The normal regulation of the Hedgehog (Hh) pathway is essential for embryonic development, stem cell maintenance, and gametogenesis for both vertebrates and invertebrates, whereas the aberrant pathway regulation can cause various developmental defects and cancers. Hence, it is important to understand the precise mechanism of how the Hh pathway is regulated.
Much of our understanding of the Hh pathway comes from studies in Drosophila but applies also to vertebrates. In Drosophila, Hh signal transduction terminates with regulation of the transcriptional activator, Cubitus interruptus (Ci). In the absence of Hh signaling, Ci is 1) processed to a repressor form via the Costal2 (Cos2) complex and suppresses the transcription of Hh target genes or 2) inhibited by binding to Cos2 and Suppressor of Fused (Su(fu)). Once the cells receive Hh ligand, however, 1) Ci processing is inhibited and 2) inhibition by Su(fu), and possibly Cos2, is countered by Fused (Fu) kinase, which ultimately transforms Ci into an activator form that goes into the nucleus and induces the transcription of Hh target genes.
How Fu alleviates Su(fu) inhibition and facilitates the activation of Ci is not well understood, and it was only discovered recently that Ci is the direct target of Fu phosphorylation. Many studies of Hh signaling have been conducted under artificial conditions, where proteins are often overexpressed, leading to findings that sometimes do not reflect in vivo events, where relative protein stoichiometry is important.
For this dissertation, I investigated how Ci activation is regulated by Su(fu) and Fu using CRISPR/Cas to generate different Ci variants expressed at physiological levels in fly wing discs. I looked at how different regions of Ci, including known phosphorylation sites, contribute to the regulation of Ci activity. From this study, I propose that different sets of Ci phosphorylation events mediated by Fu are responsible for changes in Ci-Su(fu) interactions, by altering Ci-Su(fu) interfaces, but also by changing intramolecular Ci-Ci interactions and thereby transforming Ci to an active conformation, leading to target gene activation in response to Hh.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/a872-v483 |
| Date | January 2024 |
| Creators | Kim, Hoyon |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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