One Immunological disorders of the gastrointestinal tract is inflammatory bowel disease (IBD). IBD results in recurrent and persistently elevated levels of cytokines, such as tumor necrosis factor (TNF). TNF promotes activation of the mitogen-activated protein kinase (MAPK) pathway that consists of protein kinases Raf/MEK/ERK. Kinase suppressor of Ras-1 (KSR1) binds all three MAPK cascade components Raf, MEK and ERK to facilitate activation of this pathway. While KSR1 possesses a protein kinase domain, mammalian KSR1 contains an arginine in place of an invariant lysine residue required for ATP binding. Thus evidence supporting a catalytic function of KSR1 remains controversial. Using a recombinant protein expression system in bacteria, we demonstrated that KSR1 is a functional protein kinase that undergoes serine autophosphorylation and is phosphorylates recombinant kinase-inactive MEK1 (rMEK1 K97M). In addition, immunoprecipitated FLAG-tagged wild-type KSR1 expressed in KSR1-/- cells also phosphorylated rMEK1 K97M. Finally, KSR1 promoted colon epithelial cell survival in response to TNF that was dependent on a functional KSR1 kinase domain and MEK kinase activity in vitro.
Since TNF is upregulated in human IBD, we studied the role of KSR1 during chronic inflammation by crossing KSR1-/- mice with the interleukin-10 deficient (Il10-/-) mouse model of spontaneous experimental colitis. KSR1-/-Il10-/- mice developed an accelerated severe colitis by 4 weeks of age. We determined that KSR1 expression in hematopoietic lineages mediated significant protection against colitis in Il10-/- mice. Further analysis indicated that naïve CD4+ T cells lacking KSR1 produced higher levels of IFN-γ and had a greater propensity to differentiate along the Th1 axis. Finally, administration of neutralizing antibody against IFN-γ attenuated the disease in KSR1-/-Il10-/- mice.
In summary, we have demonstrated that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF. In addition, KSR1 suppresses colitis in Il10-/- mice by regulating IFN-γ production in CD4+ T cells and promotes effector T cell developmental homeostasis. These findings indicate that KSR1 is an important molecule involved in both epithelial cell survival and immune regulation. Therefore, further investigations utilizing agents that modulate KSR1 expression to suppress inflammation may validate KSR1 as a therapeutic target for patients with IBD.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-08032010-133210 |
| Date | 03 August 2010 |
| Creators | Goettel, Jeremy Allen |
| Contributors | Matthew J. Tyska, Roy Zent, Stephen R. Hann, D. Brent Polk, Steven K. Hanks |
| 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-08032010-133210/ |
| 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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