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. 2004 Jan;166(1):201–211. doi: 10.1534/genetics.166.1.201

Knockouts of Kekkon1 define sequence elements essential for Drosophila epidermal growth factor receptor inhibition.

Diego Alvarado 1, Amy H Rice 1, Joseph B Duffy 1
PMCID: PMC1470717  PMID: 15020418

Abstract

Throughout development, cells utilize feedback inhibition of receptor tyrosine kinase (RTK) signaling as an important means to direct cellular fates. In Drosophila, epidermal growth factor receptor (EGFR) activity is tightly regulated by a complex array of autoregulatory loops, involving an assortment of inhibitory proteins. One inhibitor, the transmembrane protein Kekkon1 (Kek1) functions during oogenesis in a negative feedback loop to directly attenuate EGFR activity. Kek1 contains both leucine-rich repeats (LRRs) and an immunoglobulin (Ig) domain, two of the most prevalent motifs found within metazoan genomes. Here we demonstrate that Kek1 inhibits EGFR activity during eye development and use this role to identify kek1 loss-of-function mutations that implicate the LRRs in directing receptor inhibition. Using a GMR-GAL4, UAS kek1-GFP misexpression phenotype we isolated missense mutations in the kek1 transgene affecting its ability to inhibit EGFR signaling. Genetic, molecular, and biochemical characterization of these alleles indicated that they represent two functionally distinct classes. Class I alleles directly diminish Kek1's affinity for EGFR, while class II alleles disrupt Kek1's subcellular localization, thereby indirectly affecting its ability to associate with and inhibit the receptor. All class I alleles map to the first and second LRRs of Kek1, suggesting a primary role for these two repeats in specifying association with and inhibition of EGFR. Last, our analysis implicates glycine 160 of the second LRR in regulating EGFR binding.

