Enhancement of Tyrosyl Phosphorylation and Protein Expression of Eps8 by V-Src

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Biochimica et Biophysica Acta 1450 (1999) 341^351 www.elsevier.com/locate/bba

Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src

Ming-Chei Maa a, Jun-Ru Lai b, Ruey-Wen Lin b, Tzeng-Horng Leu b;*

a Institute of Biochemistry, Chung Shan Medical and Dental College, No. 113, Section 2, Ta-Ching Street, Taichung 402, Taiwan, R.O.C. b Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, 70101 Taiwan, R.O.C.

Received 9 February 1999; received in revised form 28 April 1999; accepted 21 May 1999

Abstract

Two eps8 isoforms, p97eps8 and p68eps8, were previously identified as substrates for receptor tyrosine kinases. Analysis of eps8 phosphotyrosine content in v-Src transformed cells (IV5) revealed that both isoforms were highly tyrosyl phosphorylated and their readiness to be phosphorylated by Src in vitro further indicated that they were putative Src substrates as well. Indeed, the enhancement of tyrosyl phosphorylation of p97eps8 detected in cells coexpressing both p97eps8 and active Src relative to that in cells expressing p97eps8 alone supported our hypothesis. The existence of common phosphotryptic peptides between in vitro 32P-labeled p97eps8 and p68eps8 indicated that these two proteins shared the same Src-mediated sites. Further in vitro binding assays demonstrated that p68eps8 was the major eps8 isoforms that could be precipitated by bacterial fusion protein containing Src SH3. Interestingly, both p68eps8 and p97eps8 were preferentially expressed in v-Src transformed cells and the presence of p68eps8 appeared to depend on Src. Since p97eps8 has been implicated in mitogenesis and tumorigenesis, its readiness to be phosphorylated and induced by v-Src might attribute to v-Src-mediated transformation. ß 1999 Elsevier Science B.V. All rights reserved.

Keywords: v-Src; eps8; Tyrosyl phosphorylation; Protein expression

1. Introduction molecules, such as PLCQ, p85 subunit of PI-3 kinase, Grb2 and Shc become associated with EGFR [1]. EGF receptor (EGFR), a 170-kDa receptor tyro- Some of these proteins are further phosphorylated sine kinase, dimerizes and becomes autophosphory- by EGFR, enzymatically activated (e.g. PLCQ [2]) lated upon EGF stimulation. Through the binding and sometimes become the binding site(s) for other between pTyr and SH2, a number of SH2-containing SH2 containing protein(s) (e.g. the Grb2 binding site on Shc [3]), thereby leading to the propagation of the EGF-induced signaling. On the other hand, proteins Abbreviations: EGF, epidermal growth factor; SH2, Src ho- that devoid of SH2 domain, but still involved in mology domain 2; SH3, Src homology domain 3; pTyr, phos- EGF-induced signaling, have also been identi¢ed. A photyrosine; PLCQ, phospholipase CQ; PI-3K, phosphoinositide prominent example is eps8 [4]. 3-kinase; PH, pleckstrin homology; aa, amino acid; CE, chicken Eps8 was initially identi¢ed as a substrate for the embryo; ECL, enhanced chemiluminescence; 2D, two-dimensional EGFR whose tyrosyl phosphorylation was increased * Corresponding author. Fax: +886-6-274-9296; in response to EGF [4]. Further studies indicated E-mail: [email protected] that eps8 could also be phosphorylated in response

