Identification of ubiquitin target proteins using cell-based arrays

被引:4
|
作者
Zhou, Tao [1 ]
Liang, Bing [1 ]
Su, Gui-Ying [1 ]
Gong, Wei-Li [1 ]
Li, Hui-Yan [1 ]
Tian, Li-Feng [1 ]
He, Kun [1 ]
Zhao, Jie [1 ]
Man, Jiang-Hong [1 ]
Li, Tao [1 ]
Li, Wei-Hua [1 ]
Zhang, Zhi-Yi [1 ]
Wang, Chen-Hui [1 ]
Li, Ai-Ling [1 ]
Liu, Hui [1 ]
Pan, Xin [1 ]
Zhang, Pei-Jing [1 ]
Jin, Bao-Feng [1 ]
Zhang, Xue-Min [1 ]
机构
[1] Natl Ctr Biomed Anal, Inst Basic Med Sci, Beijing 100850, Peoples R China
关键词
cell arrays; ubiquitin; localization; proteasome inhibitor;
D O I
10.1021/pr070299l
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A global understanding of ubiquitinated proteins in vivo is key to unraveling the biological significance of ubiquitination. There are, however, a few effective screening methods for rapid analysis of ubiquitinated proteins. In the current study, we designed a cell-based cDNA expression array combined with cell imaging for the rapid identification of polyubiquitinated proteins, which normally accumulate to form the unique "dot" structure following inhibition of ubiquitin proteasomes. The array consisted of 112 cDNAs encoding key components of major cellular pathways and potential targets of polyubiquitination. Among them, 40 proteins formed accumulation dots in response to proteasome inhibitor, MG-132, treatment. More importantly, 24 of those 40 proteins, such as MAPKAPK3, NLK, and RhoGDl2, are previously not known as the targets of ubiquitin. We further validated our findings by examining the endogenous counterparts of some of these proteins and found that those endogenous proteins form a similar "dot" structure. Immunoprecipitation assays confirmed that these accumulated proteins are polyubiquitinated. Our results demonstrate that this large-scale application of cell-based arrays represents a novel global approach in identifying candidates of the polyubiquitinated proteins. Therefore, the technique utilized here will facilitate future research on ubiquitination-regulated cell signaling.
引用
收藏
页码:4397 / 4406
页数:10
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