Abstract

Article15 February 1997free access Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor–MAP kinase–fos signalling pathway Enrica Migliaccio Enrica Migliaccio European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Search for more papers by this author Simonetta Mele Simonetta Mele Istituto di Medicina Interna e Scienze Oncologiche, University of Perugia, 06100 Perugia, Italy Search for more papers by this author Anna E. Salcini Anna E. Salcini European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Search for more papers by this author Giuliana Pelicci Giuliana Pelicci European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Search for more papers by this author Ka-Man Venus Lai Ka-Man Venus Lai Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Giulio Superti-Furga Giulio Superti-Furga EMBL, Heidelberg, Germany Search for more papers by this author Tony Pawson Tony Pawson Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Pier Paolo Di Fiore Pier Paolo Di Fiore European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Istituto di Microbiologia, University of Bari, Italy Search for more papers by this author Luisa Lanfrancone Luisa Lanfrancone European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Search for more papers by this author Pier Giuseppe Pelicci Corresponding Author Pier Giuseppe Pelicci European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Istituto di Medicina Interna e Scienze Oncologiche, University of Perugia, 06100 Perugia, Italy Search for more papers by this author Enrica Migliaccio Enrica Migliaccio European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Search for more papers by this author Simonetta Mele Simonetta Mele Istituto di Medicina Interna e Scienze Oncologiche, University of Perugia, 06100 Perugia, Italy Search for more papers by this author Anna E. Salcini Anna E. Salcini European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Search for more papers by this author Giuliana Pelicci Giuliana Pelicci European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Search for more papers by this author Ka-Man Venus Lai Ka-Man Venus Lai Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Giulio Superti-Furga Giulio Superti-Furga EMBL, Heidelberg, Germany Search for more papers by this author Tony Pawson Tony Pawson Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Pier Paolo Di Fiore Pier Paolo Di Fiore European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Istituto di Microbiologia, University of Bari, Italy Search for more papers by this author Luisa Lanfrancone Luisa Lanfrancone European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Search for more papers by this author Pier Giuseppe Pelicci Corresponding Author Pier Giuseppe Pelicci European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy Istituto di Medicina Interna e Scienze Oncologiche, University of Perugia, 06100 Perugia, Italy Search for more papers by this author Author Information Enrica Migliaccio1, Simonetta Mele2, Anna E. Salcini1, Giuliana Pelicci1, Ka-Man Venus Lai3, Giulio Superti-Furga4, Tony Pawson3, Pier Paolo Di Fiore1,5, Luisa Lanfrancone1 and Pier Giuseppe Pelicci 1,2 1European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti, 435-20141 Milan, Italy 2Istituto di Medicina Interna e Scienze Oncologiche, University of Perugia, 06100 Perugia, Italy 3Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada 4EMBL, Heidelberg, Germany 5Istituto di Microbiologia, University of Bari, Italy The EMBO Journal (1997)16:706-716https://doi.org/10.1093/emboj/16.4.706 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N-terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors. Introduction The SH2-containing Shc proteins (Pelicci et al., 1992) are cytoplasmic substrates of activated tyrosine kinases (TK) (Lotti et al., 