Regulation of the Tyrosine Kinase Substrate Eps8 Expression by Growth Factors, V-Src and Terminal Di€Erentiation

Regulation of the tyrosine kinase substrate Eps8 expression by growth factors, v-Src and terminal dierentiation

Rita Gallo1, Claudia Provenzano1, Roberta Carbone2, Pier Paolo Di Fiore,2,3, Loriana Castellani4, Germana Falcone1 and Stefano AlemaÁ 1

1Istituto di Biologia Cellulare, C.N.R., 00137 Roma, 2European Institute of Oncology, 20141 Milano, 3Istituto di Microbiologia, UniversitaÁ di Bari, 70124 Bari, and 4Dipartimento di Medicina Sperimentale e Scienze Biochimiche, UniversitaÁ di Roma Tor Vergata, 00133 Roma, Italy

SH3-containing proteins are involved in signal transduc- cells (Fazioli et al., 1993a; G Scita and PP Di Fiore, tion by a number of growth factor receptors and in the unpublished observations). The amino terminal half organization of the cytoskeleton. The recently identi®ed comprises several proline-rich regions, which are Eps8 protein, which contains an SH3 domain, is coupled potential sites for interaction with SH3 domains. At functionally and physically to the EGFR and is tyrosine variance with other signal transducers which bind in a phosphorylated by this receptor and other receptors as ligand-dependent manner through their SH2 domains well. Here, we examined the regulation of eps8 to phosphotyrosine-containing motifs, Eps8 binds expression in response to mitogenic or dierentiative stably to the juxtamembrane region of EGFR, signals. We show that Eps8 is expressed at low levels in regardless of activation of the latter (Castagnino et resting ®broblasts, but its expression is strongly induced al., 1995). Other lines of evidence have implicated Eps8 during activation by serum, phorbol esters and the v-src in the regulation of cell proliferation, since its oncogene. Conversely, expression of Eps8, but not of overexpression in EGFR-expressing cells enhances other EGFR substrates such as Shc or Eps15, is virtually mitogenic responsiveness to EGF (Fazioli et al., extinguished in non-proliferating, terminally dieren- 1993a) and Eps8 is constitutively tyrosine-phosphory- tiated murine myogenic cells. The putative role of Eps8 lated in human tumour cell lines (Matoskova et al., protein as a v-Src substrate was analysed in murine 1995). ®broblasts and in quail myogenic cells expressing a There is currently a resurgence of interest in temperature-sensitive variant of the tyrosine kinase. elucidating the downstream eectors of tyrosine Tyrosine phosphorylation of Eps8 was detected only at kinases of the src family. Much recent evidence has the permissive temperature. A non-myristylated, trans- made clear that, despite the fact that receptor and non- formation-defective mutant of v-Src did not phosphor- receptor tyrosine kinases share several signalling ylate Eps8, whereas it phosphorylated Shc. Together, pathways, there are nonetheless signi®cant dierences these ®ndings indicate that Eps8 may be a critical in the modalities of recruitment of common transdu- substrate of v-Src. They further establish Eps8 as an cers. In addition, a number of substrates for tyrosine example of a signal transducer whose expression senses phosphorylation appear to be uniquely recruited by the balance between growth and dierentiation and non-receptor tyrosine kinases (reviewed in Brown and might, therefore, be involved in the determination of Cooper, 1996). The v-src oncogene encodes a protein the phenotype. tyrosine kinase which is necessary and sucient to transform target cells infected with Rous sarcoma virus Keywords: tyrosine kinase; Eps8; Shc; dierentiation; (RSV) (Wyke and Stoker, 1987; Parsons and Weber, signal transduction 1989). Moreover, v-src, when expressed under a strong promoter, is one of the few oncogenes capable of inducing eciently and in a single-step the complete conversion of a normal into a transformed cell (Hjelle Introduction et al., 1998). The sustained activation of the v-Src tyrosine kinase results in the tyrosine phosphorylation Eps8 is a recently identi®ed substrate for the epidermal of numerous intracellular targets, many of which are growth factor receptor (EGFR) and other receptor associated with the cellular cytoskeletal network tyrosine kinases (RTKs) (Fazioli et al., 1993a; Wong et (Brown and Cooper, 1996). The transforming function al., 1994). The product of the eps8 gene exhibits several of v-src also depends on speci®c regulators of its unique structural features (Fazioli et al., 1993a; Wong enzymatic activity as well as on other gene products et al., 1994). The carboxy terminal half of the protein acting as downstream transducers of the oncoprotein