BBAMCR 14497 29-6-99 342 M.-C. Maa et al. / Biochimica et Biophysica Acta 1450 (1999) 341^351 to stimulation by various ligands including platelet- First of all, both p97eps8 and p68eps8 were highly derived growth factor and acidic ¢broblast growth tyrosyl phosphorylated in v-Src transformed cells. In factor [4]. There is a direct interaction between an immunocomplex kinase reaction, v-Src could di- EGFR and eps8 that involves the juxtamembrane rectly phosphorylate both eps8 isoforms in vitro. In- region of EGFR and the basic region of eps8, respec- terestingly, the bacterial GST fusion protein contain- tively [5]. Direct cloning of the cDNA of the EGFR ing the N-terminus of p97eps8 not only could be substrates led to the isolation of p97eps8 cDNA. And directly phosphorylated by baculovirus expressed its sequence analysis revealed the following domains Src, but also shared a common phosphotryptic pep- which might contribute to its cellular functions: an tide derived from v-Src phosphorylated p97eps8. Be- SH3 domain, a putative nuclear targeting sequence, a sides, bacterial fusion protein containing Src SH3 PH domain, several potential SH3-binding sites, and could preferentially precipitate p68eps8 in the in vitro a degenerated SH2 at the amino terminus. The eps8 binding reactions. Finally, enhanced tyrosyl phos- SH3 domain has been shown to interact with Shc [6], phorylation of p97eps8 was detected in cells coex- Shb [7], RN-tre [8], and e3B1 [9]. And interestingly, pressing both p97eps8 and active Src, but not in cells based on its deduced tertiary structure, eps8 SH3 expressing p97eps8 alone. Interestingly, in addition to domain has also been implicated in the formation tyrosyl phosphorylation, the protein levels of both of eps8 dimer [10]. Meanwhile, proteins associated eps8 isoforms were much higher in v-Src transformed with other domains of eps8 have not been identi¢ed. cells than the normal control cells. Since p97eps8 has p97eps8 has been implicated in EGF-induced mito- been implicated in mitogenesis and tumorigenesis, its genesis and tumorigenesis since its overexpression readiness to be phosphorylated and induced by v-Src in cells can enhance the EGF-dependent mitogenic may contribute to the transformation caused by v- e¡ect and focus-forming ability [4,6]. Consistent Src. with this notion was its constitutive tyrosyl phosphorylation detected in several tumor cell lines [6]. Interestingly, in some cell lines, antibodies against 2. Materials and methods p97eps8 could also recognize a 68-kDa protein (p68eps8), which has been speculated as a proteolytic 2.1. Cloning of the murine p97eps8 full-length open or an alternatively spliced product of p97eps8 [4]. The reading frame (ORF) and the generation of its exact nature of the coding sequences and the func- expression plasmid tions of p68eps8 remain to be established. In addition to mitogenesis and tumorigenesis, the reduced ex- According to the published sequences of p97eps8 pression of both eps8 isoforms in myogenic cells sug- cDNA [4], two 18-mers P1 and P2 (nt 606^623 and gest that eps8 might also be involved in terminal nt 908^923, respectively; Fig. 1) were utilized as pri- di¡erentiation [11]. mers for polymerase chain reaction (PCR) of the Rous sarcoma virus (RSV)-encoded v-Src is the cDNA derived from mouse 11-day embryo (Clon- ¢rst identi¢ed oncoprotein with tyrosine kinase activ- tech). A PCR product with 318 nucleotides was gen- ity. Its tyrosine-speci¢c protein kinase activity is pro- erated and utilized as a probe to screen the mouse ven to be essential for cellular transformation. In 11-day embryo cDNA library. Two eps8-related v-Src transformed cells, more than 20 proteins have Vgt11 clones, 2Aa and 12Aa (Fig. 1), were isolated their pTyr contents increased and are viewed as pu- from V106 plaques and shown to be completely tative v-Src substrates [12^14]. Interestingly, some of overlapped. Both clones have been sequenced with them (i.e. cortactin, Shc, and GAP) turn out to be Sanger-dideoxy method and mapped with restriction the substrates for EGFR as well [15^20]. It was enzyme digestion. The 2Aa clone contains almost all speculated that constitutive phosphorylation of these the ORF of p97eps8 except the sequences correspond- common substrates in cells expressing v-Src might ing to the C-terminal end of eps8 (amino acid (aa) contribute to cellular transformation. In this study, 668^821). In order to make up the missing protein- we present evidence, as shown below, describing that encoding region of p97eps8, another set of oligonu- eps8 isoforms were Src substrates as well. cleotides, P3 and EcoRI-tagged P4, which corre-

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The eps8 cDNA (i.e. the NaeI/HindIII digested fragment derived from the plasmid pBS-eps8) was ¢rst subcloned into the SmaI and HindIII sites of the Cla12Nco vector and then recloned into the ClaI site of an avian retroviral expression vector, RCAS A (BH) as described previously [22]. The pGEX-N/ RV which encodes the N-terminal p97eps8 (i.e. GST- N/RV, aa 1^179) is constructed as the following: the NaeI/EcoRV fragment derived from PBS-eps8 was ¢rst cloned into the SmaI site of pGEM-7Zf(+) and then the BamHI/EcoRI fragment derived from this new construct was ligated with the BamHI/ EcoRI-digested pGEX-2T.

