| Abstract |
The Notch signaling pathway is an evolutionary conserved mechanism which participates in a variety of cell fate decisions. In D. melanogaster, key-players in this signaling pathway are three single-pass transmembrane proteins and two E3 ubiquitin ligases. Notch protein, the receptor of the signaling, binds to Delta (Dl) and Serrate (Ser), which are the ligands. Following interaction, a proteolytic step is triggered which leads to the release of a part of the receptor, its translocation to the nucleus and finally the activation of target genes. Apart from transcriptional regulation, posttranscriptional modifications are important for the correct activation of the signaling.
Ubiquitination is one of those modifications. E3 ligases of ubiquitin, Neuralized (Neur) and Mindbomb1 (Mib1), are known to trigger the endocytosis of the ligands and their function is a prerequisite for the activation of Notch signaling. In this work, the role of ubiquitination during Notch signaling was studied in D. melanogaster.
Initially, the intracellular part of Dl protein was analyzed and four conserved motifs were identified. Three of them are associated with the E3 ligases. The first one was the interaction site of Neur and the second one of Mib1. The conserved lysine in the third motif was identified as the acceptor site for ubiquitination by Neur. In this part of the work it was not only proved that Dl is ubiquitinated by Neur and Mib1
directly but also that these ligases bind to and ubiquitinate the ligand in a different way.
Then, ubiquitination of the ligands was connected with their ability to signal. Using Dl mutants that could not be ubiquitinated by Neur or Mib1, we were able to prove that ubiquitination is a step before signaling and a precondition for Notch pathway to be activated.
Finally, we analyzed the subcellular localization of Dl protein. It was already known that Dl protein localizes at the cell surface and in endocytic dots and that ubiquitin ligases are responsible for the endocytosis of the ligand. In this work we proved that not only interaction but ubiquitination by ligases is needed for the endocytosis of the ligand. Furthermore, it was shown that Dl colocalizes with the endocytic markers Sara, Rab11 and Hrs. For this colocalization none of the known
endocytic motifs is responsible. Additionally, we were unable to find any differences in the rate of endocytosis triggered by Neur and Mib1.
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Ο ρόλος των ενδοκυττάριων μοτίβων της πρωτεΐνης Delta στη σηματοδότηση Notch
