RasGAP is a GTPase activating protein (GAP) that deactivates Ras by promoting Ras-GTP hydrolysis to Ras-GDP. In Drosophila melanogaster, RasGAP is required for the long-term survival of neurons in the adult brain because mutants in the RasGAP gene (vap) show an age-related neurodegenerative phenotype, with dying neurons showing morphological features of autophagy. RasGAP was shown to have a GAP-independent role within fly neurons that is dependent on its SH2 domains. The aim of this study was to identify proteins that interact with the SH2 domains of RasGAP and to understand the roles of these proteins in neuronal survival. By using tagged RasGAP affinity purification and mass spectrometry of RasGAP protein complexes from S2 cells, Sprint, a Ras effector and putative activator of the endocytic GTPase Rab5, was identified as a novel SH2-dependent RasGAP interacting protein. The interaction between Sprint and RasGAP is phosphotyrosine-dependent, since it requires tyrosine phosphorylation of Sprint. In addition, Sprint and RasGAP interaction requires the SH2 domains of RasGAP but not Sprint or the conserved site of RasGAP tyrosine phosphorylation (pTyr363), indicating an association between these two molecules. RasGAP and Sprint co-localised with Rab5-positive early endosomes and this co-localisation depended on the SH2 domains of both RasGAP and Sprint. This study demonstrates a key role for this interaction in neurodegeneration: mutation of Sprint (or Rab5) suppressed the autophagic neuronal cell death caused by the loss of RasGAP. These results indicate that the long-term survival of adult neurons in Drosophila depends on a critical balance between Ras activation and endocytosis, and that this balance is maintained by the interplay between RasGAP and Sprint.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647376 |
| Date | January 2014 |
| Creators | Rowshanravan, Behzad |
| Contributors | Hughes, David; Baron, Martin |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigation-of-the-role-of-rasgap-in-promoting-neuronal-survival-in-drosophila(e81aef19-f68b-4079-ba18-c9bc7e937e7f).html |
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