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  • Original Paper
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Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac

Oncogene volume 24, pages 4509–4520 (2005)Cite this article

Abstract

Rap1 is a Ras-related GTPase that is principally involved in integrin- and E-cadherin-mediated adhesion. Rap1 is transiently activated in response to many incoming signals via a large family of guanine nucleotide exchange factors (GEFs). The lack of potent Rap1 dominant-negative mutants has limited our ability to decipher Rap1-dependent pathways; we have therefore developed a procedure to generate such mutants consisting in the oligonucleotide-mediated mutagenesis of residues 14–19, selection of mutants presenting an enhanced interaction with Epac2 by yeast two-hybrid screening and counter-screening for mutants still interacting with Rap effectors. In detail analysis of their interaction capacity with various Rap-GEFs in the yeast two-hybrid system revealed that mutants of residues 15 and 16 interacted with Epacs, C3G and CalDAG-GEFI, whereas mutants of position 17 had selectively lost their ability to bind CalDAG-GEFI as well as, for some, C3G. In cellular models where Rap1 is activated via endogenous GEFs, the Rap1[S17A] mutant inhibits both the cAMP–Epac and EGF–C3G pathways, whereas Rap1[G15D] selectively interferes with the latter. Finally, Rap1[S17A] is able to act as a bona fide dominant-negative mutant in vivo since it phenocopies the eye-reducing and lethal effects of D-Rap1 deficiency in Drosophila, effects that are overcome by the overexpression of D-Epac or D-Rap1.

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References

  • Asha H, de Ruiter ND, Wang MG and Hariharan IK . (1999). EMBO J., 18, 605–615.

  • Bar-Sagi D and Hall A . (2000). Cell, 103, 227–238.

  • Beranger F, Aresta S, de Gunzburg J and Camonis J . (1997). Nucleic Acids Res., 25, 2035–2036.

  • Beranger F, Goud B, Tavitian A and de Gunzburg J . (1991). Proc. Natl. Acad. Sci. USA, 88, 1606–1610.

  • Biou V and Cherfils J . (2004). Biochemistry, 43, 6833–6840.

  • Bivona TG, Wiener HH, Ahearn IM, Silletti J, Chiu VK and Philips MR . (2004). J. Cell Biol., 164, 461–470.

  • Boettner B, Govek EE, Cross J and Van Aelst L . (2000). Proc. Natl. Acad. Sci. USA, 97, 9064–9069.

  • Boriack-Sjodin PA, Margarit SM, Bar-Sagi D and Kuriyan J . (1998). Nature, 394, 337–343.

  • Bos JL . (2003). Nat. Rev. Mol. Cell Biol., 4, 733–738.

  • Bos JL, de Rooij J and Reedquist KA . (2001). Nat. Rev. Mol. Cell Biol., 2, 369–377.

  • Brand AH and Perrimon N . (1993). Development, 118, 401–415.

  • Chen SY, Huff SY, Lai CC, Der CJ and Powers S . (1994). Oncogene, 9, 2691–2698.

  • Cherfils J, Menetrey J, Le Bras G, Janoueix-Lerosey I, de Gunzburg J, Garel JR and Auzat I . (1997). EMBO J., 16, 5582–5591.

  • Clyde-Smith J, Silins G, Gartside M, Grimmond S, Etheridge M, Apolloni A, Hayward N and Hancock JF . (2000). J. Biol. Chem., 275, 32260–32267.

  • de Rooij J, Boenink NM, van Triest M, Cool RH, Wittinghofer A and Bos JL . (1999). J. Biol. Chem., 274, 38125–38130.

  • de Vos AM, Tong L, Milburn MV, Matias PM, Jancarik J, Noguchi S, Nishimura S, Miura K, Ohtsuka E and Kim SH . (1988). Science, 239, 888–893.

  • Farnsworth CL and Feig LA . (1991). Mol. Cell. Biol., 11, 4822–4829.

  • Feig LA . (1999). Nat. Cell Biol., 1, E25–E27.

