Abstract
Epidermal growth factor (EGF) induces tyrosine phosphorylation of the Shc adapter protein, which plays an important role in EGF-stimulated mitogenesis. Shc stimulates Ras/mitogen-activated protein kinase (MAPK) through forming a complex with Grb2 at the phosphorylated tyrosine (Y) residue 317. In this study, we identified novel phosphorylation sites of Shc, at Y239 and Y240. To define the Shc pathway further, we used NIH 3T3 cells expressing the previously characterized mutant EGF receptor (EGF-R) which lacks all known autophosphorylation sites but retains EGF-stimulated mitogenesis with selective phosphorylation of Shc. We constructed wild-type (WT) or mutant Shc cDNAs in which Y317 or/and Y239 and Y240 are replaced with phenylalanine (F) and introduced them into NIH 3T3 cells expressing WT or mutant EGF-R. In the WT EGF-R-expressing cells, the Y239/240/317F Shc, but not Y317F or Y239/240F Shc, decreased EGF-stimulated cell growth. In the mutant EGF-R-expressing cells, Y317F Shc or Y239/240F Shc decreased EGF-stimulated cell growth significantly, though Y317F was a little more potent than Y239/240F. Although cells expressing the Y317F Shc hardly activated MAPK in response to EGF, cells expressing the Y239/240F Shc fully activated MAPK. In contrast, Y239/240F Shc, but not Y317F Shc, reduced the EGF-induced c-myc message. These results suggest that Shc activates two distinct signaling pathways, Y317 to Ras/MAPK and Y239 and Y240 to another pathway including Myc, and that both are involved in EGF-induced mitogenic signaling.
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- Barone M. V., Courtneidge S. A. Myc but not Fos rescue of PDGF signalling block caused by kinase-inactive Src. Nature. 1995 Nov 30;378(6556):509–512. doi: 10.1038/378509a0. [DOI] [PubMed] [Google Scholar]
- Basu T., Warne P. H., Downward J. Role of Shc in the activation of Ras in response to epidermal growth factor and nerve growth factor. Oncogene. 1994 Dec;9(12):3483–3491. [PubMed] [Google Scholar]
- Batzer A. G., Blaikie P., Nelson K., Schlessinger J., Margolis B. The phosphotyrosine interaction domain of Shc binds an LXNPXY motif on the epidermal growth factor receptor. Mol Cell Biol. 1995 Aug;15(8):4403–4409. doi: 10.1128/mcb.15.8.4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Batzer A. G., Rotin D., Ureña J. M., Skolnik E. Y., Schlessinger J. Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol Cell Biol. 1994 Aug;14(8):5192–5201. doi: 10.1128/mcb.14.8.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
- Buday L., Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell. 1993 May 7;73(3):611–620. doi: 10.1016/0092-8674(93)90146-h. [DOI] [PubMed] [Google Scholar]
- Burgering B. M., de Vries-Smits A. M., Medema R. H., van Weeren P. C., Tertoolen L. G., Bos J. L. Epidermal growth factor induces phosphorylation of extracellular signal-regulated kinase 2 via multiple pathways. Mol Cell Biol. 1993 Dec;13(12):7248–7256. doi: 10.1128/mcb.13.12.7248. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cai H., Szeberényi J., Cooper G. M. Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells. Mol Cell Biol. 1990 Oct;10(10):5314–5323. doi: 10.1128/mcb.10.10.5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chardin P., Camonis J. H., Gale N. W., van Aelst L., Schlessinger J., Wigler M. H., Bar-Sagi D. Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science. 1993 May 28;260(5112):1338–1343. doi: 10.1126/science.8493579. [DOI] [PubMed] [Google Scholar]
- Chen Y., Grall D., Salcini A. E., Pelicci P. G., Pouysségur J., Van Obberghen-Schilling E. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor. EMBO J. 1996 Mar 1;15(5):1037–1044. [PMC free article] [PubMed] [Google Scholar]
- Cohen G. B., Ren R., Baltimore D. Modular binding domains in signal transduction proteins. Cell. 1995 Jan 27;80(2):237–248. doi: 10.1016/0092-8674(95)90406-9. [DOI] [PubMed] [Google Scholar]