1996) and have been implicated in the transmission of activation signals from TKs to Ras proteins (Bonfini et al., 1996). Shc proteins are phosphorylated by all receptor TKs (RTKs) tested to date, including the EGF receptor (EGFR) (Pelicci et al., 1992), the platelet-derived growth factor receptor (Yokote et al., 1994), the hepatocyte growth factor receptor (Pelicci et al., 1995a), the erbB-2 receptor (Segatto et al., 1993; Ricci et al., 1995), the insulin receptor (Pronk et al., 1993; Skolnik et al., 1993), the fibroblast growth factor receptor (Vainikka et al., 1994), and the nerve growth factor receptor (Borrello et al., 1994; Stephens et al., 1994). Shc proteins are also involved in signalling from cytoplasmic TKs, since they are constitutively phosphorylated in cells that express activated Lck, Src, Fps or Sea (McGlade et al., 1992; Crowe et al., 1994; Dilworth et al., 1994; Baldari et al., 1995; Pelicci et al., 1995b). In addition, Shc proteins are rapidly phosphorylated on tyrosine after ligand stimulation of surface receptors that have no intrinsic TK activity, but are thought to signal by recruiting and activating cytoplasmic TKs (e.g. IL-2, erythropoietin, G-CSF, GM-CSF, B- and T-cell receptors, CD4, CD8) (Burns et al., 1993; Damen et al., 1993; Ravichandran et al., 1993; Baldari et al., 1995; Lanfrancone et al., 1995; Matsuguchi et al., 1994). Upon phosphorylation, Shc proteins form stable complexes with cellular tyrosine-phosphorylated polypeptides, including RTKs and receptors devoted of intrinsic TK activity. These interactions are mediated by the Shc SH2 and/or PTB domains (Pelicci et al., 1992, 1995b; Segatto et al., 1993; Blaikie et al., 1994; Borrello et al., 1994; Kavanaugh and Williams, 1994; Stephens et al., 1994; Yokote et al., 1994; Baldari et al., 1995; Lanfrancone et al., 1995; van der Geer et al., 1995). Phosphorylated Shc proteins also associate with the Grb2 adaptor protein (Clark et al., 1992; Lowenstein et al., 1992) through direct binding of the Grb2 SH2 domain to the major Shc tyrosine-phosphorylation site (Tyr317) (Rozakis-Adcock et al., 1992; Salcini et al., 1994). Grb2 is constitutively complexed with SOS (Batzer et al., 1993; Buday and Downward, 1993; Chardin et al., 1993; Egan et al., 1993; Gale et al., 1993; Olivier et al., 1993; Rozakis-Adcock et al., 1993), a ubiquitously expressed Ras guanine nucleotide exchange factor for Ras (Simon et al., 1991; Bonfini et al., 1992; Bowtell et al., 1992; Chardin et al., 1993). Recruitment of the Grb2–SOS complex results in the membrane relocalization of SOS, an event considered sufficient to induce Ras activation (Aronheim et al., 1994), thus suggesting that Shc proteins are involved in the regulation of Ras. This hypothesis is supported by the findings that Shc overexpression induces increased proliferative response, mitogen-activated (MAP) kinase and fos activation by EGF (A.E.Salcini and P.G.Pelicci, unpublished results), GM-CSF (Lanfrancone et al., 1995) or thrombin (Chen et al., 1996). Furthermore, transformation of NIH-3T3 fibroblasts by overexpression of Shc proteins depends on the presence of Tyr317 and Grb2-binding (Salcini et al., 1994), and neuronal terminal differentiation of PC12 cells by Shc overexpression is prevented by the co-expression of a Ras dominant negative mutant (Rozakis-Adcock et al., 1992), Two Shc isoforms of 52 kDa (p52shc) and 46 kDa (p46shc) have been characterized (Pelicci et al., 1992). They share a carboxy-terminal SH2 domain, an adjacent glycine/proline-rich region (CH) and, in their N-terminal region, a recently identified phosphotyrosine binding domain (PTB) (Blaikie et al., 1994; Kavanaugh and Williams, 1994; van der Geer et al., 1995). The modular organization of the p52shc/p46shc proteins is shown in Figure 1A. Consistent with a general function of Shc proteins in the