Regulation of Notch Signaling by Intracellular Trafficking
Antfolk, Daniel (2020-02-21)
Åbo Akademi - Åbo Akademi University
21.02.2020
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Publikationens permanenta adress är
https://urn.fi/URN:ISBN:978-952-12-3924-3
https://urn.fi/URN:ISBN:978-952-12-3924-3
Abstrakt
The Notch signaling pathway is one of the essential mediators of cell-cell communication during development of multicellular organisms. Notch signaling is based on receptors and ligands that interact between neighboring cells. In recent years Notch has received a lot of interest as a promising therapeutic target in cancer and several approaches to intervene with the Notch pathway are under pre-clinical and clinical trials. The challenges are prominent side effects, lack of specificity and poor knowledge of the underlying mechanisms behind deregulated Notch activity. In this thesis, several aspects of Notch regulation have been investigated.
Intermediate filaments (IFs) are cytoskeletal proteins that regulate signaling activities in addition to providing structural support for the cell. In study I, we present a novel interaction between the intermediate filament vimentin and the Notch ligand Jagged1. The use of hybrid Jagged1-Dll4 ligands demonstrates a selective regulation of Notch ligands by vimentin. Mouse embryos lacking vimentin display delayed angiogenesis with reduced branching. In vitro and ex vivo angiogenesis assays show a reduced sprouting from vimentin deficient endothelial cells, a phenotype which can be rescued by addition of immobilized Jagged1 ligands. This work implies that IFs can selectively regulate Notch ligands to balance Notch activity. In the second study, we have initiated a screening approach to identify regulators of Jagged1. A dual label approach allows for visualization and measurement of endocytosed Notch extracellular domain peptides bound to Notch ligands. A pilot screen based on cell spot microarrays (CSMA) has generated a set of potential modulators of Jagged1 endocytosis for further validation and future research. In the final part of the thesis, I present the discovery of a novel PKCζ-mediated phosphorylation site on Notch1. Phosphorylation of the identified site, S1791, leads to enhanced trafficking of Notch to the nucleus and higher Notch signaling activity. Blocking PKCζ or using a phospho-deficient form of S1791 leads to less Notch activity and localization to intracellular endosomes. Our data also imply that PKCζ- ediated phosphorylation of Notch influences differentiation of myogenic cells in vitro and neuronal cells in vivo.
In summary, the work presented in this thesis characterizes various aspects of Notch signaling modifications within the context of endocytosis of Notch receptors and ligands. These findings contribute to a better understanding of the intricacies of Notch signaling regulation and may benefit future studies targeting Notch-related diseases. Additionally, the ligand tracking approach lays the groundwork for future work to identify new modulators of ligand endocytosis.
Intermediate filaments (IFs) are cytoskeletal proteins that regulate signaling activities in addition to providing structural support for the cell. In study I, we present a novel interaction between the intermediate filament vimentin and the Notch ligand Jagged1. The use of hybrid Jagged1-Dll4 ligands demonstrates a selective regulation of Notch ligands by vimentin. Mouse embryos lacking vimentin display delayed angiogenesis with reduced branching. In vitro and ex vivo angiogenesis assays show a reduced sprouting from vimentin deficient endothelial cells, a phenotype which can be rescued by addition of immobilized Jagged1 ligands. This work implies that IFs can selectively regulate Notch ligands to balance Notch activity. In the second study, we have initiated a screening approach to identify regulators of Jagged1. A dual label approach allows for visualization and measurement of endocytosed Notch extracellular domain peptides bound to Notch ligands. A pilot screen based on cell spot microarrays (CSMA) has generated a set of potential modulators of Jagged1 endocytosis for further validation and future research. In the final part of the thesis, I present the discovery of a novel PKCζ-mediated phosphorylation site on Notch1. Phosphorylation of the identified site, S1791, leads to enhanced trafficking of Notch to the nucleus and higher Notch signaling activity. Blocking PKCζ or using a phospho-deficient form of S1791 leads to less Notch activity and localization to intracellular endosomes. Our data also imply that PKCζ- ediated phosphorylation of Notch influences differentiation of myogenic cells in vitro and neuronal cells in vivo.
In summary, the work presented in this thesis characterizes various aspects of Notch signaling modifications within the context of endocytosis of Notch receptors and ligands. These findings contribute to a better understanding of the intricacies of Notch signaling regulation and may benefit future studies targeting Notch-related diseases. Additionally, the ligand tracking approach lays the groundwork for future work to identify new modulators of ligand endocytosis.