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. 1999 Jan 4;18(1):145–155. doi: 10.1093/emboj/18.1.145

Activation of the Drosophila C3G leads to cell fate changes and overproliferation during development, mediated by the RAS-MAPK pathway and RAP1.

S Ishimaru 1, R Williams 1, E Clark 1, H Hanafusa 1, U Gaul 1
PMCID: PMC1171110  PMID: 9878058

Abstract

The cellular signal transduction pathways by which C3G, a RAS family guanine nucleotide exchange factor, mediates v-crk transformation are not well understood. Here we report the identification of Drosophila C3G, which, like its human cognate, specifically binds to CRK but not DRK/GRB2 adaptor molecules. During Drosophila development, constitutive membrane binding of C3G, which also occurs during v-crk transformation, results in cell fate changes and overproliferation, mimicking overactivity of the RAS-MAPK pathway. The effects of C3G overactivity can be suppressed by reducing the gene dose of components of the RAS-MAPK pathway and of RAP1. These findings provide the first in vivo evidence that membrane localization of C3G can trigger activation of RAP1 and RAS resulting in the activation of MAPK, one of the hallmarks of v-crk transformation previously thought to be mediated through activation of SOS.

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Selected References

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  1. Allard J. D., Chang H. C., Herbst R., McNeill H., Simon M. A. The SH2-containing tyrosine phosphatase corkscrew is required during signaling by sevenless, Ras1 and Raf. Development. 1996 Apr;122(4):1137–1146. doi: 10.1242/dev.122.4.1137. [DOI] [PubMed] [Google Scholar]
  2. Aronheim A., Engelberg D., Li N., al-Alawi N., Schlessinger J., Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell. 1994 Sep 23;78(6):949–961. doi: 10.1016/0092-8674(94)90271-2. [DOI] [PubMed] [Google Scholar]
  3. Basler K., Christen B., Hafen E. Ligand-independent activation of the sevenless receptor tyrosine kinase changes the fate of cells in the developing Drosophila eye. Cell. 1991 Mar 22;64(6):1069–1081. doi: 10.1016/0092-8674(91)90262-w. [DOI] [PubMed] [Google Scholar]
  4. Birge R. B., Fajardo J. E., Mayer B. J., Hanafusa H. Tyrosine-phosphorylated epidermal growth factor receptor and cellular p130 provide high affinity binding substrates to analyze Crk-phosphotyrosine-dependent interactions in vitro. J Biol Chem. 1992 May 25;267(15):10588–10595. [PubMed] [Google Scholar]
  5. Birge R. B., Knudsen B. S., Besser D., Hanafusa H. SH2 and SH3-containing adaptor proteins: redundant or independent mediators of intracellular signal transduction. Genes Cells. 1996 Jul;1(7):595–613. doi: 10.1046/j.1365-2443.1996.00258.x. [DOI] [PubMed] [Google Scholar]
  6. Brand A. H., Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun;118(2):401–415. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
  7. Brunner D., Oellers N., Szabad J., Biggs W. H., 3rd, Zipursky S. L., Hafen E. A gain-of-function mutation in Drosophila MAP kinase activates multiple receptor tyrosine kinase signaling pathways. Cell. 1994 Mar 11;76(5):875–888. doi: 10.1016/0092-8674(94)90362-x. [DOI] [PubMed] [Google Scholar]
  8. Clark E. A., Brugge J. S. Integrins and signal transduction pathways: the road taken. Science. 1995 Apr 14;268(5208):233–239. doi: 10.1126/science.7716514. [DOI] [PubMed] [Google Scholar]
  9. Clifford R., Schüpbach T. Molecular analysis of the Drosophila EGF receptor homolog reveals that several genetically defined classes of alleles cluster in subdomains of the receptor protein. Genetics. 1994 Jun;137(2):531–550. doi: 10.1093/genetics/137.2.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dickson B., Sprenger F., Hafen E. Prepattern in the developing Drosophila eye revealed by an activated torso--sevenless chimeric receptor. Genes Dev. 1992 Dec;6(12A):2327–2339. doi: 10.1101/gad.6.12a.2327. [DOI] [PubMed] [Google Scholar]
  11. Dickson B., Sprenger F., Morrison D., Hafen E. Raf functions downstream of Ras1 in the Sevenless signal transduction pathway. Nature. 1992 Dec 10;360(6404):600–603. doi: 10.1038/360600a0. [DOI] [PubMed] [Google Scholar]