- Decker S. J. Transmembrane signaling by epidermal growth factor receptors lacking autophosphorylation sites. J Biol Chem. 1993 May 5;268(13):9176–9179. [PubMed] [Google Scholar]
- Downward J., Parker P., Waterfield M. D. Autophosphorylation sites on the epidermal growth factor receptor. Nature. 1984 Oct 4;311(5985):483–485. doi: 10.1038/311483a0. [DOI] [PubMed] [Google Scholar]
- Eilers M., Schirm S., Bishop J. M. The MYC protein activates transcription of the alpha-prothymosin gene. EMBO J. 1991 Jan;10(1):133–141. doi: 10.1002/j.1460-2075.1991.tb07929.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gale N. W., Kaplan S., Lowenstein E. J., Schlessinger J., Bar-Sagi D. Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras. Nature. 1993 May 6;363(6424):88–92. doi: 10.1038/363088a0. [DOI] [PubMed] [Google Scholar]
- Goga A., McLaughlin J., Afar D. E., Saffran D. C., Witte O. N. Alternative signals to RAS for hematopoietic transformation by the BCR-ABL oncogene. Cell. 1995 Sep 22;82(6):981–988. doi: 10.1016/0092-8674(95)90277-5. [DOI] [PubMed] [Google Scholar]
- Gotoh N., Muroya K., Hattori S., Nakamura S., Chida K., Shibuya M. The SH2 domain of Shc suppresses EGF-induced mitogenesis in a dominant negative manner. Oncogene. 1995 Dec 21;11(12):2525–2533. [PubMed] [Google Scholar]
- Gotoh N., Tojo A., Hino M., Yazaki Y., Shibuya M. A highly conserved tyrosine residue at codon 845 within the kinase domain is not required for the transforming activity of human epidermal growth factor receptor. Biochem Biophys Res Commun. 1992 Jul 31;186(2):768–774. doi: 10.1016/0006-291x(92)90812-y. [DOI] [PubMed] [Google Scholar]
- Gotoh N., Tojo A., Muroya K., Hashimoto Y., Hattori S., Nakamura S., Takenawa T., Yazaki Y., Shibuya M. Epidermal growth factor-receptor mutant lacking the autophosphorylation sites induces phosphorylation of Shc protein and Shc-Grb2/ASH association and retains mitogenic activity. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):167–171. doi: 10.1073/pnas.91.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gotoh N., Tojo A., Shibuya M. A novel pathway from phosphorylation of tyrosine residues 239/240 of Shc, contributing to suppress apoptosis by IL-3. EMBO J. 1996 Nov 15;15(22):6197–6204. [PMC free article] [PubMed] [Google Scholar]
- Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
- Hasegawa H., Kiyokawa E., Tanaka S., Nagashima K., Gotoh N., Shibuya M., Kurata T., Matsuda M. DOCK180, a major CRK-binding protein, alters cell morphology upon translocation to the cell membrane. Mol Cell Biol. 1996 Apr;16(4):1770–1776. doi: 10.1128/mcb.16.4.1770. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heikkila R., Schwab G., Wickstrom E., Loke S. L., Pluznik D. H., Watt R., Neckers L. M. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1. 1987 Jul 30-Aug 5Nature. 328(6129):445–449. doi: 10.1038/328445a0. [DOI] [PubMed] [Google Scholar]
- Helin K., Velu T., Martin P., Vass W. C., Allevato G., Lowy D. R., Beguinot L. The biological activity of the human epidermal growth factor receptor is positively regulated by its C-terminal tyrosines. Oncogene. 1991 May;6(5):825–832. [PubMed] [Google Scholar]
- Hill C. S., Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell. 1995 Jan 27;80(2):199–211. doi: 10.1016/0092-8674(95)90403-4. [DOI] [PubMed] [Google Scholar]
- Honegger A., Dull T. J., Bellot F., Van Obberghen E., Szapary D., Schmidt A., Ullrich A., Schlessinger J. Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites. EMBO J. 1988 Oct;7(10):3045–3052. doi: 10.1002/j.1460-2075.1988.tb03169.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karunagaran D., Tzahar E., Beerli R. R., Chen X., Graus-Porta D., Ratzkin B. J., Seger R., Hynes N. E., Yarden Y. ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J. 1996 Jan 15;15(2):254–264. [PMC free article] [PubMed] [Google Scholar]