2.2. Cells and cellular lysate preparation

The clonal C3H10T1/2 murine ¢broblast cell lines (Neo and IV5) were generous gifts provided by Dr. Sarah J. Parsons and their derivation and mainte- nance were previously described [23,24]. Primary Fig. 1. Schematic illustration of various eps8 constructs. The chicken embryo (CE) cells were maintained and full-length eps8 cDNA is indicated on the top and its open transfected with retroviral vector as previously de- reading frame is marked as a closed box. Two Vgt11 clones, scribed [25,26]. Replication-competent RCAS A 12 Aa and 2 Aa, obtained by screening a mouse 11-day embryo (BH) and RCAS B (BH) retroviral vectors were cDN library are demonstrated. The full-length open reading eps8 frame of p97eps8 was constructed in Bluescript vector (pBS- used for the expression of p97 and active Src eps8) and RCAS (A) vector (RCAS-eps8) as described in Sec- (Src 518amb, a Src mutant with an amber mutation tion 2. Also indicated is pGEX-c-eps8 that encodes a bacterial at amino acid residue 518 [27]), respectively. For GST fusion protein containing C-terminal eps8 (aa 355^668). generation of CE cells overexpressing both p97eps8 The restriction enzyme cutting sites are indicated above each and active Src, cultured medium containing high-titer drawing (B, BamHI; N, NaeI; Bg, BglII; P, PvuII; R, EcoRI). P1, P2, P3 and P4 represented the primers utilized in the PCR viral stock was collected from Src transfected cells eps8 reactions described in Section 2. and incubated with p97 overexpressors. After few passages, the expression of Src and p97eps8 in these cells was con¢rmed. Cells grown to near con- spond to nt 32072 to 32091 and nt 32705 to £uency were lysed in modi¢ed RIPA bu¡er as de- 32720, respectively (Fig. 1), were utilized as primers scribed before [26] and protein concentration was for the PCR of the same mouse embryonic cDNA. determined by protein assay kit (Bio-Rad). The resulting PCR product was digested with PvuII/ EcoRI and this fragment was inserted into the 2.3. Antibody generation, immunoprecipitation and EcoRI-digested pBluescript vector along with the immunoblotting of eps8 EcoRI/PvuII-digested 2Aa to generate the full length of p97eps8-containing pBS-eps8 plasmid (Fig. 1). The Rabbit antiserum against carboxyl portion of bacterial expression plasmid (pGEX-c-eps8; Fig. 1) p97eps8 (C-eps8) was raised against a GST-eps8 bac- was constructed by cloning the BglII/EcoRI digested terially expressed fusion protein containing murine C-terminal eps8 fragment (which encodes aa 3355 to p97eps8 residues 355^668. Near 1 mg of cellular ly- 3668) into the BamHI/EcoRI sites of a GST expres- sates were immunoprecipitated with either C-eps8 sion vector pGEX-1 [21]. The construction of eu- antibody generated in our laboratory or the eps8 karyotic p97eps8 expression plasmid (i.e. RCAS- antibody provided by Dr. Pier Paolo Di Fiore [4]. eps8, Fig. 1) is brie£y described as the following. The eps8 immunocomplexes were resolved on 8%

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Fig. 2. Both p97eps8 and p68eps8 are highly tyrosyl phosphorylated in v-Src transformed cells. Equal amounts (1 mg) of cell lysates prepared from Neo (odd numbers) or IV5 (even numbers) were immunoprecipitated with rabbit polyvalent serum against eps8 either gen- erously provided by Dr. Di Fiore (eps8 Ab) or prepared in our laboratory (C-eps8). Immunoprecipitation carried out with non-speci¢c rabbit serum (PI) was used for negative control. The immunocomplexes were equally split into two parts; one-half was immunoblotted with antibody against eps8 (DiFiore's Ab) (A), and the other half was analyzed by pTyr Western immunoblotting (B). IP, immunoprecipitation.