  12. Ellis M. C., O'Neill E. M., Rubin G. M. Expression of Drosophila glass protein and evidence for negative regulation of its activity in non-neuronal cells by another DNA-binding protein. Development. 1993 Nov;119(3):855–865. doi: 10.1242/dev.119.3.855. [DOI] [PubMed] [Google Scholar]
  13. Fortini M. E., Simon M. A., Rubin G. M. Signalling by the sevenless protein tyrosine kinase is mimicked by Ras1 activation. Nature. 1992 Feb 6;355(6360):559–561. doi: 10.1038/355559a0. [DOI] [PubMed] [Google Scholar]
  14. Freeman M. Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell. 1996 Nov 15;87(4):651–660. doi: 10.1016/s0092-8674(00)81385-9. [DOI] [PubMed] [Google Scholar]
  15. Gabay L., Seger R., Shilo B. Z. MAP kinase in situ activation atlas during Drosophila embryogenesis. Development. 1997 Sep;124(18):3535–3541. doi: 10.1242/dev.124.18.3535. [DOI] [PubMed] [Google Scholar]
  16. Gaul U., Mardon G., Rubin G. M. A putative Ras GTPase activating protein acts as a negative regulator of signaling by the Sevenless receptor tyrosine kinase. Cell. 1992 Mar 20;68(6):1007–1019. doi: 10.1016/0092-8674(92)90073-l. [DOI] [PubMed] [Google Scholar]
  17. Gotoh T., Hattori S., Nakamura S., Kitayama H., Noda M., Takai Y., Kaibuchi K., Matsui H., Hatase O., Takahashi H. Identification of Rap1 as a target for the Crk SH3 domain-binding guanine nucleotide-releasing factor C3G. Mol Cell Biol. 1995 Dec;15(12):6746–6753. doi: 10.1128/mcb.15.12.6746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gotoh T., Niino Y., Tokuda M., Hatase O., Nakamura S., Matsuda M., Hattori S. Activation of R-Ras by Ras-guanine nucleotide-releasing factor. J Biol Chem. 1997 Jul 25;272(30):18602–18607. doi: 10.1074/jbc.272.30.18602. [DOI] [PubMed] [Google Scholar]
  19. Greulich H., Hanafusa H. A role for Ras in v-Crk transformation. Cell Growth Differ. 1996 Nov;7(11):1443–1451. [PubMed] [Google Scholar]
  20. Hariharan I. K., Carthew R. W., Rubin G. M. The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination. Cell. 1991 Nov 15;67(4):717–722. doi: 10.1016/0092-8674(91)90066-8. [DOI] [PubMed] [Google Scholar]
  21. Hay B. A., Wassarman D. A., Rubin G. M. Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell. 1995 Dec 29;83(7):1253–1262. doi: 10.1016/0092-8674(95)90150-7. [DOI] [PubMed] [Google Scholar]
  22. Herbst R., Carroll P. M., Allard J. D., Schilling J., Raabe T., Simon M. A. Daughter of sevenless is a substrate of the phosphotyrosine phosphatase Corkscrew and functions during sevenless signaling. Cell. 1996 Jun 14;85(6):899–909. doi: 10.1016/s0092-8674(00)81273-8. [DOI] [PubMed] [Google Scholar]
  23. Ip Y. T., Davis R. J. Signal transduction by the c-Jun N-terminal kinase (JNK)--from inflammation to development. Curr Opin Cell Biol. 1998 Apr;10(2):205–219. doi: 10.1016/s0955-0674(98)80143-9. [DOI] [PubMed] [Google Scholar]
  24. Jones S., Vignais M. L., Broach J. R. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras. Mol Cell Biol. 1991 May;11(5):2641–2646. doi: 10.1128/mcb.11.5.2641-2646.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Karess R. E., Rubin G. M. Analysis of P transposable element functions in Drosophila. Cell. 1984 Aug;38(1):135–146. doi: 10.1016/0092-8674(84)90534-8. [DOI] [PubMed] [Google Scholar]
  26. Karim F. D., Chang H. C., Therrien M., Wassarman D. A., Laverty T., Rubin G. M. A screen for genes that function downstream of Ras1 during Drosophila eye development. Genetics. 1996 May;143(1):315–329. doi: 10.1093/genetics/143.1.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Karim F. D., Rubin G. M. Ectopic expression of activated Ras1 induces hyperplastic growth and increased cell death in Drosophila imaginal tissues. Development. 1998 Jan;125(1):1–9. doi: 10.1242/dev.125.1.1. [DOI] [PubMed] [Google Scholar]
  28. Kimmel B. E., Heberlein U., Rubin G. M. The homeo domain protein rough is expressed in a subset of cells in the developing Drosophila eye where it can specify photoreceptor cell subtype. Genes Dev. 1990 May;4(5):712–727. doi: 10.1101/gad.4.5.712. [DOI] [PubMed] [Google Scholar]
