The human c-fes locus encodes a non-receptor tyrosine kinase (c-Fes) that is structurally and functionally unique. Originally, c-fes was isolated as the normal cellular homolog of sarcoma-inducing avian and feline retroviruses. However, unlike its viral oncoprotein counterparts that display constitutive tyrosine kinase activity, c-Fes exhibits restrained activity that is regulated by an undefined mechanism. Adding to its unique nature, recent studies have implicated c-Fes as a colorectal cancer-associated tumor suppressor despite its status as a proto-oncogene and tyrosine kinase.
Previous work from our group has demonstrated that c-Fes forms high molecular weight oligomers in vitro, suggesting that c-Fes catalytic activity is governed by the interconversion of c-Fes between inactive monomeric and active oligomeric forms. However, this model was based largely on in vitro data and has not been assessed in living cells. To assess the involvement of oligomerization in regulating c-Fes activity in vivo, I employed a yellow fluorescence protein (YFP)-based bimolecular fluorescence complementation (BiFC) assay. Using BiFC, I demonstrated for the first time that c-Fes forms constitutive oligomers in vivo, regardless of its activation status. In addition, I determined that both coiled-coil domains mediate the oligomerization of c-Fes. Moreover, I established that c-Fes forms coiled-coil dependent oligomers in physiologically relevant cellular contexts, suggesting a new model for c-Fes regulation where conformational changes rather than oligomerization govern c-Fes kinase activity in cells.
In colorectal cancers, loss of c-Fes expression is a common occurrence. This is not unusual, as tumorigenesis proceeds as oncogenes are activated and tumor suppressors are inactivated. To date, however, the mechanism responsible for c-fes gene repression has not been characterized. Upon determining that the absence of c-fes gene transcription was common among colorectal cancer cell lines, I used methylation inhibitor, bisulfite sequencing, and in vitro methylation analyses to establish that promoter methylation governs Fes gene and protein expression in colorectal cancers. Preliminary studies also suggest that promoter methylation governs c-Fes expression in human colon cancer surgical specimens. Taken together, the studies outlined in this thesis advance the field of c-Fes research by defining previously unknown regulatory mechanisms of both kinase activity and gene expression.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12012008-114534 |
| Date | 04 December 2008 |
| Creators | Shaffer, Jonathan Michael |
| Contributors | Martin C. Schmidt, Ph.D., Richard A. Steinman, M.D., Ph.D., Thomas E. Smithgall, Ph.D., Lin Zhang, Ph.D., William H. Walker, Ph.D. |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12012008-114534/ |
| Rights | restricted, 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 University of Pittsburgh 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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