| Abstract |
The Notch signaling pathway constitutes an evolutionarily conserved mechanism, which is activated during a variety of cell fate decisions in all metazoans. It revolves around the singlepass transmembrane Notch (N) receptor and its also transmembrane ligands, Delta (Dl) and Serrate (Ser). Following interaction of the extracellular domain of the receptor with the ligands, a proteolytic step is triggered, which leads to the release of the active intracellular part of the N receptor (Nic). Nic then travels to the nucleus, where it acts as a transcriptional co-activator and in association with other DNA binding factors activates transcription of target genes. Apart from transcription factors, a variety of proteins control the precise activation of the pathway in time and space post-transcriptionally; these include proteases, glycosyl-transferases and E3 ubiquitin ligases. In this work, the role of N ligands, Dl and Ser, as well as their two associated ubiquitin ligases, Neuralized (Neur) and Mind bomb1 (Mib1) was studied in N pathway activation in Drosophila. The role of neur was studied first. Although the gene was cloned 25 years ago, the protein was characterized as a RING type E3 ubiquitin ligase only recently and its target remained unknown. We showed that Neur is a membrane associated intracellular protein, which targets the N ligand Dl for endocytosis and degradation in a RING dependent manner. In neur loss of function mutations, Dl endocytosis is blocked and the opposite is true when neur is ectopically expressed (increased endocytosis). Neur also enhances Dl signaling activity and acts nonautonomously (in the signal-emitting cell) during N signaling. Then, the role of the two N ligands was studied during the process of lateral inhibition and it was shown that contrary to prior belief, Dl and Ser act synergistically during this process (it was thought that Dl was the sole active ligand there), and their transcriptional regulation is not important for this function. Both Dl and Ser need Neur in order to have full function during lateral inhibition and Neur physically associates with Ser and enhances its endocytosis and degradation (similar to its effects on Dl). Furthermore, we characterized Mib1, another RING type E3 ubiquitin ligase, which we showed that exhibits a redundant function with Neur during lateral inhibition. During wing margin development, Mib1 is required, because Neur is not expressed there. In the absence of both neur and mib1 signaling is completely abolished, which points to a model in which ubiquitination and endocytosis of N ligands is a prerequisite for signaling. The role of a truncated NeurΔR molecule was also analyzed in detail. This lacks the active ubiquitin ligase domain and it was thought to exhibit a negative effect in signaling. We noticed that NeurΔR is active in signal sending, but is inactive in signal reception. It can also rescue the highly neurogenic phenotype of neur loss of function mutations in embryos and adult bristles. This leads us to interesting results concerning the function of the wild type Neur protein; Neur might have a dual function during N signaling: it can activate signal sending in a RING independent manner and it can also allow cells to receive the N signal in a RING dependent manner (e.g. by reducing the cis-negative effect of the high concentration of ligands). NeurΔR seems to functionally interact with Mib1 in order to exert its signal sending activity, because in the absence of mib1, signal sending is completely abolished.
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Ο ρόλος των DSL πρωτεϊνών και των λιγασών ουβικουϊτίνης neuralized και Mind bomb 1 στη σηματοδότηση Notch