contains an SH3 domain that interacts with other activity (Brown and Cooper, 1996). The function of signalling proteins, including Shb (Karlsson et al., these not yet fully de®ned activities is important in 1995), Shc (Matoskova et al., 1995) and RN-tre determining the susceptibility to transformation of (Matoskova et al., 1996). An eector role for the dierent cell types, as demonstrated by the observation SH3 domain of Eps8 is suggested by the ®nding that it that some cells are easily transformed by v-src (AlemaÁ is able to induce maturation of Xenopus oocytes or and TatoÁ , 1987; Boettiger, 1989; Falcone et al., 1992), transcription directed by a fos promoter in mammalian others are resistant (Lipsich et al., 1984) while others undergo terminal dierentiation (AlemaÁ et al., 1985). Correspondence: S AlemaÁ In the present study, we address the question of Received 3 March 1997; revised 13 June 1997; accepted 16 June 1997 whether the Eps8 protein is a biologically relevant Eps8 and v-Src transformation RGalloet al 1930 intermediate of v-src-induced uncontrolled growth and accumulation of the 68 kDa form is strongly up- block of dierentiation. We ®nd that Eps8 protein regulated in cells transformed by erbB2, ras, raf and becomes tyrosine-phosphorylated at the permissive v-src oncogenes (not shown). temperature in cells transformed by temperature- sensitive variants of v-Src. Tyrosine phosphorylation The Eps8 gene products are up-regulated by the v-src of Eps8 protein did not occur in cells expressing a non- oncogene myristylated mutant of v-Src. We show further that the expression of Eps8 protein is directly regulated by v- To determine whether accumulation of Eps8 proteins is Src and other mitogenic signals. The accumulation of directly regulated by v-src and is not an indirect Eps8 in dividing or transformed cells and the consequence of transformation, we assayed the steady- contrasting inhibition of its expression in post- state levels of Eps8 following the activation of a ts-v- mitotic, terminally dierentiated muscle cells, strength- src mutant in a representative clonal strain of Balb/c en the notion that Eps8 is a positive regulator of 3T3 ®broblasts transformed with the MR31 retrovirus. mitogenesis and transformation. Thus, Eps8 is an When Balb/c 3T3-MR31 cells grown as subcon¯uent example of a signal transducer whose activity may be cultures at the restrictive temperature (398C) were controlled by its levels of expression according to the shifted for two days to the permissive temperature growth or dierentiation conditions. (358C) for v-Src kinase activity, we observed a progressive increase in Eps8 protein accumulation up to levels comparable to those displayed by cells continuously kept at 358C (Figure 1b). Expression of Results the 68 kDa form was particularly up-regulated (about tenfold) both in Balb/c and NIH3T3 cells. This Expression of eps8 is regulated by serum, tumour dierential accumulation was not in¯uenced by promoters and oncogenes reactivation of v-Src in cells kept in low serum (not Eps8 protein has been shown to be expressed in a shown). Steady-state levels of Shc, Eps15 and cortactin variety of cell types, including epithelial and fibro- proteins, which are known substrates of tyrosine blastic lines and some hematopoietic cells (Fazioli et kinases (McGlade et al., 1992; Fazioli et al., 1993b; al., 1993a; Wong et al., 1994; Matoskova et al., 1995). Wu et al., 1991), were not aected signi®cantly under To determine whether the levels of expression of eps8 the same culture conditions (Figure 1b, cortactin not could be modulated by mitogenic stimuli, we examined shown). This modulation occurred with a time course the expression of the Eps8 protein by Western blot similar to that resulting in morphological transforma- analysis of cell lysates derived from growing and tion. Thus, v-Src aects the levels of Eps8 protein quiescent Balb/c and NIH3T3 cells. It has been accumulation. previously shown that an anti-Eps8 serum speci®cally Our observation that expression of Eps8 was up- recognises 97 kDa and 68 kDa forms in mouse regulated in v-Src-transformed or TPA-treated cell ®broblasts (Fazioli et al., 1993a). Figure 1 shows that indeed the eps8 gene product presents two forms of 97 and 68 kDa in Balb/c 3T3 cells kept in 10% serum (Figure 1a, lane 1). Upon reduction of serum to 0.5% abc for 2-4 days, the expression of the Eps8 protein is highly reduced in Balb/c 3T3 cells (Figure 1a) or in 2D NIH3T3 cells (not shown). Under the same culture ° conditions the rate of entry into S phase of Balb/c 3T3 2D 35 4D cells, assayed by a 5 h pulse with bromo-deoxyuridine ° ° ° °

