Ο ρόλος της πρωτεΐνης Neuralized του ποντικού ως Ε3 λιγάσης ουβικουϊτίνης στη ρύθμιση του σηματοδοτικού μονοπατιού Notch

Abstract

Notch (N) signaling constitutes an evolutionary conserved mechanism that mediates cell-cell interactions in many different cellular contexts in development. Consistent with its profound and widespread roles across a range of tissues, its dysfunction results in a tremendous variety of developmental defects and adult pathologies. A great variety of regulatory proteins have been found to interact with Notch and its ligands and control signaling at multiple levels. More recent advances have highlighted that ubiquitination and endocytosis followed by endosomal sorting of both the receptor and its ligands is an essential mechanism by which Notch-mediated signaling is regulated. Two E3 ubiquitin ligases in drosophila, Neuralized (Neur) and Mind Bomb (Mib1) have been shown to play a crucial role in the regulation of Delta and Serrate protein levels. The functional analysis of these two proteins both in invertebrates and vertebrates has provided the first evidence for the role of ubiquitination in the regulation of endocytosis and degradation of Notch ligands. Intensive work from numerous laboratories has been done in order to clarify the multiple regulatory steps of ubiquitination and endocytosis that are mediated by those two ubiquitin ligases, revealing in parallel the complexity of N signaling fine-tuning in time and space. Mouse homologue of Mib1 protein is known to interact with N ligands and regulate their signaling activity, whereas mouse Neuralized-like (Neurl) protein contribution in N signaling regulation had not been elucidated. In an effort to understand the function of mouse Neurl protein we first investigated its role as an E3 ubiquitn ligase. We showed that Neurl acts as an E3 ubiquitin ligase in vitro and its activity depends on a functional RING domain that resides at the C-terminal of the protein. Neurl specifically mono-ubiquitinates the intracellular domain of Jagged1 in vitro, whereas Dll1, Dll4 and Jag2 do not act as subtrates for Neurldependent ubiquitination. However, Neurl overexpression in cells enhances the degradation of all Notch ligands that contain lysine residues in their intracellular domain. In parallel to our biochemical approach, we used the powerful tool of multidimensional protein identification technology (MudPIT) to identify proteins Neurl interacts with, after analysis of cell lines that stably express Flag-tagged Neurl .We were able to verify the interaction of Neurl with NuMA1, a large nuclear protein required for spindle organization during mitosis that our lab had previously identified as a strong Neurl interactor with the use of yeast-two-hybrid system. We also provided evidence for multiple Neurl post-translational modifications that seem to tightly regulate the protein’s function. More specifically, we showed that Neurl is targeted to the plasma membrane after its putative myristoylation and it gets rapidly degraded possibly via the proteasome. Point mutations that abolish its myristoylation and the functionality of its RING domain severely impair Neurl-dependent degradation of the Notch ligands Dll1, Dll4, Jag1 and Jag2, demonstrating the importance of these modifications in the regulation of Neurl function. Despite the regulatory effects of Neurl on all four N ligands, we were able to identify the direct interaction of Neurl only with Jag1, which is specifically mediated by NEUZ2 repeat. These results suggest that mouse Neurl can either directly or indirectly enhance the ubiquitination and subsequent degradation of N ligands, providing the first evidence for Neurl regulatory role in mammalian Notch pathway.