Receptor-Mediated Activation of Canonical Wnt Signaling
Cselenyi, Christopher Stephen
:
2008-07-26
Abstract
Wnt/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.