  29. Kitayama H., Sugimoto Y., Matsuzaki T., Ikawa Y., Noda M. A ras-related gene with transformation suppressor activity. Cell. 1989 Jan 13;56(1):77–84. doi: 10.1016/0092-8674(89)90985-9. [DOI] [PubMed] [Google Scholar]
  30. Knudsen B. S., Feller S. M., Hanafusa H. Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk. J Biol Chem. 1994 Dec 30;269(52):32781–32787. [PubMed] [Google Scholar]
  31. Knudsen B. S., Zheng J., Feller S. M., Mayer J. P., Burrell S. K., Cowburn D., Hanafusa H. Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins. EMBO J. 1995 May 15;14(10):2191–2198. doi: 10.1002/j.1460-2075.1995.tb07213.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lai C. C., Boguski M., Broek D., Powers S. Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins. Mol Cell Biol. 1993 Mar;13(3):1345–1352. doi: 10.1128/mcb.13.3.1345-1352.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Li Q., Hariharan I. K., Chen F., Huang Y., Fischer J. A. Genetic interactions with Rap1 and Ras1 reveal a second function for the fat facets deubiquitinating enzyme in Drosophila eye development. Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12515–12520. doi: 10.1073/pnas.94.23.12515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lu X., Chou T. B., Williams N. G., Roberts T., Perrimon N. Control of cell fate determination by p21ras/Ras1, an essential component of torso signaling in Drosophila. Genes Dev. 1993 Apr;7(4):621–632. doi: 10.1101/gad.7.4.621. [DOI] [PubMed] [Google Scholar]
  36. Marra M. A., Hillier L., Waterston R. H. Expressed sequence tags--ESTablishing bridges between genomes. Trends Genet. 1998 Jan;14(1):4–7. doi: 10.1016/S0168-9525(97)01355-3. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Marshall C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995 Jan 27;80(2):179–185. doi: 10.1016/0092-8674(95)90401-8. [DOI] [PubMed] [Google Scholar]
  39. Matsuda M., Tanaka S., Nagata S., Kojima A., Kurata T., Shibuya M. Two species of human CRK cDNA encode proteins with distinct biological activities. Mol Cell Biol. 1992 Aug;12(8):3482–3489. doi: 10.1128/mcb.12.8.3482-3489.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mayer B. J., Hamaguchi M., Hanafusa H. A novel viral oncogene with structural similarity to phospholipase C. Nature. 1988 Mar 17;332(6161):272–275. doi: 10.1038/332272a0. [DOI] [PubMed] [Google Scholar]
  41. Miklos G. L., Rubin G. M. The role of the genome project in determining gene function: insights from model organisms. Cell. 1996 Aug 23;86(4):521–529. doi: 10.1016/s0092-8674(00)80126-9. [DOI] [PubMed] [Google Scholar]
  42. Minden A., Karin M. Regulation and function of the JNK subgroup of MAP kinases. Biochim Biophys Acta. 1997 Oct 24;1333(2):F85–104. doi: 10.1016/s0304-419x(97)00018-8. [DOI] [PubMed] [Google Scholar]
  43. Nievers M. G., Birge R. B., Greulich H., Verkleij A. J., Hanafusa H., van Bergen en Henegouwen P. M. v-Crk-induced cell transformation: changes in focal adhesion composition and signaling. J Cell Sci. 1997 Feb;110(Pt 3):389–399. doi: 10.1242/jcs.110.3.389. [DOI] [PubMed] [Google Scholar]
  44. Noselli S. JNK signaling and morphogenesis in Drosophila. Trends Genet. 1998 Jan;14(1):33–38. doi: 10.1016/S0168-9525(97)01320-6. [DOI] [PubMed] [Google Scholar]
  45. Olivier J. P., Raabe T., Henkemeyer M., Dickson B., Mbamalu G., Margolis B., Schlessinger J., Hafen E., Pawson T. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell. 1993 Apr 9;73(1):179–191. doi: 10.1016/0092-8674(93)90170-u. [DOI] [PubMed] [Google Scholar]
  46. Quilliam L. A., Huff S. Y., Rabun K. M., Wei W., Park W., Broek D., Der C. J. Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8512–8516. doi: 10.1073/pnas.91.18.8512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Raabe T., Riesgo-Escovar J., Liu X., Bausenwein B. S., Deak P., Maröy P., Hafen E. DOS, a novel pleckstrin homology domain-containing protein required for signal transduction between sevenless and Ras1 in Drosophila. Cell. 1996 Jun 14;85(6):911–920. doi: 10.1016/s0092-8674(00)81274-x. [DOI] [PubMed] [Google Scholar]
  48. Resh M. D. Myristylation and palmitylation of Src family members: the fats of the matter. Cell. 1994 Feb 11;76(3):411–413. doi: 10.1016/0092-8674(94)90104-x. [DOI] [PubMed] [Google Scholar]
