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Investigating the Regulation of c-Fes Non-Receptor Tyrosine Kinase Activation and Gene Expression

Shaffer, Jonathan Michael (2008) Investigating the Regulation of c-Fes Non-Receptor Tyrosine Kinase Activation and Gene Expression. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Shaffer, Jonathan Michaeljms80@pitt.eduJMS80
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSmithgall, Thomas Etsmithga@pitt.eduTSMITHGA
Committee MemberZhang, LinZhangLx@upmc.eduLIZ22
Committee MemberSchmidt, Martin Cmcs2@pitt.eduMCS2
Committee MemberSteinman, Richard Asteinman@pitt.eduSTEINMAN
Committee MemberWalker, William Hwalkerw@pitt.eduWALKERW
Date: 4 December 2008
Date Type: Completion
Defense Date: 19 November 2008
Approval Date: 4 December 2008
Submission Date: 1 December 2008
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Biochemistry and Molecular Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: bimolecular fluorescence complementation; c-Fes; colorectal cancer; non-receptor tyrosine kinase; promoter methylation; tyrosine kinase regulation
Other ID:, etd-12012008-114534
Date Deposited: 10 Nov 2011 20:07
Last Modified: 19 Dec 2016 14:37


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