SDS-polyacrylamide gels, transferred to nitrocellu- 2.5. Phosphoamino acid analysis lose membranes and probed with rabbit polyvalent serum against phosphotyrosine or eps8. Binding of The 32P-labeled p97eps8 was excised from the gel, rabbit antiserum to membrane was detected with eluted out, precipitated with trichloroacetic acid, protein A labeled with 125I or conjugated with horse- washed with ethanol and hydrolyzed in 100 Wlof radish peroxidase (ECL; Amersham). 6.0 M HCl at 110³C for 30 min [28]. Hydrolyzed 32P-labeled p97eps8 (V500 cpm) was mixed with 2.4. In vitro kinase reactions 0.3 Wg of each authentic phosphoserine, phosphothreonine and phosphotyrosine, and resolved by 2D p97eps8 was immunoprecipitated from C3H10T1/2 thin layer electrophoresis [28]. Ninhydrin staining of cells while v-Src was immunoprecipitated from IV5 authentic phosphoamino acids was used as a refer- cells. These two immunoprecipitates were either in- ence for autoradiographic identi¢cation of labeled cubated alone or together in the kinase bu¡er (5 mM phosphoamino acids. MgCl2, 5 mM MnCl2, 20 mM PIPES (pH 7.5), 50 WM ATP) containing 10 WCi [Q-32P]ATP (Dupont/ 2.6. Two-dimensional phosphotryptic NEN) for 15 min at room temperature. Incubations peptide mapping were terminated by addition of sample bu¡er. After 5-min boiling, the labeled products were resolved by The 32P-incorporated p97eps8 and p68eps8 were ex- SDS/PAGE and visualized by autoradiography. The tracted from an SDS-polyacrylamide as described same experiment was also carried out by incubation above. Each protein was then digested with trypsin of both the bacterial expressed GST-N/RV fusion and analyzed by 2D-thin layer chromatography as protein and the puri¢ed baculovirus-expressed hu- described previously [26]. Brie£y, the tryptic peptides man c-Src (UBI, Lake placid, New York). of each digestion (V1000 cpm) were resolved on

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Fig. 3. v-Src was capable of phosphorylating p97eps8 in vitro. Eps8 and Src immunocomplexes were derived from Neo and IV5, respectively. These two immunocomplexes were either incubated alone or mixed together, and subjected to in vitro kinase reactions. The 32P-labeled proteins were analyzed by SDS/PAGE and detected by autoradiography (20 min exposure) (A). The position of p97eps8 is indicated on the left. A potential 32P-labeled p68eps8 in the Src immunocomplex is marked with an asterisk. The 32P-labeled p97eps8 was excised and extracted for phosphoamino acid analysis by 2D electrophoresis (the ¢rst dimension was in pH 1.9 bu¡er, and the second dimension was in pH 3.5 bu¡er) (B). pS, phosphoserine; pT, phosphothreonine; pY, phosphotyrosine. cellulose thin-layer plates by electrophoresis in the 3. Results ¢rst dimension (15 min at 1500 V in pH 1.9 bu¡er [28]) and ascending chromatography in the second 3.1. Eps8 was highly tyrosyl phosphorylated in v-Src dimension (in phospho chromatography bu¡er transformed cells [28]). To test the possibility that eps8 may be a substrate 2.7. In vitro solution binding assay for Src, total lysates prepared from normal (Neo) or v-Src transformed cells (IV5) were immunoprecipi- Bacterially expressed GST fusion proteins contain- tated with eps8 polyclonal antibodies that either pro- ing Src SH3 and SH2 alone or both were immobi- vided by Dr. Di Fiore (eps8, Fig. 2) or generated in lized on glutathione-Sepharose 4B beads as described our laboratory (C-eps8). The anti-eps8 immunopre- previously [25]. Then beads containing various cipitates were then examined for protein tyrosyl amounts of each fusion protein were incubated phosphorylation by anti-phosphotyrosine (anti- with 400 Wg of cellular lysates prepared from IV5 pTyr) immunoblotting. As demonstrated in Fig. 2B, cells. After 12 h of gentle mixing at 4³C, complexes while both p97eps8 and p68eps8 isoforms were highly were gently washed with modi¢ed RIPA bu¡er once tyrosyl phosphorylated in IV5 cells, little tyrosyl and Tris-saline bu¡er (150 mM NaCl, 50 mM Tris- phosphorylation of these proteins was observed in HCl, pH 7.4) three times and Western blotted with normal cells. Furthermore, anti-eps8 immunoblotting monoclonal anti-eps8 antibody purchased from revealed that p97eps8 was the major form of eps8 ex- Transduction Laboratories (Kentucky, USA) and de- pressed in normal cells and about twice as much was tected by ECL. expressed in v-Src transformed cells (Fig. 2A, lanes

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3.2. v-Src can directly phosphorylate p97eps8 in vitro