transmission of signals from activated TKs, p52shc and p46shc are ubiquitously expressed. However, antibodies raised against the Shc SH2 domain, also detect an ∼66 kDa polypeptide, expressed mainly in epithelial cells (Pelicci et al., 1992). We report here the structural characterization and preliminary functional properties of p66shc. Figure 1.Partial DNA and primary amino acid sequence of the λK9 cDNA. (A) Schematic representation and limited restriction enzyme map of the λK9 and λGF11 cDNA sequences. The box represents the longest ORF starting from the ATG positions 195 (ATG1; λK9) and 83 (ATG2; λGF11) with the PTB, SH2 and the collagen-homologous (CH) 1 and 2 regions (CH1 and CH2) indicated. The ATGs at amino acid positions 525 and 660 of the λK9 sequence and 83 and 218 of the λGF11 (ATG2 and ATG3, respectively) (see text) are also indicated. P, PvuII; A, AvaI; B, BamHI. (B) Nucleotide and deduced amino acid sequences of the λK9 5′ extremity. The nucleotide sequence corresponds to the λK9 clone from nucleotides 1–566. The translation of the λK9 sequence from the first in-frame ATG (ATG1 at position 195; bold type) is shown below the nucleotide sequence. The 5′ in-frame termination codon at position 75 is underlined. Nucleotide and amino acid positions are indicated on the right of each lane. The region of the λK9 sequence common to that of the λGF11 is underlined (the bold ATG at nucleotide position 525 corresponds to ATG2 of λK9 and λGF11). (C) Comparison of the λK9 collagen-related sequence (CH2) with that of the α1-collagen chain (Coll). |, identical residues; :, conserved residues. Download figure Download PowerPoint Results Isolation and sequence analysis of the λK9 cDNA clone To isolate cDNAs representative of p66, a human cDNA library prepared from p66-expressing cells was screened with a DNA probe representative of the Shc SH2 domain. Twenty-two clones were isolated from the 500 000 phage plaques of the Calu1 cDNA library. The insert of one clone (λK9) was longer than the λGF11 clone which encodes p52shc/p46shc, and differed from λGF11 (Pelicci et al., 1992) in its 5′ extremity at restriction mapping analysis (Figure 1A). The λK9 insert was subcloned into the pGEM-3 vector (pK9 plasmid) and sequenced. Analysis of the λK9 DNA sequence and comparison with that of λGF11 revealed that the two clones were identical from nucleotide position 525 of λK9 and nucleotide position 76 of λGF11 to their ends. The sequences of the 5′ extremity of the λK9 clone, including its unique 524 bp region, is shown in Figure 1B. The longest open reading frame (ORF) predicted from clone λK9 is 1749 bp with the first in-frame ATG at position 195 (ATG1 in Figure 1B) and an in-frame TGA at position 1944 (not shown). ATG195 is probably the translation initiation codon of λK9, since it is flanked by sequences (TCAACTATGG) that match the Kozak consensus for translation initiation in eukaryotes (Kozak, 1989) and is preceded by an in-frame TGA codon at nucleotide position 75 (Figure 1B). The protein predicted from the λK9 clone sequence is 583 amino acids in length, and has a molecular mass of 62 898 Da. Comparison between the λK9 and the λGF11 (p52shc/p46shc) proteins disclosed an amino-terminal region of 110 amino acids unique to λK9, which continued into a common region starting at residue 1 of λGF11 and residue 111 of λK9, and terminated at the ends of both proteins. The first amino acid common to both proteins is the Met translation initiation codon of p52shc (see the partial amino acid sequence of λK9 shown in Figure 1B and ATG2 in Figure 1A). A search in the genebank database for sequences homologous to the amino-terminal-unique region of K9 identified human α1 collagen (20% amino acid identity and 33% similarity; Figure 1C). This collagen-homologous λK9 region was rich in glycine (24%) and proline (37%) residues, both of which are also abundant in the collagen α1 chain (57% and 46% in the λK9-homologous collagen α1 chain region, respectively). Some 31% of glycine and 50% of proline α1 collagen residues are conserved in the λK9 collagen-homologous region. Because the p52shc/p46shc protein contains a glycine/proline-rich region that is also partially homologous with the collagen α1 chain (CH region), we renamed it CH1 and called the λK9 region CH2 (Figure 1A). Not only do these two regions fail to display a high degree of homology, but they share homology with two adjacent regions of the collagen α1 chain which overlap for 34 amino acids (bold sequence in Figure 1C). Identification of the λK9 cDNA clone as the p66shc cDNA To ascertain whether the λK9 clone is, indeed, representative of the transcript encoding p66, the expression of λK9 corresponding mRNA was determined by RNase protection experiments and compared with p66shc expression. A 498 bp RNA probe containing a 454 bp region unique to λK9 (probe R1, Figure 2A) gave a strong 454 bp protection fragment in murine NIH-3T3 cells stably transfected with a λK9 expression vector (NIH-p66) and in a series of human p66-expressing cell samples (the A-172, NCTC, SK-N-MC and Calu1 cell lines and in mesothelial primary cells) (compare cell samples of Figure 2B, left panel and Figure 3). A fainter protection fragment was observed in cell lines that express lower levels of p66 (K562, GTL16 and PEER; Figures 2B and 3). tRNA and mRNAs from mouse NIH-3T3 fibroblasts or human cells that do not express p66 (U937, HL-60, KG1) were not protected by the R1 probe (Figures 2B and 3). Similar results were obtained with a second λK9 RNA probe that spans the junction between the unique λK9 sequences and the sequences common to λK9 and λGF11 (probe R2, Figure 2A). The 281 bp R2 probe yielded a 240 bp fully protected fragment in cells expressing p66shc (NIH-p66 fibroblasts and A-172 cells), a 40 bp protection fragment in p52shc/p46shc expressing cells [NIH-3T3 transfected with the λGF11 cDNA (NIH-p52/p46) A-172 and HL-60 cells] and no protection of tRNA and RNA from mouse cells (NIH-3T3 cells). The 40 bp protection fragment corresponds to the partial protection of the R2 probe by the p52shc/p46shc transcript (Figure 2B, right panel). Figure 2.RNase protection analysis of the expression of the p66shc encoding transcript. (A) Schematic representation of the used RNase probes (R1 and R2) and their positions with respect to the λK9 cDNA. (B) RNAs derived from the indicated cell samples were hybridized to the R1 (left panel) or R2 (right panel) RNA antisense probes. The protected fragments are shown by arrows; the origin (p66; p52/p46) of the fragment is also indicated. Riboprobes contained variable lengths of vector plasmids (10–40 bp) and therefore migrated slightly slower than fully protected fragments. Molecular weight markers are given on the right. tRNA, transfer RNA used as control of RNase digestion. Download figure Download PowerPoint Figure 3.Western blotting analysis of p66shc, p52shc and p46shc expression. Whole-cell lysates were prepared from the indicated cell lines and 50 μg of total protein separated by 10% SDS–PAGE, transferred to nitrocellulose and immunoblotted with anti-SH2 (upper panel) or anti-CH2 (lower panel) Shc antibodies (αSH2 and αCH2). The SHC proteins (p66, p52 and p46) are indicated on the left. Download figure Download PowerPoint We next generated a polyclonal antibody against the CH2 portion of λK9 (αCH2, see Materials and methods). A bacterial glutathione S-transferase (GST)-CH2 fusion protein was produced by inserting the sequence of the λK9 cDNA encoding amino acids 1–110, corresponding to its CH2 region, into the bacterial expression plasmid pGEX-2T to generate polyclonal antibodies in rabbit. The resulting serum reacted