crk-SH3 binding motif in C3G. The crystal structure of the Crk SH3 domain complexed with the 10-amino-acid binding motif of C3G (53) showed that the peptide bound in the same axial orientation as that of a 9-amino-acid Sos1 proline-rich peptide to the carboxyl-terminal SH3 domain of Grb2(28, 29) . The hydrophobic surface of the Crk SH3 domain bound the two coplanar positioned proline residues, Pro and Pro, of the polyproline type II helix in a conformational mode referred to as external packing (29). The second set of coplanar positioned residues, Leu and Lys, contact the SH3 domain of Crk in a mode referred to as internal packing(29) . The Lys residue, which is tightly coordinated by acidic residues in the RT loop of the Crk SH3 domain, is the key determinant of binding orientation, affinity, and specificity. Amino acids carboxyl-terminal to Lys do not appear to strongly affect binding(53, 54) .

However, a characteristic binding motif is not synonymous with an in vivo interaction. Although Eps15/15R and C3G share the same proline-rich binding motif, we failed to coimmunoprecipitate Eps15/15R with c-Crk or v-Crk from cell lysates. In addition, stimulation of the EGFR had no impact on the ability of native Eps15/15R to bind Crk. Only amino-terminal GST- or T7 gene10 protein-tagged Eps15/15R bound to Crk; we thus assume that the native conformations of Eps15 or Eps15R do not expose their Crk-SH3 binding motifs, whereas amino-terminal extensions or truncations might permit a surface presentation of these motifs.

In vivo, the conformation of Eps15 might change during transient interaction with the EGFR kinase, fostering an association with v-Crk within a heterotrimeric complex. Our data, using in vitro-translated, S-radiolabeled Eps15 and v-Crk proteins, supports this concept. Presumably, the EGFR interacts with the amino terminus of Eps15 through its kinase domain and the SH2 domain of v-Crk through its autophosphorylated tail; this allows the SH3 domain of Crk to bind to a proline-rich motif (prm2) of Eps15 (see model, Fig. 7). The EGFR contains at residue 992 a possible Crk-SH2 phosphotyrosine binding motif, Y-L-I-P(13, 21) . Eps15 association with the active EGFR-v-Crk complex was abolished in the presence of competing peptides containing the Crk SH3 domain proline-rich binding motif. Therefore, we assume that the transition state of Eps15 with the active EGFR kinase might expose the proline-rich motif in Eps15 permitting a ``capture'' by the v-Crk SH3 domain. Our inability to confirm an in vivo association between v-Crk and Eps15 may be largely due to a disruption of the tenuous Eps15-v-Crk-EGFR complex by the immunoprecipitation reaction.


Figure 7: Model of v-Crk mediated stable association of Eps15 with the stimulated EGF receptor. Stimulation of the EGFR leads to the recruitment of v-Crk and Eps15 from the cytoplasm to the membrane. The SH2 domain of Crk binds to a specific phosphotyrosine binding motif (pYXXP) in the carboxyl-terminal tail of the autophosphorylated receptor whereas the SH3 domain of Crk interacts with the proline-rich motif (PALPPK) in the carboxyl-terminal end of Eps15. The amino-terminal end of Eps15 contains a candidate tyrosine phosphorylation motif (Y) which gets phosphorylated by the EGFR kinase.



Stimulation of the EGFR should lead to the recruitment of Crk and Eps15 from the cytosol to the membrane. The biological consequences of a possible convergence of Eps15 and Crk on EGFR signaling remain to be investigated. Crk might decrease turnover of the receptor-substrate complex. In this respect, Crk might foster a prolonged association of Eps15 and the EGFR, altering the signal propagating from the complex. Similarly, adaptor proteins are likely to act as a bridge between the receptor, its substrate(s), and downstream effectors. Crk and Eps15 might be crucial links in the mitogenic signal emanating from the EGFR.

  
Table: X


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant CA44356 (to H. H.) and Training Grant CA09673 (to B. K.), a postdoctoral fellowship for research training in cancer from the International Agency for Research on Cancer, WHO (to T. O.), a physician postdoctoral research fellowship from the American Cancer Society (to R. H. G.), and research scholarships from the Swiss National Foundation for Science and the Bernese Society of Cancer in Switzerland (to C. S.). 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.

§
To whom correspondence and reprint requests should be addressed: Laboratory of Molecular Oncology, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327- 8802; Fax: 212-327-7943.

The abbreviations used are: SH2, Src homology 2; SH3, Src homology 3; Crk, CT10-related kinase; EGF, epidermal growth factor; EGFR, EGF receptor; Eps15, EGFR pathway substrate clone 15; Eps15R, Eps15-related; RIPA, radioimmune precipitation buffer; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s); GST, glutathione S-transferase; prm, proline-rich motif; aa, amino acid.

K. K. Teng, H. Landers, E. J. Fajardo, D. K. Mahadeo, H. Hanafusa, B. L. Hempstead, and R. B. Birge, submitted for publication.

C. Schumacher, data not shown.

R. B. Birge, unpublished results.


ACKNOWLEDGEMENTS

Useful suggestions and technical assistance provided by Eduardo Fajardo, Akiko Hata, Stephan Feller, and Mutsuko Ohuchi are gratefully acknowledged. We thank Ray Birge for comments and help on the manuscript.


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