The increased phosphorylation of eps8 isoforms in v-Src transformed cells can be attributed to either direct or indirect phosphorylation mediated by v- Src. To di¡erentiate these two possibilities, eps8 immunoprecipitates prepared from normal cells (Neo) were either incubated alone or with IV5-derived v- Src immunoprecipitates in the presence of [Q-32P]ATP and subjected to in vitro kinase assays. As shown in Fig. 3A, in the absence of v-Src, eps8 immunoprecipitates prepared from normal cells did not exhibit any obvious 32P-labeled proteins. By contrast, v-Src and its associated proteins became 32P-labeled in v- Src immunoprecipitates. When eps8 and v-Src immunocomplexes were incubated together, p97eps8 became 32P-labeled. To exclude the possibility that p97eps8 is phosphorylated by the Src-associated ser- ine/threonine kinase and not by Src itself, phosphoamino acid analysis of in vitro 32P-labeled p97eps8 was carried out. As shown in Fig. 3B, only 32P-labeled phosphotyrosine was detected. This result suggested that v-Src was capable of directly phosphorylating p97eps8. A similar result was detected when Src immunocomplex was substituted by baculovirus ex- Fig. 4. Baculovirus-expressed Src is capable of phosphorylating pressed Src to carry out the in vitro kinase reactions the bacterial GST fusion protein containing the N-terminus of (data not shown). Further evidence came from the in p97eps8 in vitro. In vitro kinase reactions were carried out with the GST fusion protein containing the N-terminus of p97eps8 vitro phosphorylation of the bacterial GST fusion eps8 (GST-N/RV) in the presence or absence of baculovirus-ex- protein containing the N-terminus of p97 (GST- pressed Src (Bac Src). The 32P-labeled proteins were analyzed N/RV; with amino acids 1^179) by baculovirus ex- by SDS/PAGE and detected by autoradiography (10 min expo- pressed Src (Fig. 4A). Analysis of the phosphotryptic sure) (A). The tryptic phosphopeptide map of baculovirus Src map of the 32P-incorporated GST-N/RV mediated by phosphorylated GST-N/RV fusion protein was prepared (1000 baculovirus expressed Src revealed the co-migration cpm) (B, upper panel). To con¢rm the identity of the major phosphopeptide, the recovered arrow-pointed one (120 cpm) of the major phosphotryptic peptide (Fig. 4B, upper was mixed with the tryptic peptides derived from v-Src phos- panel) with the one generated from v-Src phospho- phorylated p97eps8 (500 cpm) (B, lower panel). For comparison, rylated p97eps8 (Fig. 4B, middle panel; spot d). We the tryptic phosphopeptide map of v-Src phosphorylated p97eps8 concluded that p97eps8 was indeed directly phos- is also demonstrated (1000 cpm) (B, middle panel). Unambigu- phorylated by Src, at least, on a Tyr residue located ously, the arrow-pointed spot is identical to spot d as desig- nated in Fig. 5. The asterisks represent the position of origin in at its N-terminus. all panels. 3.3. The existence of p68eps8 in v-Src immunocomplexes 2 and 4). And surprisingly, the expression of p68eps8 was dependent on the presence of v-Src (Fig. 2A). Carefully examined the 32P-labeled proteins in v- None of eps8 was detected in the immunocomplexes Src immunoprecipitates, a protein with the molecular precipitated by preimmunized antibody, although a weight close to 68 kDa was detected (Fig. 3A, band few non-speci¢c tyrosyl phosphorylated proteins marked with asterisk). This prompted us to speculate were recognized (Fig. 2, lanes 5 and 6). the possibility that this protein might be p68eps8.To