specifically with the λK9 cDNA products that had been translated in vitro and transiently expressed in COS-1 cells, as well as with a polypeptide of similar size in the Calu1 cell line (not shown). The anti-CH2 antibody also recognized a protein of ∼66 kDa in all cell samples that expressed p66 (Figure 3, lower panel). Moreover, the translation products of the λK9 cDNA were recognized by the anti-SH2 Shc antibody both in vitro and in vivo (not shown). Taken together, these data indicate that the λK9 clone encodes for the p66shc isoform. Origin of the p66 cDNA The origin of the human p66shc transcript was investigated by isolating the human Shc locus and mapping the various Shc exons. A genomic library from normal human embryo lung fibroblasts (WI38) was screened with the Shc SH2 DNA probe and two overlapping λ-clones (λ-JO and λ-PA1) were isolated (not shown). The inserts from these phages spanned ∼15 kb of human DNA (Figure 4A). To map Shc exons, the two λ-genomic clones were digested with the EcoRI, XbaI and BamHI restriction enzymes and hybridized to different portions of the Shc cDNA. The hybridizing restriction fragments were subcloned into the plasmid vector pGEM3 and the exon/intron boundaries sequenced. The restriction enzyme map and exon/intron organization of the Shc locus are reported in Figure 4A. The Shc locus contains 13 exons. Exon 1 is non-coding and contains only sequences that correspond to the p52shc/p46shc transcripts (λGF11 cDNA from nucleotides 1–75). The 5′ 521 bp of exon 2 contain sequences corresponding to the CH2 p66shc region and includes both the 5′ untranslated region and the p66shc ATG1. The 3′ 168 bp of exon 2 contain sequences corresponding to the 56 amino acids common to the p66shc and p52shc/p46shc isoforms (including ATG2 and ATG3) (exon 2a). Exons 3 to 13 encode the remaining p66shc and p52shc/p46shc common sequences. It appears, therefore, that only the 168 bp 3′ portion of exon 2 (exon 2a) is incorporated within the mature transcript encoding p52shc/p46shc, as found in the λGF11 cDNA clone. An acceptor consensus site for splicing was found 5′ to the Shc exon 2a (Figure 4B), suggesting that the λGF11 transcript is formed by the juxtaposition of exons 1 and 2a. In contrast, transcripts encoding p66shc would incorporate the entire exon 2. Figure 4.(A) Organization of the murine and human Shc locus and exon assembly of Shc transcripts. A limited restriction enzyme map of the mouse and human loci is shown at the top. Boxes indicate Shc exons and the exon numbers are given above [numbering is temporary since the Shc cap site(s) has not been mapped]. A schematic representation of the exon assembly in the p66shc and p52shc/p46shc encoding transcripts is given below. Exons are indicated by boxes and the splicing events are shown by the broken zig-zag line. The position of the three Shc ATGs (see text) is given. E, EcoRI; B, BamHI; X, XbaI. (B) Nucleotide sequences of exon 1–2a and exon 1–2b donor/acceptor splice junctions in both the human (Hu.) and mouse (Mu.) loci. The donor and acceptor sites are underline; exons 1 and 2 and intervening sequences (intron 1 and the retained intron) are indicated. Download figure Download PowerPoint A similar Shc genomic organization and Shc transcript assembly was found in mice. Alignment of the predicted mouse p66shc sequence, as derived from analysis of mouse p66 cDNAs (not shown), with human p66shc showed a high degree of amino acid identity and identical overall organization of the two proteins (not shown). In the mouse Shc locus, exon 1 encodes p52shc/p46shc unique sequences, the 5′ end of exon 2 encodes p66shc unique sequences, exon 2a–13 sequences common to all Shc isoforms (Figure 4A). An acceptor consensus site for splicing is also found 5′ to the mouse Shc exon 2a at the same position as in the human Shc