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. 2003 Sep;165(1):159–169. doi: 10.1093/genetics/165.1.159

The AF-6 homolog canoe acts as a Rap1 effector during dorsal closure of the Drosophila embryo.

Benjamin Boettner 1, Phoebe Harjes 1, Satoshi Ishimaru 1, Michael Heke 1, Hong Qing Fan 1, Yi Qin 1, Linda Van Aelst 1, Ulrike Gaul 1
PMCID: PMC1462758  PMID: 14504224

Abstract

Rap1 belongs to the highly conserved Ras subfamily of small GTPases. In Drosophila, Rap1 plays a critical role in many different morphogenetic processes, but the molecular mechanisms executing its function are unknown. Here, we demonstrate that Canoe (Cno), the Drosophila homolog of mammalian junctional protein AF-6, acts as an effector of Rap1 in vivo. Cno binds to the activated form of Rap1 in a yeast two-hybrid assay, the two molecules colocalize to the adherens junction, and they display very similar phenotypes in embryonic dorsal closure (DC), a process that relies on the elongation and migration of epithelial cell sheets. Genetic interaction experiments show that Rap1 and Cno act in the same molecular pathway during DC and that the function of both molecules in DC depends on their ability to interact. We further show that Rap1 acts upstream of Cno, but that Rap1, unlike Cno, is not involved in the stimulation of JNK pathway activity, indicating that Cno has both a Rap1-dependent and a Rap1-independent function in the DC process.

