Browsing by Subject "Epsin"

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    The role of receptor transport in notch signaling.
    (2010-12) Sorensen-Kamakian, Erika Beth
    The Notch signaling pathway is important for cell differentiation and proliferation. The tight regulation of Notch signaling is critical, as aberrant signaling is associated with human cancers. Recent observations suggest that endocytosis is critical in modulating both the activation and down-regulation of Notch signaling events. Although endocytosis is clearly important, it is unclear how endocytosis regulates the Notch signaling pathway. To directly investigate the relationship between Notch internalization and signaling, we developed a quantitative Notch uptake assay using mammalian tissue culture cells. siRNA-mediated depletion studies reveal that Notch endocytosis is clathrin-dependent and requires epsin1, AP2, and Nedd4. Moreover, we show by coimmunopreipitation analysis that epsin1 interaction with Notch is ubiquitin-dependent. Contrary to current models, we demonstrate, by reporter assay, that internalization defects lead to elevated Notch signaling. These results indicate that signal activation occurs independently of Notch endocytosis. In addition to identifying endocytic components critical to regulating Notch signaling, we evaluated the relationship between AAK1 and Numb, two conserved factors implicated in Notch signaling. We find that AAK1, a key endocytic kinase, interacts with and phosphorylates Numb. Mutation of the AAK1 phosphorylation site restricts Numb localization and changes Numb migration when resolved by SDS-PAGE. To test if Numb phosphorylation plays a role in receptor internalization, we employ uptake assays using both immunofluoroscence and a quantitative Elisa-based approach. We find that overexpression of mutant Numb potently disrupts transferrin receptor internalization. Collectively, these observations indicate that phosphorylation is a general mechanism that regulates Numb activity by modulating its distribution within the cell.