exon 2a (Figure 4B). Overexpression of the p66shc cDNA does not induce transformation of NIH-3T3 fibroblasts Overexpression of the p52shc/p46shc isoforms induces transformation of NIH-3T3 fibroblasts, as determined by their acquired capacity to grow in semi-solid media and to form tumours in nude mice (Pelicci et al., 1992). The biological activity of the p66shc and the p52shc/p46shc isoforms was compared by expressing the p66shc and p52shc/p46shc cDNAs into NIH-3T3 fibroblasts and evaluating the capacity of the resulting transformants to form colonies in soft agar. NIH-3T3 fibroblasts express all three Shc isoforms. As expression of the p66shc cDNA into NIH-3T3 fibroblasts yielded a marked increase in the level of p66shc and p52shc and, to a lesser extent, p46shc (NIH-p66 clone; Figure 5A); all three isoforms are presumably encoded by the p66shc cDNA. Indeed, the protein predicted by the translation of the p66shc cDNA contains three in-frame ATGs, the second and third corresponding to the first and second of the p52shc/p46shc predicted protein (Figure 1A). To determine the potential of isoform translation of the λGF11 and λK9 cDNAs, ATGs were variously mutagenized. In vitro translation of the λGF11(p52shc/p46shc cDNA) yielded two polypeptides of 52 and 46 kDa that reacted specifically against the anti-SH2 Shc antibody (not shown). Mutation of the ATG3 of λGF11 to TTG (GF11TTG) abrogates translation of p46, while mutagenesis of ATG2 (GF13) abrogates translation of p52 (Pelicci et al., 1992). Expression of the GF11TTG cDNA in NIH-3T3 cells resulted in expression of the p52shc isoform (NIH-p52 clone), while expression of the GF13 cDNA resulted in the production of the p46shc protein (NIH-p46 clone) (Figure 5A). These results suggest that p52shc and p46shc isoforms are translated from the same transcript by alternative usage of two in-frame ATGs. In vitro translation of the λK9 (pK9 plasmid) yielded p66 and p52 proteins and, to a lesser extent, a p46 polypeptide, all of which reacted with the anti-SH2 antibody (not shown). Simultaneous mutagenesis of ATG2 and ATG3 of λK9 (K9TTGs) did not affect translation of the p66 polypeptide, but abrogated p52 and p46 expression, as assayed by both in vitro translation (not shown) and stable expression in NIH-3T3 fibroblasts (NIH-p66-TTGs clone; Figure 5A). Taken together, these data provide evidence that the three in-frame ATGs are all used as translation initiation sites both in vitro and in vivo and that the λK9 cDNA encode all three Shc isoforms. Figure 5.Potential of isoform translation of the λGF11 and λK9 cDNAs. The following cDNAs were expressed into NIH-3T3 cells (A and B): GF11, encoding p52shc/p46shc (Pelicci et al., 1992); K9, encoding p66shc; GF11TGG, derived from the GF11 cDNA by phenylalanine/alanine substitution of ATG3 (Pelicci et al., 1992); GF13, starting between ATG2 and ATG3; K9TTGs, derived from the K9 cDNA by phenylalanine/alanine substitution of ATG2 and ATG3. (A) Western blotting analysis of Shc expression of NIH-3T3 cells stably transfected with the LXSN expression vector (NIH-SN) or with the same vector expressing the GF11 (NIH-p52/p46), GF13 (NIH-p46), GF11TGG (NIH-p52), K9 (NIH-p66), K9TTGs (NIH-p66-TTGs) cDNAs. (B) Western blotting analysis of Shc-overexpressing clones. Lysates from one control NIH-3T3 clones (NIH-SN) and clones overexpressing p52shc/p46shc (NIH-SHC-13 and NIH-SHC-9), p66shc/p52shc (NIH-p66-8), p66shc (NIH-p66-TTGs5 and NIH-p66-TTGs7) were blotted against the anti-SH2 Shc antibody (αSH2). Download figure Download PowerPoint To test for transformation capability, NIH-3T3 clones overexpressing the wild-type p52shc/p46shc (NIH-SHC-9 and NIH-SHC-13), the wild-type p66shc (NIH-p66-2 and NIH-p66-8), or a mutant version of p66 with phenylalanine substitutions at the second and third ATGs (NIH-p66-TTGs5 and