FCS Low 2D serum 4D 2D+FCS 2D 2D TPA (BrdU), was 64% in 10% FCS and 3.6% in cells 35 39 39 39 FCS +TPA — 97 cultured for 2 days in 0.5% FCS. The levels of Eps8 — 68 accumulation of the Eps8 protein could be restored to those observed in growing ®broblasts by stimulating — 66 resting cells with 10% serum for 2 days (70% BrdU- Shc — 52 — 46 positive cells) (Figure 1a, lane 4). Exposure of growing cells to the tumour promoter phorbol 12-0-tetradeca- Eps15 — 142 noyl phorbol 13-acetate (TPA) for 2 days resulted in a 1 2 3 4 5 6 7 8 9 10 11 pronounced up-regulation of Eps8 protein in Balb/c Balb/c 3T3 Balb/c 3T3-MR31 NIH 3T3 3T3 (Figure 1a, lane 5) and NIH3T3 ®broblasts Figure 1 Regulation of Eps8 protein expression levels in normal (Figure 1c). The speci®c eect of culture conditions and v-src-transformed Balb/c 3T3 cells. (a) Balb/c 3T3 cells were on the levels of expression of Eps8 was con®rmed by cultured on collagen-coated dishes in 10% FCS (lane 1) or in monitoring the expression levels of both Shc (Pelicci et 0.5% FCS for 2 (lane 2) and 4 days (lane 3). Lane 4 refers to cells al., 1992) and Eps15 (Fazioli et al., 1993b) proteins, that had been maintained in 0.5% FCS for 2 days and then exposed to 10% FCS for two more days. Lane 5 refers to cells which remained mostly unchanged (Figure 1). 77 cultured in 10% FCS in the presence of 10 M TPA for 2 days. In an attempt to demonstrate that the up-regulation (b) Balb/c 3T3 cells transformed by a temperature-sensitive of Eps8 is a phenomenon observable in growing cells mutant of v-Src (ts-MR31) were propagated in 10% FCS at and possibly common to transformed cells, lysates of either 358C (lane 6) or at 398C for 2 (lane 7) and 4 days (lane 9). various oncogene-transformed NIH3T3 cells were Lane 8 refers to cells kept at 398C for 4 days and then shifted to 358C for 2 days. (c) NIH3T3 cells cultured in 10% FCS in the analysed by Western blot for relative levels of the 77 absence (lane 1) or the presence (lane 2) of 10 M TPA for 2 Eps8 protein. Whereas in growing NIH3T3 cells, the days. Expression levels of Eps8, Shc and Eps15 proteins were predominant form of Eps8 is the 97 kDa species, the determined by immunoblotting of total lysates Eps8 and v-Src transformation RGalloet al 1931 lines prompted us to investigate whether the regulation presence of the DNA synthesis inhibitor cytosine b-D- of Eps8 was exerted at the transcriptional level. To this arabinofuranoside (Ara-C) to eliminate undifferen- end, total RNAs extracted from control and v-src- tiated cells, as well as in primary murine satellite cells transformed Balb/c 3T3-MR31 cells grown at 358C and (MSC) (Figure 3a and b). The decrease in the amount 398C and from control and TPA-treated NIH3T3 cells of both forms of Eps8 paralleled the increase in were analysed by Nothern blot (Figure 2). Two accumulation of the muscle dierentiation marker transcripts of approximately 4.7 and 4.0 kb were myosin heavy chain (MHC). Levels of Eps15 and Shc found in all cell types (Fazioli et al., 1993a). No proteins were not signi®cantly aected by muscle signi®cant eect of temperature was evident in control dierentiation, with the exception of the 66 kDa form Balb/c 3T3 cells, whereas both v-Src-transformation of of Shc which was markedly up-regulated concomitantly Balb/c 3T3 cells and TPA-treatment of NIH3T3 cells with the attainment of the post-mitotic state. Because greatly augmented the accumulation of both tran- dierentiated myotubes do not re-enter the cell cycle in scripts, in close agreement with that observed for the response to mitogen stimulation, we investigated Eps8 proteins. A poor correlation between the ratios of whether eps8 silencing persisted under these condi- the two transcripts and of the two protein forms, tions. Indeed, the down-regulation of Eps8 levels was however, was observed in BalB/c 3T3 cells, possibly maintained in fully dierentiated MSC after re- resulting from dierential rates of translation or addition of serum for 2 days (Figure 3b, lane 4). We processing. also monitored the expression of Eps8 in a transformed line of C2C12 murine myoblasts that stably expresses a ts-v-src mutant gene (C2C12 tsMR31) and exhibits Silencing of eps8 upon terminal dierentiation of murine temperature-dependent transformation and block of myogenic cells dierentiation (MC Gauzzi and SA., unpublished). At Skeletal muscle terminal dierentiation entails the high serum concentrations (GM) transformed C2C12 coordination of muscle-speci®c gene expression and tsMR31 myoblasts expressed both forms of Eps8 in withdrawal from the cell cycle. Because expression of larger amounts than proliferating parental C2C12 cells eps8 is controlled by signals leading to mitogenesis or (Figure 3c, compare lane 1 and 2). In the absence of transformation we sought to determine whether serum (DM) at the permissive temperature the expression of eps8 was regulated during differentia- accumulation of Eps8 was in part reduced (Figure 3c, tion. To this purpose we employed two systems of lane 3), although it remained higher than in parental dierentiating murine myoblasts. Skeletal myogenesis cells kept in GM (Figure 3c, lane 1), consistent with in the C2C12 cell line and in primary satellite cells the upregulation of Eps8 exerted by v-Src. Upon (MSC) can be induced by depriving cycling myoblasts terminal dierentiation at the restrictive temperature, of mitogens. This leads, within three days in culture, to however, expression of Eps8 was virtually extinguished the formation of multinucleated myotubes and to (Figure 3c, lane 4), and could only be resumed expression of a vast array of muscle-speci®c proteins. marginally by the addition of mitogens to post-mitotic Expression of both the 97 kDa and 68 kDa proteins myotubes or activation of v-Src upon shift to 358C was strongly down-regulated during terminal differ- (Figure 3c, lanes 5 and 6). entiation of C2C12 cells, both in the absence and in the Tyrosine phosphorylation of Eps8 proteins in ts-v-src transformed ®broblasts and myoblasts

