Research Collection Doctoral Thesis Characterization of Drosophila Lnk an adaptor protein involved in growth control Author(s): Werz, Christian Publication Date: 2009 Permanent Link: https://doi.org/10.3929/ethz-a-006073429 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH Nr. 18640 Characterization of Drosophila Lnk – An Adaptor protein involved in growth control ABHANDLUNG Zur Erlangung des Titels DOKTOR DER WISSENSCHAFTEN der ETH ZÜRICH vorgelegt von CHRISTIAN WERZ Dipl. Biol. Geboren am 13.02.1977 von Neckarsulm, Deutschland Angenommen auf Antrag von Prof. Dr. Ernst Hafen Prof. Dr. Konrad Basler Prof. Dr. Markus Affolter Zürich 2009 Table of Contents Table of Contents Summary: .................................................................................................................. 4 Zusammenfassung: .................................................................................................. 5 Introduction: ............................................................................................................. 7 The fundamental process of growth control ............................................................ 7 The Insulin/IGF and TOR pathway.......................................................................... 9 Signaling downstream of the receptor................................................................... 11 Insulin signaling in Drosophila ............................................................................... 17 Phenotypes associated with impaired Insulin signaling in Drosophila ................... 21 ey-FLP screen to identify new genes involved in growth control ........................... 22 The mammalian SH2B-family of adaptor proteins................................................. 23 Adaptor proteins.................................................................................................... 26 Results: ................................................................................................................... 28 The Drosophila SH2B Family Adaptor Lnk Acts in Parallel to Chico in the Insulin Signaling Pathway .................................................................................................... 29 Additional Results .................................................................................................. 55 1. Phenotypic analysis........................................................................................... 55 1.1 Loss or reduction of functionanalysis .............................................................. 55 1.1.1 All lnk alleles reduce dry weight of adult flies to the same extent.............. 55 1.1.2 lnk RNAi in S2 cells does not affect cell size............................................. 56 1.2 Gain of function analysis ................................................................................. 58 1.2.1 Over expression of lnk rescues the phenotypes displayed by homozygous mutant flies but causes lethality and rough eyes at higher levels....................... 58 1.2.2 Over expression of lnk in specific tissues.................................................. 60 1.3 The genomic rescue construct restores the reduction in dry weight and defects in oogenesis due to lnk loss of function................................................................. 65 2 Table of Contents 2.1 lnk is expressed ubiquitously in early development and localizes to the membrane and intracellular clusters in cells ...................................................... 66 2.2 Lnk localizes to the membrane and intracellular clusters in S2 cells............... 68 3. Interaction studies ............................................................................................. 71 3.1 chico;lnk double mutants are severely delayed in development and die in prepupal stage ................................................................................................... 71 3.2 cbl does not interact genetically with lnk ......................................................... 72 3.3 PD experiments to identify interactors of Lnk .................................................. 75 3.3.1 Identification of proteins that are pulled down with HA-Lnk....................... 75 3.3.2 Heterozygosity for hrs does not modify lnk loss- and gain of function phenotypes ........................................................................................................ 78 3.3.3 Identification of proteins interacting with HA-Lnk in response to activated Insulin signaling ................................................................................................. 79 3.3.4 Heterozygosity for slik has no influence on lnk over expression phenotypes ........................................................................................................................... 80 General Discussion ................................................................................................ 82 Outlook .................................................................................................................... 90 Material and Methods ............................................................................................. 91 References .............................................................................................................. 97 Acknowledgements .............................................................................................. 107 Curriculum Vitae ................................................................................................... 108 3 Summary Summary One of the essential and most tightly regulated processes an organism has to accomplish throughout development is the control of growth and proliferation in order to reach and maintain the appropriate size. Until today, the underlying mechanisms are still far from being fully understood. It has previously been shown that, among other signaling cascades, the insulin signaling pathway plays a crucial role in controlling growth. In Drosophila , binding of either one of the seven Insulin like peptides initiates the autophosphorylation of the receptor, which in turn leads to subsequent phosphorylation of a variety of downstream signaling molecules such as Chico, PI3K, PKB and PDK1. As a result, the insulin signaling pathway regulates cellular growth, proliferation, apoptosis and transcription. Strikingly, mutants of positive core components of the insulin receptor pathway lead to common phenotypes such as decreased body size due to smaller and less cells, female sterility, developmental delay and increased total lipid levels. In an unbiased screen for genes affecting organ size based on the eyFLP-FRT system, we found that mutations in a gene called lnk result in flies with a smaller head, suggesting a growth promoting role for lnk . The lnk gene encodes an adaptor protein, containing a PH-domain, an SH2-domain and a highly conserved C-terminal tyrosine phosphorylation site. The phenotypes of lnk mutant flies were reminiscent of the phenotypes observed in mutants of the insulin pathway, suggesting an important function for Lnk in promoting the insulin signal. In mammals, three proteins sharing the same protein structure to Drosophila Lnk have been described, SH2B1, SH2B2 and SH2B3 referred to as the SH2B family of adaptor proteins. The members of this protein family have been shown to regulate receptor tyrosine pathways either by direct binding to the receptor or by interaction with one of the multiple signaling proteins such as Grb2, PI3K and c-Cbl. In this work we present the characterization of Drosophila lnk . By analysing the mutant phenotypes displayed by homozygous lnk animals, genetic interaction experiments and molecular readouts for insulin signaling activity we were able to place lnk into the Insulin pathway between the receptor and PI3K . 4 Zusammenfassung Zusammenfassung Während der Entwicklung eines mehrzelligen Organismus ist einer der grundlegendsten und am strengsten kontrollierten Prozesse die Regulation des Wachstums. Bisher sind wir jedoch noch weit davon entfernt, den Prozess der Wachstumskontrolle vollständig zu verstehen. Der Insulinsignalweg nimmt, neben anderen wichtigen Signalkaskaden, eine entscheidende Rolle in der Regulierung von Zellwachstum und –proliferation ein. In Drosophila wird durch die Bindung von sogenannten ‚Insulin like peptides‘ die Autophosphorylierung des Insulinrezeptors ausgelöst, was wiederum die Phosphorylierung einer Vielzahl von nachgeschalteten Signalmolekülen wie Chico, PI3K, PKB und PDK1 zur Folge hat. Der Insulinsignalweg reguliert neben der Zellgröße und Proliferation auch den Programmierten Zelltod und die Transkription bestimmter Gene. Auffallender weise bewirken Mutationen in den Hauptkomponenten des Insulinsignalwegs einheitliche Phänotypen, wie eine reduzierte Körpergröße, hervorgerufen durch kleinere und weniger Zellen, Sterilität der Weibchen, Entwicklungsverzögerung und erhöhte Lipid werte. In einem auf dem eyFLP-FRT System basierenden Screen zur Identifikation von neuen Genen, welche die Organgröße beeinflussen, haben wir Mutationen im sogenannten lnk Gen gefunden. Diese Mutationen führen zu Fliegen mit einem kleineren Kopf, was darauf hindeutet, dass lnk Wachstum positiv beeinflusst. Das lnk Gen kodiert für ein Adaptorprotein. welches eine PH Domäne, eine SH2 Domäne und eine hochkonservierten C-terminalen