- Kavanaugh W. M., Williams L. T. An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science. 1994 Dec 16;266(5192):1862–1865. doi: 10.1126/science.7527937. [DOI] [PubMed] [Google Scholar]
- Kim H. K., Kim J. W., Zilberstein A., Margolis B., Kim J. G., Schlessinger J., Rhee S. G. PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254. Cell. 1991 May 3;65(3):435–441. doi: 10.1016/0092-8674(91)90461-7. [DOI] [PubMed] [Google Scholar]
- Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
- Lai K. M., Olivier J. P., Gish G. D., Henkemeyer M., McGlade J., Pawson T. A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase. Mol Cell Biol. 1995 Sep;15(9):4810–4818. doi: 10.1128/mcb.15.9.4810. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Land H., Parada L. F., Weinberg R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 1983 Aug 18;304(5927):596–602. doi: 10.1038/304596a0. [DOI] [PubMed] [Google Scholar]
- Li N., Batzer A., Daly R., Yajnik V., Skolnik E., Chardin P., Bar-Sagi D., Margolis B., Schlessinger J. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 1993 May 6;363(6424):85–88. doi: 10.1038/363085a0. [DOI] [PubMed] [Google Scholar]
- Li N., Schlessinger J., Margolis B. Autophosphorylation mutants of the EGF-receptor signal through auxiliary mechanisms involving SH2 domain proteins. Oncogene. 1994 Dec;9(12):3457–3465. [PubMed] [Google Scholar]
- Lowenstein E. J., Daly R. J., Batzer A. G., Li W., Margolis B., Lammers R., Ullrich A., Skolnik E. Y., Bar-Sagi D., Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. doi: 10.1016/0092-8674(92)90167-b. [DOI] [PubMed] [Google Scholar]
- Marais R., Wynne J., Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell. 1993 Apr 23;73(2):381–393. doi: 10.1016/0092-8674(93)90237-k. [DOI] [PubMed] [Google Scholar]
- Margolis B. L., Lax I., Kris R., Dombalagian M., Honegger A. M., Howk R., Givol D., Ullrich A., Schlessinger J. All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor. J Biol Chem. 1989 Jun 25;264(18):10667–10671. [PubMed] [Google Scholar]
- Marshall C. J. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev. 1994 Feb;4(1):82–89. doi: 10.1016/0959-437x(94)90095-7. [DOI] [PubMed] [Google Scholar]
- Matuoka K., Shibata M., Yamakawa A., Takenawa T. Cloning of ASH, a ubiquitous protein composed of one Src homology region (SH) 2 and two SH3 domains, from human and rat cDNA libraries. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9015–9019. doi: 10.1073/pnas.89.19.9015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McGlade J., Cheng A., Pelicci G., Pelicci P. G., Pawson T. Shc proteins are phosphorylated and regulated by the v-Src and v-Fps protein-tyrosine kinases. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8869–8873. doi: 10.1073/pnas.89.19.8869. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mulcahy L. S., Smith M. R., Stacey D. W. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature. 1985 Jan 17;313(5999):241–243. doi: 10.1038/313241a0. [DOI] [PubMed] [Google Scholar]
- Müller R., Bravo R., Burckhardt J., Curran T. Induction of c-fos gene and protein by growth factors precedes activation of c-myc. Nature. 1984 Dec 20;312(5996):716–720. doi: 10.1038/312716a0. [DOI] [PubMed] [Google Scholar]
- Pawson T. Protein modules and signalling networks. Nature. 1995 Feb 16;373(6515):573–580. doi: 10.1038/373573a0. [DOI] [PubMed] [Google Scholar]
- Pelicci G., Lanfrancone L., Grignani F., McGlade J., Cavallo F., Forni G., Nicoletti I., Grignani F., Pawson T., Pelicci P. G. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell. 1992 Jul 10;70(1):93–104. doi: 10.1016/0092-8674(92)90536-l. [DOI] [PubMed] [Google Scholar]