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Selected References

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  1. Arai A., Nosaka Y., Kanda E., Yamamoto K., Miyasaka N., Miura O. Rap1 is activated by erythropoietin or interleukin-3 and is involved in regulation of beta1 integrin-mediated hematopoietic cell adhesion. J Biol Chem. 2000 Dec 21;276(13):10453–10462. doi: 10.1074/jbc.M004627200. [DOI] [PubMed] [Google Scholar]
  2. Asha H., de Ruiter N. D., Wang M. G., Hariharan I. K. The Rap1 GTPase functions as a regulator of morphogenesis in vivo. EMBO J. 1999 Feb 1;18(3):605–615. doi: 10.1093/emboj/18.3.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boettner B., Govek E. E., Cross J., Van Aelst L. The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin. Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):9064–9069. doi: 10.1073/pnas.97.16.9064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boettner B., Herrmann C., Van Aelst L. Ras and Rap1 interaction with AF-6 effector target. Methods Enzymol. 2001;332:151–168. doi: 10.1016/s0076-6879(01)32199-7. [DOI] [PubMed] [Google Scholar]
  5. Bokoch G. M., Quilliam L. A., Bohl B. P., Jesaitis A. J., Quinn M. T. Inhibition of Rap1A binding to cytochrome b558 of NADPH oxidase by phosphorylation of Rap1A. Science. 1991 Dec 20;254(5039):1794–1796. doi: 10.1126/science.1763330. [DOI] [PubMed] [Google Scholar]
  6. Bos J. L., de Rooij J., Reedquist K. A. Rap1 signalling: adhering to new models. Nat Rev Mol Cell Biol. 2001 May;2(5):369–377. doi: 10.1038/35073073. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Buchert M., Schneider S., Meskenaite V., Adams M. T., Canaani E., Baechi T., Moelling K., Hovens C. M. The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain. J Cell Biol. 1999 Jan 25;144(2):361–371. doi: 10.1083/jcb.144.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Caron E., Self A. J., Hall A. The GTPase Rap1 controls functional activation of macrophage integrin alphaMbeta2 by LPS and other inflammatory mediators. Curr Biol. 2000 Aug 24;10(16):974–978. doi: 10.1016/s0960-9822(00)00641-2. [DOI] [PubMed] [Google Scholar]
  10. Caron Emmanuelle. Cellular functions of the Rap1 GTP-binding protein: a pattern emerges. J Cell Sci. 2003 Feb 1;116(Pt 3):435–440. doi: 10.1242/jcs.00238. [DOI] [PubMed] [Google Scholar]
  11. Cook S. J., Rubinfeld B., Albert I., McCormick F. RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblasts. EMBO J. 1993 Sep;12(9):3475–3485. doi: 10.1002/j.1460-2075.1993.tb06022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harden N., Ricos M., Ong Y. M., Chia W., Lim L. Participation of small GTPases in dorsal closure of the Drosophila embryo: distinct roles for Rho subfamily proteins in epithelial morphogenesis. J Cell Sci. 1999 Feb;112(Pt 3):273–284. doi: 10.1242/jcs.112.3.273. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Knox Andrea L., Brown Nicholas H. Rap1 GTPase regulation of adherens junction positioning and cell adhesion. Science. 2002 Feb 15;295(5558):1285–1288. doi: 10.1126/science.1067549. [DOI] [PubMed] [Google Scholar]
  15. Linnemann T., Geyer M., Jaitner B. K., Block C., Kalbitzer H. R., Wittinghofer A., Herrmann C. Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily. J Biol Chem. 1999 May 7;274(19):13556–13562. doi: 10.1074/jbc.274.19.13556. [DOI] [PubMed] [Google Scholar]
  16. Maly F. E., Quilliam L. A., Dorseuil O., Der C. J., Bokoch G. M. Activated or dominant inhibitory mutants of Rap1A decrease the oxidative burst of Epstein-Barr virus-transformed human B lymphocytes. J Biol Chem. 1994 Jul 22;269(29):18743–18746. [PubMed] [Google Scholar]
  17. Matsuo T., Takahashi K., Kondo S., Kaibuchi K., Yamamoto D. Regulation of cone cell formation by Canoe and Ras in the developing Drosophila eye. Development. 1997 Jul;124(14):2671–2680. doi: 10.1242/dev.124.14.2671. [DOI] [PubMed] [Google Scholar]
  18. Mochizuki N., Ohba Y., Kiyokawa E., Kurata T., Murakami T., Ozaki T., Kitabatake A., Nagashima K., Matsuda M. Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i) Nature. 1999 Aug 26;400(6747):891–894. doi: 10.1038/23738. [DOI] [PubMed] [Google Scholar]
  19. Okada S., Matsuda M., Anafi M., Pawson T., Pessin J. E. Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes. EMBO J. 1998 May 1;17(9):2554–2565. doi: 10.1093/emboj/17.9.2554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Quilliam L. A., Castro A. F., Rogers-Graham K. S., Martin C. B., Der C. J., Bi C. M-Ras/R-Ras3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPase-activating protein, interacts with the putative Ras effector AF6. J Biol Chem. 1999 Aug 20;274(34):23850–23857. doi: 10.1074/jbc.274.34.23850. [DOI] [PubMed] [Google Scholar]
  21. Rangarajan Savithri, Enserink Jorrit M., Kuiperij H. Bea, de Rooij Johan, Price Leo S., Schwede Frank, Bos Johannes L. Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor. J Cell Biol. 2003 Feb 10;160(4):487–493. doi: 10.1083/jcb.200209105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reedquist K. A., Ross E., Koop E. A., Wolthuis R. M., Zwartkruis F. J., van Kooyk Y., Salmon M., Buckley C. D., Bos J. L. The small GTPase, Rap1, mediates CD31-induced integrin adhesion. J Cell Biol. 2000 Mar 20;148(6):1151–1158. doi: 10.1083/jcb.148.6.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sawada Y., Nakamura K., Doi K., Takeda K., Tobiume K., Saitoh M., Morita K., Komuro I., De Vos K., Sheetz M. Rap1 is involved in cell stretching modulation of p38 but not ERK or JNK MAP kinase. J Cell Sci. 2001 Mar;114(Pt 6):1221–1227. doi: 10.1242/jcs.114.6.1221. [DOI] [PubMed] [Google Scholar]
  24. Stronach B. E., Perrimon N. Stress signaling in Drosophila. Oncogene. 1999 Nov 1;18(45):6172–6182. doi: 10.1038/sj.onc.1203125. [DOI] [PubMed] [Google Scholar]
  25. Tsukamoto N., Hattori M., Yang H., Bos J. L., Minato N. Rap1 GTPase-activating protein SPA-1 negatively regulates cell adhesion. J Biol Chem. 1999 Jun 25;274(26):18463–18469. doi: 10.1074/jbc.274.26.18463. [DOI] [PubMed] [Google Scholar]
  26. Van Aelst L. Two-hybrid analysis of Ras-Raf interactions. Methods Mol Biol. 1998;84:201–222. doi: 10.1385/0-89603-488-7:201. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Zhu J. Julius, Qin Yi, Zhao Mingming, Van Aelst Linda, Malinow Roberto. Ras and Rap control AMPA receptor trafficking during synaptic plasticity. Cell. 2002 Aug 23;110(4):443–455. doi: 10.1016/s0092-8674(02)00897-8. [DOI] [PubMed] [Google Scholar]
  29. Zwartkruis F. J., Bos J. L. Ras and Rap1: two highly related small GTPases with distinct function. Exp Cell Res. 1999 Nov 25;253(1):157–165. doi: 10.1006/excr.1999.4695. [DOI] [PubMed] [Google Scholar]
  30. van den Berghe N., Cool R. H., Horn G., Wittinghofer A. Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N). Oncogene. 1997 Aug 14;15(7):845–850. doi: 10.1038/sj.onc.1201407. [DOI] [PubMed] [Google Scholar]

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