Kaposi's sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castlemen's disease (MCD). This ×-herpesvirus infects endothelial cells lining the vascular spaces of KS lesions, the so-called spindle cells and B cells in PEL. KSHV encodes proteins that mimic the actions of cellular proteins and override normal cell cycle checkpoints. Viral Bcl-2 and Flice-like inhibitory protein (v-FLIP) contribute to increased cell survival, while a virally-encoded G protein-coupled receptor and viral IL-6 regulate cell growth.
K-cyclin, the cyclin D2 homolog, has non-cell cycle-related transcriptional regulatory properties. Data from our laboratory demonstrates that K-cyclin associates with the transcriptionally active S1 fraction of chromatin along with other proteins like p300, polymerase II and transcription factor IID (TFIID) known to be involved in transcription. K-cyclin/cdk6 complexes can bind and phosphorylate p300, and by doing so activate NF-ÛB dependent transcription. This is significant because our lab's work shows that K-cyclin's activity contributes to the constitutive activation of the NF-kappaB transcriptional pathway in HHV8-associated tumors. K-cyclin contains a protein docking site near the amino terminus, termed the MRAIL motif. This conserved site allows cellular cyclins to interact with proteins containing RXL motifs. We questioned whether the MRAIL motif was essential for the K-cyclin/p300 interaction and the subsequent activation of NF-kappaB-dependent transcription. To answer this question, using site-directed mutagenesis we generated alanine mutants of the amino acid residues comprising the MRAIL motif. We have found that the mutation of any single amino acid in the MRAIL motif is sufficient to disrupt the ability of K-cyclin to activate NF-kappaB-dependent transcription using an NF-kappaB luciferase reporter assay.
The three most important latency genes, v-FLIP, K-cyclin and LANA are under the control of the same promoter, with both LANA and K-cyclin having emerging roles as transcriptional regulators. As K-cyclin and LANA are both transcribed from a single promoter, are expressed at the same time in the viral life cycle and are both found in the nucleus, we sought to determine whether they physically interact. We have identified a novel association between K-cyclin and LANA, a multifunctional viral protein involved in transcription by interacting directly with DNA and through protein-protein interactions with other transcription factors like AP-1, Sp1 and pRb. This interaction was detected at endogenous levels of both proteins in a PEL cell line by coimmunoprecipitation analysis in both directions. Finally, studies in transfected COS-7L cells suggest that K-cyclin-LANA interation is not dependent on any other viral proteins. These findings suggest that K-cyclin is a transcriptional regulator and that this association may play a role in the signature NF-kappaB activation found in KSHV-infected cells.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-06222007-085004 |
| Date | 30 July 2007 |
| Creators | Duell, Stephanie |
| Contributors | Jin Chen, PhD, Philip Browning, MD, Stephen Brandt, MD |
| 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-06222007-085004/ |
| 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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