NIH-p66-TTGs7), or control NIH-3T3 clones (NIH-SN and NIH-SN-5) were plated in triplicate at various concentrations in 0.3% agar medium supplemented with 20% serum, and colonies scored after 14 days. Western blot analysis of the various Shc-overexpressing clones and one of the two control clones (NIH-SN) is reported in Figure 5B. The two NIH-SHC clones formed colonies in soft agar at the expected frequencies (Pelicci et al., 1992), whereas the control NIH-SN clones, the NIH-p66 and NIH-p66-TTGs clones did not (Table I). It, therefore, seems that the p66shc isoform does not retain the transforming activity of the p52shc/p46shc isoforms. Table 1. Cloning efficiencya Cells Cloning efficiency (%) NIH-SN 0.5 NIH-SHC-9 >3 NIH-p66-2 <0.001 NIH-p66-8 <0.001 NIH-p66-TGGs5 <0.001 NIH-p66TGGs7 <0.001 Cloning efficiency was monitored in control NIH-3T3 clones (NIH-SN-1 and NIH-SN-5) and NIH-3T3 clones expressing p52shc/46shc (NIH-SHC-9 and NIH-SHC-13), wild-type p66shc (NIH-p66-2 and NIH-p66-8) and mutant p66TGGs (NIH-p66-TGGs5 and NIH-p66-TGGs7) cDNAs. Colonies were examined 14 days after cells were seeded in triplicate at 103, 104 and 105 cells per culture dish. Each result is the average of five separate experiments. p66shc is phosphorylated upon EGF stimulation and binds to activated EGFR and Grb2 In order to investigate the molecular basis of the lack of transforming potential of p66shc, its ability to mimic the effects of p52shc/p46shc on known signal transduction mechanisms was investigated by comparing the capacity of p66shc and p52shc/p46shc to bind activated EGFR, to be phosphorylated upon EGF stimulation, to bind Grb2 and activate MAP kinases. The p66shc (K9TGGs cDNA) and p52shc/p46shc (GF11 cDNA) coding sequences were cloned under the control of the adenovirus promoter in a plasmid containing the SV40 origin of replication (pMT2) and transiently transfected into COS-1 cells. At 16 h after transfection, the cells were washed and kept in serum-free media for an additional 24 h and either treated for 5 min or not with 30 ng/ml of EGF. Lysates prepared from serum-starved or EGF-stimulated cells were analysed for Shc protein expression (Figure 6B) and immunoprecipitated with anti-Shc antibodies. The immunoprecipitates were immunoblotted with anti-phosphotyrosine or anti-Grb2 antibodies (Figure 6A), EGF stimulation induced a comparable increase in the phosphotyrosine content of p66shc and p52shc/46shc proteins (Figure 6A, upper panel). A novel phosphotyrosine-containing polypeptide of ∼175 kDa stably complexed with both p66shc and p52shc/46shc upon EGF stimulation (Figure 6A, upper panel). Probing of the same anti-Shc immunoprecipitates with anti-EGFR antibodies revealed that the 175 kDa polypeptide is indeed the EGFR (data not shown). In addition, phosphorylated p66shc and p52/46shc formed stable complexes with Grb2 in EGF-stimulated cells (Figure 6A, lower panel). It can, therefore, be concluded that p66shc is phosphorylated by activated EGFR and binds to activated EGFR and Grb2 similarly to p52shc/p46shc. Figure 6.Tyrosine phosphorylation and Grb2 binding of p52shc/p46shc and p66shc. (A) Anti-phosphotyrosine Western blotting of anti-Shc (αSH2) immunoprecipitates from lysates of Cos-1 cells transfected with the pMT2-p66-TTGs and pMT2-p52/p46 expression vectors and subsequently treated (+) or not (−) with EGF (upper panel). The same blot was reprobed with anti-Grb2 antibodies (α Grb2) (lower panel). The EGFR, p66shc, p52shc and p46shc polypeptides and the αGrb2 25 kDa immunoreactive polypeptide are indicated by arrows. Immunoglobulin cross-reactive polypeptides are also indicated (Ig). (B) Western blotting analysis of Shc proteins expression in Cos-1 cells transfected with the pMT2-p66-TTGs and pMT2-p52/p46 expression vectors and treated (+) or not (−) with EGF. Download figure Download Pow