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Fig. 5. Comparative tryptic phosphopeptide maps for in vitro Src-phosphorylated p97eps8 and p68eps8. The 32P-incorporated p97eps8 (A) and p68eps8 (B) harvested from the in vitro kinase reaction (Fig. 3) were digested with trypsin. The resulting phosphopeptides (V1000 cpm) were separated into two dimensions by electrophoresis (pH 1.9) and chromatography on thin layer plates and detected by autoradiography as described in Section 2. (C) Tryptic map of the mixture of peptides in A and B. (D) Schematic drawing of the eps8-derived phosphotryptic peptides mediated by Src. The asterisk represents the position of origin in each panel. prove this speculation, the in vitro 32P-labeled p97eps8 the interaction between these two proteins. Since and the asterisk-marked protein were eluted from the there were putative SH3-binding motifs in p97eps8, gel, subjected to trypsin digestion and phosphotryp- we wondered whether it was possible that through tic peptide map analysis. As shown in Fig. 5, the these sites eps8 isoforms were capable of associating common phosphotryptic peptides shared by these with Src SH3 domain. To address this question, two proteins (spots a, b, c, e) indicating that the whole cell lysates prepared from v-Src transformed asterisk-marked protein should correspond to cells were incubated with GST-SrcSH2, GST- p68eps8 since they shared the common phosphory- SrcSH3, or GST-SrcSH3SH2. The proteins bound lated sites mediated by v-Src. Also, the detection of to these bacterial fusion proteins were then resolved p68eps8 in v-Src immunocomplexes suggested the as- with SDS/PAGE and immunoblotted with anti-eps8 sociation between p68eps8 and v-Src in vivo (see Sec- antibody. As demonstrated in Fig. 6, though tiny tion 4). amounts of p97eps8 were associated with SrcSH3, p68eps8 was the major eps8 isoforms that could com- 3.4. v-Src can preferentially associate with p68eps8 in plex with SrcSH3. By contrast, no association was vitro through its SH3 domain detected between eps8 isoforms and SrcSH2 and no synergistic e¡ect was observed in SrcSH3SH2. This Though no available co-immunoprecipitation data implicated that SH3 was the domain within v-Src supporting the association between eps8 isoforms that could mediate the interaction between eps8 iso- and v-Src, the detection of p68eps8 in v-Src immuno- forms and Src. precipitates in the in vitro kinase reaction did suggest

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levels and their pTyr contents revealed that only in the presence of active Src can p97eps8 become highly tyrosyl phosphorylated (Fig. 7A,B, lanes 5 and 6). This result suggests that indeed active Src can mediate p97eps8 phosphorylation in vivo. The failure of detection of chicken endogenous p97eps8 might be due to the antibodies used in this study cannot recognize the chicken counterpart and/or there was no p97eps8 expression in chicken cells.

4. Discussion

In this study, based on the following observation, we demonstrated that both eps8 isoforms were Src substrates. These were: (1) v-Src immunocomplexes and baculovirus-expressed c-Src could directly phosphorylate both eps8 isoforms in vitro; (2) the detection of p68eps8 in v-Src immunoprecipitates; (3) association with p68eps8 with SrcSH3; and (4) expression of v-Src or active Src could lead to the enhancement of p97eps8 tyrosyl phosphorylation in vivo. Since EGFR and Src are two common active tyrosine ki- Fig. 6. Solution binding of eps8 to Src SH3 domain in v-Src nases involved in neoplastic growth and both are transformed cells. Lysates (400 Wg) from IV5 cells were incu- capable of phosphorylating the two eps8 isoforms, bated with GST-SrcSH3 2 Wg (lane 2); 4 Wg (lane 3); GST- eps8 SrcSH3SH2 8 Wg (lane 5); GST-SrcSH2 6 Wg (lane 6) as de- the constitutive tyrosyl phosphorylation of p97 scribed in Section 2. The precipitated proteins in each group detected in several tumor cell lines may be mediated along with IV5 lysates (15 Wg) (lane 1) and GST-SrcSH3 4 Wg by these kinases or their relatives. Comparing to the alone (lane 4) were resolved on SDS/PAGE and analyzed by eps8 immunoprecipitates alone, the failure of detect- Western immunoblotting with monoclonal eps8 antibody pur- ing the enhancement of 32P-labeled p68eps8 in the chased from Transduction Laboratories. The positions of various GST fusion proteins are marked with asterisks. presence of Src was likely due to the low abundance of p68eps8 in Neo lysates that was utilized for the preparation of eps8 immunocomplexes (Fig. 3A, 3.5. Active Src can mediate the phosphorylation of left and middle lanes). A 32P-labeled protein with p97eps8 in vivo the molecular weight of 68 kDa, however, was detected in the Src immunocomplexes prepared from Once p97eps8 was con¢rmed to be a direct substrate IV5 cell lysates (Fig. 3A, middle lane; protein for v-Src in vitro, the next question turned out to be marked with an asterisk). Further 2D phosphotryptic whether an active Src was capable of mediating peptide analysis of the in vitro 32P-labeled p97eps8 p97eps8 phosphorylation in vivo. To address this and the putative p68eps8 revealed that both shared question, a retrovirus expression system was utilized most of the tryptic peptides (Fig. 5) although Src- to express p97eps8 alone or p97eps8 and active Src (i.e. mediated sites were preferentially located on d for Src518am) both in chicken embryo (CE) cells. As p97eps8 and c, e for p68eps8 respectively. Since d was shown in Fig. 7A, multiple tyrosyl phosphorylated also detected in the tryptic map derived from GST proteins were detected in cells overexpressing both fusion protein containing the ¢rst 179 N-terminal p97eps8 and active Src as compared to that in paren- amino acids of p97eps8 phosphorylated by baculovi- tal CE cells and cells overexpressing p97eps8 alone rus expressed Src (Fig. 4B), thus, it implicated that (Fig. 7A, lanes 1^3). Further analysis of the eps8 most likely p68eps8 did not contain this region or this

