Type of Document Dissertation Author Cselenyi, Christopher Stephen Author's Email Address firstname.lastname@example.org URN etd-07212008-143939 Title Receptor-Mediated Activation of Canonical Wnt Signaling Degree PhD Department Cell and Developmental Biology Advisory Committee
Advisor Name Title Robert Coffey Committee Chair Chin Chiang Committee Member Ela Knapik Committee Member Ethan Lee, Advisor Committee Member Jennifer Pietenpol Committee Member Susan Wente Committee Member Keywords
- G proteins
- Diseases -- Animal models
Date of Defense 2008-07-16 Availability unrestricted AbstractWnt/beta-catenin signaling controls various cell fates in metazoan development and is misregulated in several cancers and developmental disorders. Binding of a Wnt ligand to its transmembrane coreceptors, Frizzled (Fz) and LRP5/6, inhibits phosphorylation and degradation of the transcriptional coactivator beta-catenin, which then translocates to the nucleus to regulate target gene expression. To understand how Wnt signaling prevents beta-catenin degradation, I focused on the Wnt coreceptor LRP6, which is required for signal transduction and is sufficient to activate Wnt signaling when overexpressed. LRP6 has been proposed to stabilize beta-catenin by stimulating degradation of Axin, a scaffold protein required for beta-catenin degradation. In certain systems, however, Wnt-mediated Axin turnover is not detected until after beta-catenin has been stabilized. Thus, LRP6 may also signal through a mechanism distinct from Axin degradation. To establish a biochemically tractable system to test this hypothesis, I expressed and purified the LRP6 intracellular domain from bacteria and show that it promotes beta-catenin stabilization and Axin degradation in Xenopus egg extract. Using an Axin mutant that does not degrade in response to LRP6, I demonstrate that LRP6 can stabilize beta-catenin in the absence of Axin turnover. Through experiments in egg extract and reconstitution with purified proteins, I identify a mechanism whereby LRP6 stabilizes beta-catenin independently of Axin degradation by directly inhibiting GSK3's phosphorylation of beta-catenin.
In addition to studies of LRP6, I explore the role of the other Wnt coreceptor Fz, which has been suggested to be a G protein coupled receptor. Through biochemical studies in Xenopus egg extract, I demonstrate that Galphao, Galphai, Galphaq, and Gbetagamma promote beta-catenin stabilization by inhibiting GSK3’s phosphorylation of beta-catenin.
Independently of studies on Wnt signaling, I find that two enzymes involved in glycosylation, NAGK and DPAGT1, regulate anteroposterior patterning in Xenopus embryogenesis. I discover that these enzymes involved in N-glycosylation specifically regulate FGF-mediated events in Xenopus development. Because partial loss-of-function mutations in global regulators of N-glycosylation cause a group of human developmental disorders called Congenital Disorders of Glycosylation (CDGs), I suggest the use of Xenopus as a model organism to study the molecular etiology of CDGs.
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