° ° To investigate tyrosine phosphorylation of Eps8

proteins, Balb/c 3T3-MR31 or NIH3T3-MR31 cells ° ° were kept either at the permissive temperature or at the restrictive temperature for 2 days. The tyrosine kinase v-Src was activated in cultures kept at 398Cby Balb/c 35 Balb/c 39 Balb/c-MR31 35 Balb/c-MR31 39 NIH3T3 NIH3T3+TPA temperature shift to 358C for 2 and 24 h. When — 28S lysates from these cells were immunoprecipitated with Eps8 anti-Eps8 serum, bands of 97 kDa and 68 kDa were detected in blots that reacted with anti-phosphotyrosine antibody as early as 2 h after v-Src activation (Figure 4a), suggesting that both forms of Eps8 can be tyrosine-phosphorylated in the presence of an active v- Src. Comparable levels of tyrosine phosphorylation of GAPDH Eps8 were detected both in high and low serum concentrations (not shown). We next estimated the percentage of Eps8 molecules that are tyrosine- 1 2 3 4 5 6 phosphorylated in vivo in both NIH3T3- and Balb/c Figure 2 Accumulation of eps8 gene transcripts is modulated by 3T3-MR31 cells. To this end we adopted a previously TPA and transformation by v-src. RNAs (12 mg/lane) extracted from the following cells were examined by Northern analysis: described methodology (Matoskova et al., 1995). We Balb/c 3T3 (lanes 1 and 2) and Balb/c 3T3-MR31 (lane 3 and 4) estimated that about 10% of the total Eps8 pool was cells grown at 358C (lanes 1 and 3) or 398C (lanes 2 and 4); constitutively tyrosine-phosphorylated at the permis- control (lane 5) or TPA-treated (lane 6) NIH3T3 cells. Note that sive temperature in both cell types, whereas about 5% the 4.0 kb mRNA species in control cells only becomes visible of the total pool was phosphorylated following 24 h of with longer exposures. Total RNAs were harvested from cells in semicon¯uent cultures in 10% FCS. Filters were hybridised with v-Src activation in cells previously kept at the probes for eps8 and GAPDH restrictive temperature for 2 days (not shown) Eps8 and v-Src transformation RGalloet al 1932

a b c GM ° DM ° GM DM DM DM+FCS 35 ° ° ° ° °

GM DM 3D 3D DM/AraC 5D 3D+FCS GM DM 3D DM 4D DM 3D+FCS C2C12-35 35 35 39 39 39

Eps8 — 97 — 68

MHC — 200

1 2 3 4 MSC Shc — 66 — 52 — 46

Eps15 — 142

1 2 3 4 5 1 2 3 4 5 6 C2C12 C2C12 tsMR31 Figure 3 Expression of Eps8 is extinguished during muscle dierentiation. (a) Western blot analysis of total lysates from C2C12 myoblasts grown at 378C in GM (lane 1), dierentiated in DM for 3 (lanes 2, 3 and 5) or 5 days (lane 4) in the absence (lane 2) or in the presence of 50 mM Ara C (lanes 3, 4 and 5) to eliminate residual undierentiated cells. Lane 5 refers to cells that had been maintained in DM for 3 days and then exposed to 10% FCS for 48 h. (b) Western blot analysis of total lysates from primary mouse satellite cells (MSC) maintained in GM (lane 1) or in DM for 3 (lane 2) and 4 (lane 3) days. Lane 4 refers to MSC maintained in DM for 3 days and then exposed to 10% FCS for 24 h. (c) Western blot analysis of total lysates from control C2C12 myoblasts at 358C in GM (lane 1) and from C2C12 tsMR31 cells (lanes 2 ± 6) maintained at 358C for 2 days in GM (lane 2) or in DM (lane 3) and dierentiated at 398C in DM (lanes 4 ± 6). Lanes 5 and 6 refer to cells dierentiated in DM at 398C for 3 days and then either challenged with 10% FCS for 24 h (lane 5) or shifted to 358C for 24 h (lane 6). Amounts of Eps8, myosin heavy chain (MHC), Shc and Eps15 proteins were determined