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Fig. 7. Active Src can mediate p97eps8 tyrosyl phosphorylation in vivo. Cellular lysates (lanes 1^3) and p97eps8 immunocomplexes (lanes 4^6) were resolved in an SDS/PAGE and analyzed by Western immunoblotting with either pTyr antibody (A) or C-eps8 antibody (B). The position of p97eps8 and IgG are indicated on the right. Lanes 1 and 4, CE cells; lanes 2 and 5, CE cells expressing p97eps8 ; lanes 3 and 6, CE cells expressing both active Src (Src 518amb) and p97eps8. region was preferentially phosphorylated in p97eps8. most likely candidate. Further experiments are now These results not only suggested that Src could phos- in progress to identify and characterize the Src-medi- phorylate these two eps8 isoforms at the same tyro- ated sites on p97eps8. sine residues, but also con¢rmed that these two eps8 The detection of p68eps8 in v-Src immunocom- isoforms were indeed derived from the same gene plexes in the in vitro kinase reaction suggested the product. association of p68eps8 and v-Src in vivo. Indeed, we Previously, Gallo et al. described the enhancement demonstrated that it was the Src SH3, not SH2 of p97eps8 tyrosyl phosphorylation in response to Src which mediated this interaction (Fig. 6) and the stoi- activation [11] and the similar result was observed in chiometry was estimated to be 2^3% in our experi- this study. However, one cannot exclude the possi- ments. This low stoichiometry could be interpreted bility that other Src-activated tyrosine kinases ac- by the weak interaction between Src SH3 and the tually do this job. To clarify this point, we demon- proline-rich sequence. Thus, we had di¤culty in de- strated that both Src immunocomplexes and puri¢ed tecting both eps8 isoforms in the v-Src immunocom- baculovirus-expressed Src could indeed mediate plexes in the traditional co-immunoprecipitation ex- p97eps8 phosphorylation and a tyrosine residue lo- periments. Since association with the Src SH3 or cated at p97eps8 N-terminus turned out to be one of SH2 domain usually leads to tyrosyl phosphorylation the sites mediated by Src. Based on the conclusion of various Src substrates [30], therefore, it is likely derived from Songyang et al. E/D-E/D-I/V/L-Y-G/E- that the same mechanism results in the tyrosyl phos- E-F-D/E was the speci¢c sequence that can be phosphorylation of eps8. In supporting this hypothesis, phorylated by Src [29]. However, no such sequence we observed that baculovirus expressed Src can me- was present at the N-terminus of p97eps8. Neverthe- diate the phosphorylation of GST-N/R. However, less, sequences composing Tyr-12 (G-Y-G), Tyr-15 the phosphorylation stoichiometry was low (0.4 mol (V-Y-P), Tyr-22 (G-Y-G), Tyr-45 (L-Y-E) showed Pi/mol; data not shown), and this result might be limited homology with the Src phosphorylation con- due to the absence of Pro-rich sequences in the sensus sequences and among them, Tyr-45 was the N-terminal 179 amino acids of p97eps8. Further ex-