  • Feig LA and Cooper GM . (1988). Mol. Cell. Biol., 8, 3235–3243.

  • Ferguson KM and Higashijima T . (1991). Methods Enzymol., 195, 188–192.

  • Franke B, Akkerman JW and Bos JL . (1997). EMBO J., 16, 252–259.

  • Gao X, Satoh T, Liao Y, Song C, Hu CD, Kariya Ki K and Kataoka T . (2001). J. Biol. Chem., 276, 42219–42225.

  • Hancock JF, Cadwallader K, Paterson H and Marshall CJ . (1991). EMBO J., 10, 4033–4039.

  • Hariharan IK, Carthew RW and Rubin GM . (1991). Cell, 67, 717–722.

  • Hay BA, Maile R and Rubin GM . (1997). Proc. Natl. Acad. Sci. USA, 94, 5195–5200.

  • Hogan C, Serpente N, Cogram P, Hosking CR, Bialucha CU, Feller SM, Braga VM, Birchmeier W and Fujita Y . (2004). Mol. Cell. Biol., 24, 6690–6700.

  • Ichiba T, Hashimoto Y, Nakaya M, Kuraishi Y, Tanaka S, Kurata T, Mochizuki N and Matsuda M . (1999). J. Biol. Chem., 274, 14376–14381.

  • Ishimaru S, Williams R, Clark E, Hanafusa H and Gaul U . (1999). EMBO J., 18, 145–155.

  • Jung V, Wei W, Ballester R, Camonis J, Mi S, Van Aelst L, Wigler M and Broek D . (1994). Mol. Cell. Biol., 14, 3707–3718.

  • Karess RE and Rubin GM . (1984). Cell, 38, 135–146.

  • Karim FD and Rubin GM . (1998). Development, 125, 1–9.

  • Katagiri K, Ohnishi N, Kabashima K, Iyoda T, Takeda N, Shinkai Y, Inaba K and Kinashi T . (2004). Nat. Immunol., 5, 1045–1051.

  • Kawasaki H, Springett GM, Toki S, Canales JJ, Harlan P, Blumenstiel JP, Chen EJ, Bany IA, Mochizuki N, Ashbacher A, Matsuda M, Housman DE and Graybiel AM . (1998). Proc. Natl. Acad. Sci. USA, 95, 13278–13283.

  • Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y and Noda M . (1989). Cell, 56, 77–84.

  • Knox AL and Brown NH . (2002). Science, 295, 1285–1288.

  • Lafuente EM, van Puijenbroek AA, Krause M, Carman CV, Freeman GJ, Berezovskaya A, Constantine E, Springer TA, Gertler FB and Boussiotis VA . (2004). Dev. Cell, 7, 585–595.

  • Lai CC, Boguski M, Broek D and Powers S . (1993). Mol. Cell. Biol., 13, 1345–1352.

  • Liao Y, Kariya K, Hu CD, Shibatohge M, Goshima M, Okada T, Watari Y, Gao X, Jin TG, Yamawaki-Kataoka Y and Kataoka T . (1999). J. Biol. Chem., 274, 37815–37820.

  • Liao Y, Satoh T, Gao X, Jin TG, Hu CD and Kataoka T . (2001). J. Biol. Chem., 276, 28478–28483.

  • Maillet M, Robert SJ, Cacquevel M, Gastineau M, Vivien D, Bertoglio J, Zugaza JL, Fischmeister R and Lezoualc'h F . (2003). Nat. Cell Biol., 5, 633–639.

  • Mirey G, Balakireva M, L'Hoste S, Rosse C, Voegeling S and Camonis J . (2003). Mol. Cell. Biol., 23, 1112–1124.

  • Mochizuki N, Yamashita S, Kurokawa K, Ohba Y, Nagai T, Miyawaki A and Matsuda M . (2001). Nature, 411, 1065–1068.

  • Nancy V, Wolthuis RM, de Tand MF, Janoueix-Lerosey I, Bos JL and de Gunzburg J . (1999). J. Biol. Chem., 274, 8737–8745.