To further test the ability of vSrc to induce tyrosine sucient to induce high levels of phosphorylation of a phosphorylation of the Eps8 protein and since the number of cellular substrates (not shown). Cell lysates available antibodies do not recognise avian Eps8 were immunoprecipitated with anti-Eps8 or anti-Shc proteins, Eps8 was constitutively or transiently ex- antibodies and the resolved immunoprecipitates were pressed in quail myoblasts transformed with ts-mutants blotted with anti-p-Tyr antibody. Phosphorylation of of v-Src (QMb-LA29). We have previously shown that Eps8 was strictly temperature-dependent in QMb-LA29- in these cells the level of P-Tyr-containing cellular transformed myoblasts irrespective of Eps8 being proteins is temperature-dependent both in myoblasts expressed transiently (Figure 4b, left panel) or constitu- and in myotubes shifted to the permissive temperature tively (not shown). Tyrosine phosphorylation of Eps8 for v-Srs (AlemaÁ and TatoÁ , 1994; Castellani et al., 1995, was detected as early as 30 min after shifting cells to the 1996). Since structural modi®cations of speci®c domains permissive temperature and was very evident after 4 h of v-Src have been shown to alter the tyrosine (Figure 4b), when major morphological alterations phosphorylation of v-Src substrates (Kamps and become visible (Castellani et al., 1995). Under the same Sefton, 1986; Wu et al., 1991; Fincham et al., 1995; conditions, tyrosine phosphorylation of the 52 kDa Shc Verderame et al., 1995; Brown and Cooper, 1996) we protein was also observable after activation of v-Src also sought to determine whether a membrane localisa- (Figure 4c, left panel). Note that although quail Shc tion of v-Src was required for tyrosine phosphorylation proteins migrate faster than their mammalian counter- of Eps8 and Shc proteins. To this end we employed quail parts and the antibody used identi®ed preferentially the myoblasts infected by a non-myristylated, transforma- 52 kDa form, we have retained the canonic designation tion-defective version of the ts-LA29 v-Src protein (McGlade et al., 1992; see Verderame et al., 1995). As (QMb-LA29A2) (Catling et al., 1993). QMb-LA29 and shown in Figure 4b (right panel), Western blot analysis -LA29A2 were transiently transfected with an eps8 showed no detectable tyrosine-phosphorylated Eps8 expression vector and then either kept at 358Cor protein in extracts from cells expressing the non- allowed to dierentiate at 418C for 2 days. A third set of myristylated form of v-Src. In contrast, tyrosine- cells after dierentiation at 418C was shifted to 358C for phosphorylation of Shc was observed under all 4 h. Both QMb-LA29 and -LA29A2 expressed v-Src conditions whereby the tyrosine kinase was active, mutant proteins at levels easily detectable by immuno- regardless of its membrane association (Figure 4c, right ¯uorescence analysis with the anti-Src mAb 327 and panel). Eps8 and v-Src transformation RGalloet al 1933 Discussion EGFRs in a ligand-independent fashion. Overexpres- sion of Eps8 in ®broblasts enhances EGF-dependent The Eps8 protein is a recently identi®ed tyrosine kinase mitogenic signals (Fazioli et al., 1993a) and promotes a substrate that carries an SH3 domain and binds to transformed phenotype (Matoskova et al., 1995). Furthermore, Eps8 and the signal transducer Shc are constitutively tyrosine-phosphorylated in many tumori- a genic cell lines, suggesting a key role in neoplastic growth for these intracellular transducers (Pelicci et al., /2 hrs /24 hrs /2 hrs /24 hrs

° ° ° ° 1995; Matoskova et al., 1995). Here, we have demonstrated that Eps8 expression is highly regulated

35 35 35 35 in murine mesenchymal cells. Whereas resting 3T3 cells ° ° ° ° ° ° ° °

35 39 39 39 35 39 39 39 express very low levels of Eps8, we found that mitogenic activators such as serum, TPA and selected — 97 Blot: α Eps8 oncoproteins, notably the v-Src tyrosine kinase, all — 68 induce Eps8 expression. We have shown that eps8 transcripts accumulate in Blot: α P-Tyr — 97 TPA-treated or v-Src-transformed 3T3 ®broblasts at — 68 levels comparable to those attained by Eps8 proteins. 1 2 3 4 5 6 7 8 Possible mechanisms for the overall increase in mRNA IP: α Eps8 accumulation include increased rate of transcription or enhanced transcript stability. We also observed a b striking relative increase of the 4.0 kb mRNA species, which correlated with increased levels of expression of /4 hrs