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Selected References

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  1. Bai J., Chiu W., Wang J., Tzeng T., Perrimon N., Hsu J. The cell adhesion molecule Echinoid defines a new pathway that antagonizes the Drosophila EGF receptor signaling pathway. Development. 2001 Feb;128(4):591–601. doi: 10.1242/dev.128.4.591. [DOI] [PubMed] [Google Scholar]
  2. Derheimer Frederick A., MacLaren Christina M., Weasner Brandon P., Alvarado Diego, Duffy Joseph B. Conservation of an inhibitor of the epidermal growth factor receptor, Kekkon1, in dipterans. Genetics. 2004 Jan;166(1):213–224. doi: 10.1534/genetics.166.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fehon R. G., Kooh P. J., Rebay I., Regan C. L., Xu T., Muskavitch M. A., Artavanis-Tsakonas S. Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila. Cell. 1990 May 4;61(3):523–534. doi: 10.1016/0092-8674(90)90534-l. [DOI] [PubMed] [Google Scholar]
  4. Ghiglione C., Carraway K. L., 3rd, Amundadottir L. T., Boswell R. E., Perrimon N., Duffy J. B. The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis. Cell. 1999 Mar 19;96(6):847–856. doi: 10.1016/s0092-8674(00)80594-2. [DOI] [PubMed] [Google Scholar]
  5. Ghiglione Christian, Amundadottir Laufey, Andresdottir Margret, Bilder David, Diamonti John A., Noselli Stéphane, Perrimon Norbert, Carraway III Kermit L. Mechanism of inhibition of the Drosophila and mammalian EGF receptors by the transmembrane protein Kekkon 1. Development. 2003 Sep;130(18):4483–4493. doi: 10.1242/dev.00617. [DOI] [PubMed] [Google Scholar]
  6. Hime G. R., Dhungat M. P., Ng A., Bowtell D. D. D-Cbl, the Drosophila homologue of the c-Cbl proto-oncogene, interacts with the Drosophila EGF receptor in vivo, despite lacking C-terminal adaptor binding sites. Oncogene. 1997 Jun 5;14(22):2709–2719. doi: 10.1038/sj.onc.1201223. [DOI] [PubMed] [Google Scholar]
  7. Klueg Kristin M., Alvarado Diego, Muskavitch Marc A. T., Duffy Joseph B. Creation of a GAL4/UAS-coupled inducible gene expression system for use in Drosophila cultured cell lines. Genesis. 2002 Sep-Oct;34(1-2):119–122. doi: 10.1002/gene.10148. [DOI] [PubMed] [Google Scholar]
  8. Kobe B., Deisenhofer J. A structural basis of the interactions between leucine-rich repeats and protein ligands. Nature. 1995 Mar 9;374(6518):183–186. doi: 10.1038/374183a0. [DOI] [PubMed] [Google Scholar]
  9. Kobe B., Deisenhofer J. The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci. 1994 Oct;19(10):415–421. doi: 10.1016/0968-0004(94)90090-6. [DOI] [PubMed] [Google Scholar]
  10. Kobe B., Kajava A. V. The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol. 2001 Dec;11(6):725–732. doi: 10.1016/s0959-440x(01)00266-4. [DOI] [PubMed] [Google Scholar]
  11. Kumar J. P., Moses K. EGF receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification. Cell. 2001 Mar 9;104(5):687–697. doi: 10.1016/s0092-8674(01)00265-3. [DOI] [PubMed] [Google Scholar]
  12. Kumar J. P., Tio M., Hsiung F., Akopyan S., Gabay L., Seger R., Shilo B. Z., Moses K. Dissecting the roles of the Drosophila EGF receptor in eye development and MAP kinase activation. Development. 1998 Oct;125(19):3875–3885. doi: 10.1242/dev.125.19.3875. [DOI] [PubMed] [Google Scholar]
  13. Lesokhin A. M., Yu S. Y., Katz J., Baker N. E. Several levels of EGF receptor signaling during photoreceptor specification in wild-type, Ellipse, and null mutant Drosophila. Dev Biol. 1999 Jan 1;205(1):129–144. doi: 10.1006/dbio.1998.9121. [DOI] [PubMed] [Google Scholar]
  14. Musacchio M., Perrimon N. The Drosophila kekkon genes: novel members of both the leucine-rich repeat and immunoglobulin superfamilies expressed in the CNS. Dev Biol. 1996 Aug 25;178(1):63–76. doi: 10.1006/dbio.1996.0198. [DOI] [PubMed] [Google Scholar]
  15. Nilson L. A., Schüpbach T. EGF receptor signaling in Drosophila oogenesis. Curr Top Dev Biol. 1999;44:203–243. doi: 10.1016/s0070-2153(08)60471-8. [DOI] [PubMed] [Google Scholar]
  16. Pai L. M., Barcelo G., Schüpbach T. D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis. Cell. 2000 Sep 29;103(1):51–61. doi: 10.1016/s0092-8674(00)00104-5. [DOI] [PubMed] [Google Scholar]
  17. Peifer M., Orsulic S., Sweeton D., Wieschaus E. A role for the Drosophila segment polarity gene armadillo in cell adhesion and cytoskeletal integrity during oogenesis. Development. 1993 Aug;118(4):1191–1207. doi: 10.1242/dev.118.4.1191. [DOI] [PubMed] [Google Scholar]
  18. Pruess Manuela, Fleischmann Wolfgang, Kanapin Alexander, Karavidopoulou Youla, Kersey Paul, Kriventseva Evgenia, Mittard Virginie, Mulder Nicola, Phan Isabelle, Servant Florence. The Proteome Analysis database: a tool for the in silico analysis of whole proteomes. Nucleic Acids Res. 2003 Jan 1;31(1):414–417. doi: 10.1093/nar/gkg105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Queenan A. M., Ghabrial A., Schüpbach T. Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and the embryo. Development. 1997 Oct;124(19):3871–3880. doi: 10.1242/dev.124.19.3871. [DOI] [PubMed] [Google Scholar]
  20. Schweitzer R., Howes R., Smith R., Shilo B. Z., Freeman M. Inhibition of Drosophila EGF receptor activation by the secreted protein Argos. Nature. 1995 Aug 24;376(6542):699–702. doi: 10.1038/376699a0. [DOI] [PubMed] [Google Scholar]
  21. Schweitzer R., Shilo B. Z. A thousand and one roles for the Drosophila EGF receptor. Trends Genet. 1997 May;13(5):191–196. doi: 10.1016/s0168-9525(97)01091-3. [DOI] [PubMed] [Google Scholar]
  22. Stevens L. Twin peaks: Spitz and Argos star in patterning of the Drosophila egg. Cell. 1998 Oct 30;95(3):291–294. doi: 10.1016/s0092-8674(00)81759-6. [DOI] [PubMed] [Google Scholar]
  23. Tio M., Ma C., Moses K. spitz, a Drosophila homolog of transforming growth factor-alpha, is required in the founding photoreceptor cells of the compound eye facets. Mech Dev. 1994 Oct;48(1):13–23. doi: 10.1016/0925-4773(94)90002-7. [DOI] [PubMed] [Google Scholar]
  24. Wasserman J. D., Freeman M. An autoregulatory cascade of EGF receptor signaling patterns the Drosophila egg. Cell. 1998 Oct 30;95(3):355–364. doi: 10.1016/s0092-8674(00)81767-5. [DOI] [PubMed] [Google Scholar]
  25. Xu T., Rubin G. M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development. 1993 Apr;117(4):1223–1237. doi: 10.1242/dev.117.4.1223. [DOI] [PubMed] [Google Scholar]

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