BBAMCR 14497 29-6-99 350 M.-C. Maa et al. / Biochimica et Biophysica Acta 1450 (1999) 341^351 periments demonstrating reduction or abolishment of his critical comments on this manuscript. This eps8 tyrosyl phosphorylation in cells expressing SH3- work is supported by National Science Council defective Src are needed to support this hypothesis. Grant to M.-C.M. (NSC 88-2314-B-040-020) and Meanwhile, analysis of amino acid sequences of eps8 grants of NHRI to T.-H.L. (DOH-86-HR-625, revealed several proline-rich regions potential for Src DOH-87-HR-625 and DOH-88-HR-625). SH3 binding. However, none of them resembled the known optimal Src-SH3 binding motif, such as RPLPXXP [31,32]. Synthesis of these proline-rich References peptides and determination of their Src SH3 binding a¤nity can pinpoint the one responsible for Src SH3 [1] T. Pawson, J. Schlessinger, SH2 and SH3, Curr. Trends Biol. binding. 3 (1993) 434^442. In addition to tyrosyl phosphorylation, the ele- [2] B. Margolis, S.G. Rhee, S. Felder, M. Mervic, R. Lyall, A. Levitzki, A. Ullrich, A. Zilberstein, J. Schlessinger, EGF vated expression of both eps8 isoforms was also ob- induces tyrosine phosphorylation of phospholipase C-II: a served in v-Src transformed cells (Fig. 2A). Further potential mechanism for EGF receptor signaling, Cell 57 analysis of the Northern blots prepared from normal (1989) 1101^1107. and v-Src transformed cells revealed that the abun- [3] M. Rozakis-Adcock, J. McGlade, G. Mbamalu, G. Pelicci, dance of two major eps8 transcripts paralleled with R. Daly, W. Li, A. Batzer, S. Thomas, J. Brugge, P.G. Pel- that of their encoded proteins (data not shown). icci, J. Schlessinger, T. Pawson, Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activa- Thus, the di¡erential expression of eps8 isoforms in tion of the Ras pathway by tyrosine kinases, Nature 360 normal and v-Src transformed cells might be in part (1992) 689^692. due to transcriptional regulation. Since overexpres- [4] F. Fazioli, L. Minichiello, V. Matoska, P. Castagnino, T. sion of p97eps8 has been implicated in the enhance- Miki, W.T. Wong, P.P. Di Fiore, Eps8, a substrate for the ment of EGF-induced mitogenesis and tumori- epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals, EMBO J. 12 (1993) 3799^3808. genesis, we speculated that Src-mediated eps8 [5] P. Castagnino, Z. Biesova, W.T. Wong, F. Fazioli, G.N. overexpression might minimize the requirement of Gill, P.P. Di Fiore, Direct binding of eps8 to the juxtamem- growth factors (or serum) needed for cellular growth brane domain of EGFR is phosphotyrosine- and SH2-inde- and contributed to the oncogenesis induced by Src. pendent, Oncogene 10 (1995) 723^729. This hypothesis is now under investigation. [6] B. Matoskova, W.T. Wong, A.E. Salcini, P.G. Pelicci, P.P. The signi¢cance of tyrosyl phosphorylation and/or Di Fiore, Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation, Mol. Cell. eps8 expression of p97 in mitogenesis and tumorigene- Biol. 15 (1995) 3805^3812. sis has been implicated by Matoskova et al. [6]. [7] T. Karlsson, Z. Songyang, E. Landgren, C. Lavergne, P.P. Based on our ¢ndings in this study, it was likely Di Fiore, M. Ana¢, T. Pawson, L.C. Cantley, L. Claesson- that Src-mediated tyrosyl phosphorylation and pro- Welsh, M. Welsh, Molecular interactions of the Src homol- tein expression of eps8, especially the p68eps8, may be ogy 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain pro- involved in v-Src-induced transformation. Further teins, Oncogene 10 (1995) 1475^1483. studies of the levels of eps8 tyrosyl phosphorylation [8] B. Matoskova, W.T. Wong, N. Nomura, K.C. Robbins, P.P. and protein expression in cell lines expressing various Di Fiore, RN-tre speci¢cally binds to the SH3 domain of v-Src mutants that are transformation defective will eps8 with high a¤nity and confers growth advantage to be needed to support this speculation. NIH3T3 upon carboxy-terminal truncation, Oncogene 12 (1996) 2679^2688. [9] Z. Biesova, C. Piccoli, W.T. Wong, Isolation and characterization of e3B1, an eps8 binding protein that regulates cell Acknowledgements growth, Oncogene 14 (1997) 233^241. [10] K.V. Kishan, G. Scita, W.T. Wong, P.P. Di Fiore, M.E. We would like to thank Drs. Pier Paolo Di Fiore, Newcomer, The SH3 domain of Eps8 exists as a novel in- W.T. Wong and Sarah J. Parsons for providing eps8 tertwined dimer, Nat. Struct. Biol. 4 (1997) 739^743. [11] R. Gallo, C. Provenzano, R. Carbone, P.P. Di Fiore, L. and Src antibodies, respectively, and Dr. J. Thomas Castellani, G. Falcone, S. Alema, Regulation of the tyrosine Parsons for the RCAS-Src518amb plasmid DNA. kinase substrate Eps8 expression by growth factors, v-Src We are also grateful to Dr. Hsing-Jien Kung for and terminal di¡erentiation, Oncogene 15 (1997) 1929^1936.

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