/4 hrs the 68 kDa protein form in the same cells. Whereas at ° ° present we can only speculate as to how the two Eps8 proteins are generated, a satisfactory explanation is GM DM 35 GM DM 35 ° ° ° ° ° ° that the two mRNA species arise from alternative 35 41 41 35 41 41 splicing and code separately for the 97 kDa and Blot: α Eps8 — 97 68 kDa forms. The alternative possibility that the 68 kDa species represents a speci®c cleavage product of the 97 kDa species was addressed by forced Blot: α P-Tyr — 97 expression of a 97 kDa Eps8-myc isoform in Balb/c 1 2 3 4 5 6 3T3-MR31 cells. Eps8-myc levels were not up-regulated IP: α Eps8 by activation of the ts-allele of v-src, nor was a 68 kDa species detected (unpublished data), and hence, in all c probability, the 68 kDa species is unlikely to result from post-translational modi®cations. In addition, we demonstrate that expression of Eps8 /4 hrs /4 hrs ° ° in both primary and established murine myoblasts is turned o following mitogen removal and onset of GM DM 35 GM DM 35 ° ° ° ° ° ° terminal dierentiation, suggesting that Eps8 may 35 41 41 35 41 41 function in myoblasts to prevent them from commit- Blot: α Shc — 52 ment to permanent withdrawal from the cell cycle. — 46 Murine myogenic cells and the signals that induce their dierentiation are, so far, unique in their ability to α — 52 Blot: P-Tyr — 46 suppress expression of Eps8. It will be interesting to see 1 2 3 4 5 6 whether this kind of regulation operates in differ- IP: α Shc entiated cell types belonging to other lineages. Whereas levels of expression of the 52 and 46 kDa Shc proteins Figure 4 Phosphorylation of Eps8 and Shc proteins in v-src- were not in¯uenced by growth conditions or transfor- transformed cells. (a) ts-MR31-transformed Balb/c 3T3 (left panel) and NIH3T3 cells (right panel) were grown in 10% FCS mation, the 66 kDa isoform was markedly up- at 358C (lanes 1 and 5) or at 398C for 2 days (lanes 2 and 6) and regulated in muscle cells concomitantly with the subsequently shifted to 358C for 2 (lanes 3 and 7) and 24 h (lanes attainment of the post-mitotic state. The sequence of 4 and 8). Cells were lysed and immunoprecipitated with anti-Eps8 the 66 kDa form overlaps that of the 52 kDa isoform serum. Immunoprecipitates were immunoblotted with anti-Eps8 and contains a unique N-terminal region. Unlike the or anti-phosphotyrosine (P-Tyr) antibodies. (b and c) Quail myoblasts transformed by the ts-LA29 strain of Rous sarcoma other two protein species the 66 kDa form does not virus (QMb-LA29) (lanes 1 ± 3, left panels) or expressing a non- increase EGF activation of MAP kinases and inhibits myristylated, transformation-defective version of the v-Src kinase fos promoter activation (Migliaccio et al., 1997). (QMb-LA29A2) (lanes 4 ± 6, right panels) were transiently Regulation of this isoform expression is, therefore, transfected with pCEV-eps8 vector, propagated at 358CinGM (lanes 1 and 4) or dierentiated at 418C in DM for 2 days (lanes 2 expected to in¯uence the cellular response to growth and 5) and then shifted to 358C in DM for 4 h (lanes 3 and 6). factors in dierent cell types. Cell were lysed and immunoprecipitated with anti-Eps8 (b)or Eps8 appears to belong to the class of v-Src anti-Shc (c) antibodies. Immunoprecipitates were immunoblotted substrates that are also phosphorylated by activated with anti-phosphotyrosine (P-Tyr) and with anti-Eps8 (b) or anti- RTKs and are most likely involved in mitogenic Shc antibodies (c). Mobilities of the Eps8 and Shc proteins are indicated. Note that in b, left panel, the 97 kDa Eps8 protein signalling pathways. A second class of substrates appears as a closely-spaced doublet includes proteins that are speci®cally phosphorylated Eps8 and v-Src transformation RGalloet al 1934 in v-src-transformed cells and these are often membrane, co-localising with F-actin and the F-actin associated to focal adhesions and the cytoskeleton binding protein cortactin. Furthermore, both Eps8 and (Brown and Cooper, 1996). Regardless of its belonging cortactin are readily re-located to podosomes and to to either class, several lines of evidence support the the actin bodies (our unpublished results) generated contention that Eps8 may be a critical substrate of v- upon activation of v-Src in quail myotubes (Castellani Src. First, tyrosine-phosphorylated Eps8 is a relatively et al., 1995, 1996). These observations and previous abundant protein in v-Src-transformed cells. The extent work (Wu and Parsons, 1993; Okamura and Resh, of Eps8 tyrosine-phosphorylation by v-Src (5 ± 10% of 1995) suggest a similar subcellular localisation of Eps8, total Eps8 protein pool) is comparable to that found in cortactin and v-Src in cytoskeleton-associated struc- human tumour cell lines (Matoskova et al., 1995), or tures. Although it is still not clear whether Eps8 is a after activation of overexpressed RTKs by ligands direct substrate of v-Src, the proximity of the two (Fazioli et al., 1993a; Matoskova et al., 1995). Second, proteins at speci®c cellular districts suggests that this Eps8 proteins are phosphorylated in a transformation- may be the case. The ®nding that Eps8 protein was not speci®c manner. The tyrosine-phosphorylation of a phosphorylated in non-myristylated mutant-infected number of identi®ed v-Src substrates, notably talin, cells, is therefore expected on the basis of the