  • Nassar N, Horn G, Herrmann C, Scherer A, McCormick F and Wittinghofer A . (1995). Nature, 375, 554–560.

  • Ohba Y, Kurokawa K and Matsuda M . (2003). EMBO J., 22, 859–869.

  • Papin C, Denouel A, Calothy G and Eychene A . (1996). Oncogene, 12, 2213–2221.

  • Pizon V, Chardin P, Lerosey I, Olofsson B and Tavitian A . (1988). Oncogene, 3, 201–204.

  • Powers S, O'Neill K and Wigler M . (1989). Mol. Cell. Biol., 9, 390–395.

  • Price LS, Hajdo-Milasinovic A, Zhao J, Zwartkruis FJ, Collard JG and Bos JL . (2004). J. Biol. Chem., 279, 35127–35132.

  • Reedquist KA, Ross E, Koop EA, Wolthuis RM, Zwartkruis FJ, van Kooyk Y, Salmon M, Buckley CD and Bos JL . (2000). J. Cell Biol., 148, 1151–1158.

  • Rubin GM, Kidwell MG and Bingham PM . (1982). Cell, 29, 987–994.

  • Schweighoffer F, Cai H, Chevallier-Multon MC, Fath I, Cooper G and Tocque B . (1993). Mol. Cell. Biol., 13, 39–43.

  • Shi S, Noda M and Kitayama H . (2004). Oncogene, 23, 8711–8719.

  • Sigal IS, Gibbs JB, D'Alonzo JS, Temeles GL, Wolanski BS, Socher SH and Scolnick EM . (1986). Proc. Natl. Acad. Sci. USA, 83, 952–956.

  • Transy C and Legrain P . (1995). Mol. Biol. Rep., 21, 119–127.

  • Traver S, Bidot C, Spassky N, Baltauss T, De Tand MF, Thomas JL, Zalc B, Janoueix-Lerosey I and Gunzburg JD . (2000). Biochem. J., 350 (Part 1), 19–29.

  • Van Aelst L, Barr M, Marcus S, Polverino A and Wigler M . (1993). Proc. Natl. Acad. Sci. USA, 90, 6213–6217.

  • van den Berghe N, Cool RH, Horn G and Wittinghofer A . (1997). Oncogene, 15, 845–850.

  • Vossler MR, Yao H, York RD, Pan MG, Rim CS and Stork PJ . (1997). Cell, 89, 73–82.

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Acknowledgements

We are indebted to Drs J Flanders and J Hancock for plasmids, to Pr M Matsuda for C3G expression vectors and related antibodies, to Drs U Gaul, E Hafen and I Hariharan for Drosophila constructs and strains, to Dr F Lezoualc'h for the CHO-5HT4b cell line, and to Dr M Balakireva for Drosophila lines as well as critical reading of the manuscript. This work was funded in part by a grant from the Association pour la Recherche contre le Cancer. A Dupuy and S L'Hoste were successively supported by doctoral fellowships from the Ministère de la Recherche and the Association pour la Recherche contre le Cancer. G Gaudriault was the recipient of a postdoctoral fellowship from the Association pour la Recherche contre le Cancer.

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  1. Georges Gaudriault

    Present address: ObeTherapy Biotechnology, 4 rue Pierre Fontaine, 91058, Evry, France

  2. Georges Gaudriault and Jean de Gunzburg: These authors contributed equally to this work

Authors and Affiliations

  1. Inserm U528, Institut Curie, 26 rue d'Ulm, 75005, Paris, France

    Aurélien G Dupuy, Sébastien L'Hoste, Jacques Camonis, Georges Gaudriault & Jean de Gunzburg

  2. CNRS UPR 9063, Avenue de la Terrasse, 91198, Gif-sur-Yvette Cedex, France

    Jacqueline Cherfils

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  1. Aurélien G Dupuy
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Correspondence to Jean de Gunzburg.

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Dupuy, A., L'Hoste, S., Cherfils, J. et al. Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac. Oncogene 24, 4509–4520 (2005). https://doi.org/10.1038/sj.onc.1208647

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