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Interaction with TSG101 modulates the ubiquitination of KLIP1

Abstract
Tumor susceptibility gene TSG101 plays an important role in cellular functions including intracellular protein sorting, vesicular trafficking, and transcription regulation. Our previous results from yeast two-hybrid screening show that TSG101 interacts with a novel transcriptional repressor protein, KLIP1. In this study, we demonstrated in vivo interaction between TSG101 and KLIP1 in nucleus of 293 cells using co-immunoprecipitation assay and confocal imaging. In addition, we found KLIP1 could be modified in a modality of either poly- and mono-ubiquitination when exogenously expressed in 293 cells in conjunction with either wild type His-tagged ubiquitin or a mutant His-tagged ubiquitin (K0-Ub) which has no capability of forming polyubiquitin chain. Furthermore, we found that TSG101 could increase 60 kDa-KLIP1, but decrease 71 kDa-KLIP1 levels of monoubiquitinated KLIP1 protein species in a dose dependent manner. These results indicate that TSG101 might regulate KLIP1 protein function through affecting its monoubiquitin modification status. Further investigation using wildtype pHA-KLIP1 and mutant pHA-KLIP1-M6 containing mutation in its 6 lysine residues for possible ubiquitin modification revealed that wildtype HA-KLIP1, but not HA-KLIP1-M6, could inhibit transcription activity of thymidine kinase (TK) promoter. In conclusion, our results support that TSG101 interacts and acts as a transcriptional co-repressor of KLIP1 by keeping it in 60 kDa-monoubiquitinated status in the nucleus, where KLIP1 functions as a transcription repressor for TK promoter. Further experiment using mutant HA-KLIP1 expression plasmid containing single mutation in the 6 lysine sites should reveal the exact location of ubiquitin-modified lysine site for monoubiquitinated species of KLIP1 protein.

Identiferoai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0829108-152942
Date29 August 2008
CreatorsHung, Kuo-Hsuan
ContributorsYi-Ren Hong, Jiin-Tsuey Cheng, Chi-Huei Wang
PublisherNSYSU
Source SetsNSYSU Electronic Thesis and Dissertation Archive
LanguageCholon
Detected LanguageEnglish
Typetext
Formatapplication/pdf
Sourcehttp://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0829108-152942
Rightsnot_available, Copyright information available at source archive

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