p36, vinculin and cortactin, is poorly correlated with subcellular localisation of the mutant v-Src protein. transformation by various mutants of v-Src kinase. The potential link between phosphorylation of Eps8 Other substrates, however, such as FAK and pp120cas and the morphological changes associated with cell satisfy the criterion of not being phosphorylated in transformation is currently under investigation. cells expressing the transformation-defective, non- In conclusion, our results indicate that Eps8 may be myristylated mutant of the kinase (Brown and one of the critical signal transduction molecules Cooper, 1996). Eps8 was not phosphorylated in avian associated with and possibly required in the transmis- cells expressing non-myristylated v-Src but was sion of the signals generated by v-Src, that lead to cell phosphorylated in both murine and avian cells transformation as well as to block of myogenic transformed by ts-v-src. Moreover, Eps8 proteins dierentiation. The reported ability of Eps8 in were readily phosphorylated after a temperature shift modulating cell proliferation, the association of its to the permissive temperature. Shc proteins, on the tyrosine phsphorylation with the transformed state and other hand, were tyrosine-phosphorylated in cells the induction of its expression in response to growth expressing the transformation-defective non-myristy- promoting stimuli, converge to present it as a highly lated allele of v-Src, thus suggesting that phosphoryla- suitable candidate to link a variety of growth- tion of this substrate is not sucient for cell regulatory pathways, including cell dierentiation. transformation, although it may be required. Accord- ingly, in chicken cells expressing host-range or transformation-defective v-src alleles, p52shc was highly phosphorylated on tyrosine whenever a kinase-active Materials and methods src is expressed, regardless of whether the cells are Materials transformed or not (Verderame et al., 1995). Is Eps8 directly phosphorylated by v-Src? The 12-0-tetradecanoyl phorbol 13-acetate (TPA) and a observation that phosphorylation of Eps8 takes place monoclonal antibody to phosphotyrosine (PT-66) were with at least the same kinetics of other substrates may from Sigma; anti-Src mAb 327 was kindly provided by J be relevant to this point. Tyrosine phosphorylation, Brugge and anti-cortactin mAb 4F11 by T Parsons; anti- Myc mAb 9E10 was a gift from A Cattaneo; a polyclonal however, is apparently uncoupled from direct binding anti-Shc was from Transduction Labs; polyclonal sera of v-Src to Eps8 proteins. In immunoprecipitation speci®c for the eps8 and eps15 gene products were experiments we could neither isolate a v-Src/Eps8 generated as previously described (Fazioli et al., 1993a complex, nor did v-Src and Eps8 interact in vitro via and b); a polyclonal anti-skeletal muscle myosin serum was their SH3 domains (our unpublished results). While the developed in house using puri®ed chicken muscle myosin as lack of a stable detergent-resistant association between immunogen. Eps8 and v-Src does not preclude their contact in vivo, it also leaves the possibility open that v-Src does not Cell culture and transfection directly phosphorylate Eps8 but does so through other cellular tyrosine kinases which are activated in v-src- NIH3T3 cells (provided by E Westin), Balb/c 3T3 cells and al v-src transformants derived from them were maintained transformed cells, perhaps through autocrine secretion in Eagle's Minimal Essential Medium (MEM) supplemen- of growth factors. However, we note that Eps8 is ted with 10% FCS and grown in 5% CO2 at 378C. NIH- phosphorylated by v-Src with comparable eciency in erbB2, NIH-ras, and NIH-raf cells (kindly provided by O low-serum, arguing against the possibility that its Segatto) were maintained in Dulbecco's modi®ed Eagle's phosphorylation requires the cooperation of kinases medium (DMEM) supplemented with 10% fetal calf serum activated by mitogens present in serum. p190, a (FCS). C2C12 myoblasts were maintained in DMEM GTPase activator of Rho, and Shc are examples of supplemented with 20% FCS (growth medium, GM). substrates that display high levels of tyrosine Primary murine satellite cells (MSC) were provided by M phosphorylation in v-src-transformed cells, yet have Crescenzi and were maintained in DMEM containing 10% not been shown to construct stable detergent-resistant FCS and 20 ng/ml ®broblast growth factor. Muscle dierentiation was induced by incubating cultures at 70% interactions with the kinase (Brown and Cooper, 1996). con¯uence in DMEM containing 10 mg of insulin and 5 mg Alternatively, it is possible that Eps8 is made accessible of transferrin per ml (dierentiation medium, DM) for to v-Src phosphorylation following interaction with three days. Clonal strains of C2C12 myogenic cells and other intermediate proteins. A signi®cant fraction of NIH3T3 and Balb/c 3T3 ®broblasts expressing v-src were the Eps8 protein pool is found associated to the plasma obtained by infection with the retroviral construct MR31, Eps8 and v-Src transformation RGalloet al 1935 carrying the ts-src mutant gene from LA31-RSV (kindly ing a 1.2 Kb avian GAPDH (glyceraldehyde-3-phosphate- provided by A La Rocca), followed by selection of dehydrogenase) cDNA (obtained from C Schneider). transformed colonies in soft agar at the permissive Quantitative