- Pronk G. J., de Vries-Smits A. M., Buday L., Downward J., Maassen J. A., Medema R. H., Bos J. L. Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras. Mol Cell Biol. 1994 Mar;14(3):1575–1581. doi: 10.1128/mcb.14.3.1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rollins B. J., Stiles C. D. Serum-inducible genes. Adv Cancer Res. 1989;53:1–32. doi: 10.1016/s0065-230x(08)60277-8. [DOI] [PubMed] [Google Scholar]
- Rotin D., Margolis B., Mohammadi M., Daly R. J., Daum G., Li N., Fischer E. H., Burgess W. H., Ullrich A., Schlessinger J. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma. EMBO J. 1992 Feb;11(2):559–567. doi: 10.1002/j.1460-2075.1992.tb05087.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roussel M. F., Shurtleff S. A., Downing J. R., Sherr C. J. A point mutation at tyrosine-809 in the human colony-stimulating factor 1 receptor impairs mitogenesis without abrogating tyrosine kinase activity, association with phosphatidylinositol 3-kinase, or induction of c-fos and junB genes. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6738–6742. doi: 10.1073/pnas.87.17.6738. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rozakis-Adcock M., Fernley R., Wade J., Pawson T., Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 1993 May 6;363(6424):83–85. doi: 10.1038/363083a0. [DOI] [PubMed] [Google Scholar]
- Rozakis-Adcock M., McGlade J., Mbamalu G., Pelicci G., Daly R., Li W., Batzer A., Thomas S., Brugge J., Pelicci P. G. Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature. 1992 Dec 17;360(6405):689–692. doi: 10.1038/360689a0. [DOI] [PubMed] [Google Scholar]
- Salcini A. E., McGlade J., Pelicci G., Nicoletti I., Pawson T., Pelicci P. G. Formation of Shc-Grb2 complexes is necessary to induce neoplastic transformation by overexpression of Shc proteins. Oncogene. 1994 Oct;9(10):2827–2836. [PubMed] [Google Scholar]
- Sasaoka T., Langlois W. J., Bai F., Rose D. W., Leitner J. W., Decker S. J., Saltiel A., Gill G. N., Kobayashi M., Draznin B. Involvement of ErbB2 in the signaling pathway leading to cell cycle progression from a truncated epidermal growth factor receptor lacking the C-terminal autophosphorylation sites. J Biol Chem. 1996 Apr 5;271(14):8338–8344. doi: 10.1074/jbc.271.14.8338. [DOI] [PubMed] [Google Scholar]
- Sasaoka T., Rose D. W., Jhun B. H., Saltiel A. R., Draznin B., Olefsky J. M. Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. J Biol Chem. 1994 May 6;269(18):13689–13694. [PubMed] [Google Scholar]
- Satoh T., Endo M., Nakafuku M., Akiyama T., Yamamoto T., Kaziro Y. Accumulation of p21ras.GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7926–7929. doi: 10.1073/pnas.87.20.7926. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Seetharam L., Gotoh N., Maru Y., Neufeld G., Yamaguchi S., Shibuya M. A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF. Oncogene. 1995 Jan 5;10(1):135–147. [PubMed] [Google Scholar]
- Skolnik E. Y., Lee C. H., Batzer A., Vicentini L. M., Zhou M., Daly R., Myers M. J., Jr, Backer J. M., Ullrich A., White M. F. The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J. 1993 May;12(5):1929–1936. doi: 10.1002/j.1460-2075.1993.tb05842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spencer C. A., Groudine M. Control of c-myc regulation in normal and neoplastic cells. Adv Cancer Res. 1991;56:1–48. doi: 10.1016/s0065-230x(08)60476-5. [DOI] [PubMed] [Google Scholar]
- Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
- Walton G. M., Chen W. S., Rosenfeld M. G., Gill G. N. Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals self-phosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates. J Biol Chem. 1990 Jan 25;265(3):1750–1754. [PubMed] [Google Scholar]
- Wary K. K., Mainiero F., Isakoff S. J., Marcantonio E. E., Giancotti F. G. The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell. 1996 Nov 15;87(4):733–743. doi: 10.1016/s0092-8674(00)81392-6. [DOI] [PubMed] [Google Scholar]