The Wnt signaling pathway plays essential roles in a wide variety of biological processes including early animal development, cell fate determination, cell proliferation, organogenesis, and stem-cell renewal. Deregulation of the Wnt pathway can lead to human disease (e.g. developmental defects and cancers). Our lab had previously demonstrated that X-linked Inhibitor of Apoptosis (XIAP) is required for Wnt signaling via a mechanism that involves XIAP-facilitated dissociation of Gro/TLE from TCF/Lef. We had shown that XIAP is recruited onto TCF/Lef complexes upon Wnt pathway activation and ubiquitinates Gro/TLE-bound TCF/Lef. Ubiquitinated Gro/TLE has decreased affinity for TCF/Lef, allowing beta-catenin to bind. Inhibition of GSK3 by lithium causes XIAP to be recruited to TCF/Lef, although the precise mechanism was unknown.
We found that XIAP is phosphorylated by GSK3, a known component of the Wnt pathway. Using mass spectrometry analysis with purified proteins, we identified two GSK3 phosphorylation sites on XIAP that are strongly phosphorylated. Mutational analysis of these two sites indicate that they are required by XIAP to fully activate Wnt signaling, as assessed by reporter assays in cultured mammalian cells and axis duplication assays in Xenopus embryos. Using purified proteins, we found that the XIAP phosphomutants have similar ubiquitination activity as wild-type XIAP. In cultured mammalian cells, however, the XIAP phosphomutants have a markedly decreased capacity to ubiquitinate Gro/TLE. We also showed that the phosphomutants also have a decreased affinity for Gro/TLE.
We propose a model in which phosphorylation is necessary for the interaction between XIAP and Gro/TLE to activate Wnt signaling. GSK3 phosphorylates XIAP at T180 and S239 to facilitate its binding to Gro/TLE. XIAP can then ubiquitinate and remove Gro/TLE for subsequent binding of beta-catenin to form the transcriptionally active complex.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07072016-135625 |
| Date | 25 July 2016 |
| Creators | Hang, Brian I |
| Contributors | Stephen R. Hann, Jin Chen, Michael K. Cooper, Ryoma Ohi |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-07072016-135625/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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