analysis was carried out using a Molecular temperature. A clonal strain of Balb/c-MR31 cells stably Dynamics PhosphorImager 400A with Image Quant expressing an Eps8-myc-tagged protein was established by version 3.2 software. transfection of an eps8-myc expression vector derived by myc-epitope tagging of the Eps8 protein encoded by the Whole-cell extracts and Western blot analysis previously described pCEV-eps8 vector (Fazioli et al., 1993a) and selection with G418 (0.8 mg/ml). Cells were brie¯y rinsed with phosphate-buered saline Polyclonal population of ts-src-transformed quail myo- (PBS), containing 0.5 mM orthovanadate and collected blasts were established from primary cultures infected with with 0.2 ml/35 mm plate of SDS sample buer (8 M viral stocks of ts-LA29, a temperature-sensitive mutant of Urea, 0.14 M b-mercaptoethanol, 0.04 M DTT, 2% SDS, RSV, as previously described (QMb-LA29) (Falcone et al., 0.075 M Tris-Cl, pH 8.0). SDS ± PAGE (2 ± 20 mgoftotal 1991; AlemaÁ and TatoÁ , 1994). Quail myoblasts infected by a proteins per well) was carried out according to Laemmli non-myristylated, transformation-defective version of the ts- 1970. Western blots were carried out as described by LA29 v-Src protein (RCAN-29A2) (Catling et al., 1993) were Towbin et al., (1979) using horseradish peroxidase- kindly provided by F TatoÁ . Transformed myoblasts stably conjugated goat anti-rabbit and anti-mouse antibodies expressing Eps8 protein (QMb-LA29-eps8) were obtained by and revealed using the ECL chemiluminescence detection transfection of QMb-LA29 cells with pCEV-eps8 and system (Amersham). selection in G418 (0.8 mg/ml). Infected quail myoblasts were propagated on collagen-coated dishes in DMEM supplemen- Analysis of Eps8 and Shc tyrosine phosphorylation ted with 10% FCS, 10% tryptose phosphate broth, 1% chicken serum, referred to as growth medium (GM) at 358C Immunoprecipitations of Eps8 and Shc proteins were (permissive temperature). Dierentiation was induced by performed as described (Fazioli et al., 1993a; Matoskova plating 105 cells on 35 mm collagen-coated dishes in GM et al., 1995). Brie¯y, cultures were washed twice with PBS and, the following day, by shifting the cultures to 418C andstoredfrozenat7808C. Lysis was performed in (restrictive temperature) for 2 ± 3 days in F14 medium, 1.5 ml/100 mm dish of lysis buer (30 mM Tris-Cl pH 7.5, supplemented with 2% foetal calf serum (referred to as 0.1 M NaC1, 1% Triton X100, 2 mM MgCl2,1.5mM dierentiation medium, DM); for reactivation of ts-Src in EGTA, 10 mM NaF, 1 mM orthovanadate, 40 mg/ml dierentiated myotubes, a parallel set of plates kept at 418C leupeptin, 40 mg/ml aprotinin, 40 mg/ml soybean trypsin was shifted to 358C for the appropriate lengths of time. inhibitor, 1 mM PMSF, 10% glycerol) and cell extracts Transient transfections in v-Src-transformed quail myoblasts were centrifuged at 15 000 g for 15 min at 48C. Cell (1 mg of DNA per 35 mm plate or 5 mg per 100 mm plate) extracts (0.5 ± 2 mg of total proteins), normalised for were carried out by using an optimised calcium phosphate protein content by the BCA colorimetric method, were transfection procedure (Chen and Okayama, 1987). incubated with either polyclonal anti-Eps8 serum or The proliferation rate of growing and serum-derived Balb/ polyclonal anti-Shc serum for four hours and 50 mlof c 3T3 cells was assayed by measuring the percentage of nuclei 50% protein A-agarose slush for 1 h at 48C. Immunopre- which incorporated BrdU. 105 cells were plated in duplicate cipitates were washed twice with lysis buer, once with on 35 mm dishes in MEM-10% FCS and the day after 20 mM Tris-Cl pH 7.5 and boiled in 50 mlofSDSsample shifted to fresh MEM-10% FCS or MEM-0.5% FCS for 2 buer. Immunoprecipitates were analysed on SDS ± PAGE, days. Cells were then fed for 5 h with the same media also and blotted with speci®c antibodies as indicated. Sequential containing 10 mM BrdU. BrdU-labelled cultures were ®xed anti-phosphotyrosine immunoprecipitations for quantita- for 15 min with ice-cld 95% ethanol/5% acetic acid, treated tion of phosphorylation were carried out as described in for 10 min with 1.5 N HC1, rinsed in PBS and stained with Matoskova et al., 1995) an anti-BrdU monoclonal antibody (Amersham) for 1 h.

RNA isolation and Northern blot ananlysis Total RNA was prepared by the Ultraspec RNA isolation Acknowledgements system (Biotecx). 12 mg aliquots of the obtained RNA were We would like to thank Oreste Segatto, Giuliana Pelicci, resolved in 0.9% agarose/2.2 M formaldehyde gels. Thomas Parsons, Joan Brugge, Marco Crescenzi, Anna La Transfer to nitrocellulose membranes and high stringency Rocca and Franco TatoÁ for generous gifts of reagents, and hybridisation were carried out according to standard Delio Mercanti for skilful assistance in antibody produc- procedures. Probes were labelled with a random primed tion. RG was supported by fellowships from CNR and DNA labelling kit. For detection of transcripts, inserts of Fondazione A Buzzati-Traverso. CP is an AIRC fellow. the following plasmids were cut with the appropriate This work was supported by grants from Comitato restriction enzymes and used as probes: pCEV-eps8, Promotore Telethon, CNR (PF-ACRO), AIRC and containing a 3.6 Kb mouse eps8 cDNA a plasmid contain- European Community (BIOMED-2)

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