  49. Robinow S., White K. Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development. J Neurobiol. 1991 Jul;22(5):443–461. doi: 10.1002/neu.480220503. [DOI] [PubMed] [Google Scholar]
  50. Roth M. B., Zahler A. M., Stolk J. A. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol. 1991 Nov;115(3):587–596. doi: 10.1083/jcb.115.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sakai R., Iwamatsu A., Hirano N., Ogawa S., Tanaka T., Mano H., Yazaki Y., Hirai H. A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J. 1994 Aug 15;13(16):3748–3756. doi: 10.1002/j.1460-2075.1994.tb06684.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Simon M. A., Dodson G. S., Rubin G. M. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell. 1993 Apr 9;73(1):169–177. doi: 10.1016/0092-8674(93)90169-q. [DOI] [PubMed] [Google Scholar]
  54. Songyang Z., Shoelson S. E., McGlade J., Olivier P., Pawson T., Bustelo X. R., Barbacid M., Sabe H., Hanafusa H., Yi T. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol Cell Biol. 1994 Apr;14(4):2777–2785. doi: 10.1128/mcb.14.4.2777-2785.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Staehling-Hampton K., Hoffmann F. M., Baylies M. K., Rushton E., Bate M. dpp induces mesodermal gene expression in Drosophila. Nature. 1994 Dec 22;372(6508):783–786. doi: 10.1038/372783a0. [DOI] [PubMed] [Google Scholar]
  56. Tamkun J. W., Deuring R., Scott M. P., Kissinger M., Pattatucci A. M., Kaufman T. C., Kennison J. A. brahma: a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SWI2. Cell. 1992 Feb 7;68(3):561–572. doi: 10.1016/0092-8674(92)90191-e. [DOI] [PubMed] [Google Scholar]
  57. Tanaka S., Hanafusa H. Guanine-nucleotide exchange protein C3G activates JNK1 by a ras-independent mechanism. JNK1 activation inhibited by kinase negative forms of MLK3 and DLK mixed lineage kinases. J Biol Chem. 1998 Jan 16;273(3):1281–1284. doi: 10.1074/jbc.273.3.1281. [DOI] [PubMed] [Google Scholar]
  58. Tanaka S., Morishita T., Hashimoto Y., Hattori S., Nakamura S., Shibuya M., Matuoka K., Takenawa T., Kurata T., Nagashima K. C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3443–3447. doi: 10.1073/pnas.91.8.3443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tanaka S., Ouchi T., Hanafusa H. Downstream of Crk adaptor signaling pathway: activation of Jun kinase by v-Crk through the guanine nucleotide exchange protein C3G. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2356–2361. doi: 10.1073/pnas.94.6.2356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Teng K. K., Lander H., Fajardo J. E., Hanafusa H., Hempstead B. L., Birge R. B. v-Crk modulation of growth factor-induced PC12 cell differentiation involves the Src homology 2 domain of v-Crk and sustained activation of the Ras/mitogen-activated protein kinase pathway. J Biol Chem. 1995 Sep 1;270(35):20677–20685. doi: 10.1074/jbc.270.35.20677. [DOI] [PubMed] [Google Scholar]
  61. Therrien M., Chang H. C., Solomon N. M., Karim F. D., Wassarman D. A., Rubin G. M. KSR, a novel protein kinase required for RAS signal transduction. Cell. 1995 Dec 15;83(6):879–888. doi: 10.1016/0092-8674(95)90204-x. [DOI] [PubMed] [Google Scholar]
  62. Tomlinson A., Ready D. F. Cell fate in the Drosophila ommatidium. Dev Biol. 1987 Sep;123(1):264–275. doi: 10.1016/0012-1606(87)90448-9. [DOI] [PubMed] [Google Scholar]
  63. Vossler M. R., Yao H., York R. D., Pan M. G., Rim C. S., Stork P. J. cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell. 1997 Apr 4;89(1):73–82. doi: 10.1016/s0092-8674(00)80184-1. [DOI] [PubMed] [Google Scholar]
  64. York R. D., Yao H., Dillon T., Ellig C. L., Eckert S. P., McCleskey E. W., Stork P. J. Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature. 1998 Apr 9;392(6676):622–626. doi: 10.1038/33451. [DOI] [PubMed] [Google Scholar]
  65. Yoshida Y., Kawata M., Miura Y., Musha T., Sasaki T., Kikuchi A., Takai Y. Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes. Mol Cell Biol. 1992 Aug;12(8):3407–3414. doi: 10.1128/mcb.12.8.3407-3414.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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