Identification of Ubiquitin Target Proteins Using Cell-Based Arrays
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- 作者：Tao Zhou ; Bing Liang ; Gui-Ying Su ; Wei-Li Gong ; Hui-Yan Li ; Li-Feng Tian ; Kun He ; Jie Zhao ; Jiang-Hong Man ; Tao Li ; Wei-Hua Li ; Zhi-Yi Zhang ; Chen-Hui Wang ; Ai-Ling Li ; Hui Liu ; Xin Pan ; Pei-Jing Zhan
- 刊名：Journal of Proteome Research
- 出版年：2007
- 出版时间：November 2007
- 年：2007
- 卷：6
- 期：11
- 页码：4397 - 4406
- 全文大小：876K
- 年卷期：v.6,no.11(November 2007)
- ISSN：1535-3907
文摘
A global understanding of ubiquitinated proteins in vivo is key to unraveling the biological significanceof ubiquitination. There are, however, a few effective screening methods for rapid analysis ofubiquitinated proteins. In the current study, we designed a cell-based cDNA expression array combinedwith cell imaging for the rapid identification of polyubiquitinated proteins, which normally accumulateto form the unique "dot" structure following inhibition of ubiquitin proteasomes. The array consistedof 112 cDNAs encoding key components of major cellular pathways and potential targets ofpolyubiquitination. Among them, 40 proteins formed accumulation dots in response to proteasomeinhibitor, MG-132, treatment. More importantly, 24 of those 40 proteins, such as MAPKAPK3, NLK, andRhoGDI2, are previously not known as the targets of ubiquitin. We further validated our findings byexamining the endogenous counterparts of some of these proteins and found that those endogenousproteins form a similar "dot" structure. Immunoprecipitation assays confirmed that these accumulatedproteins are polyubiquitinated. Our results demonstrate that this large-scale application of cell-basedarrays represents a novel global approach in identifying candidates of the polyubiquitinated proteins.Therefore, the technique utilized here will facilitate future research on ubiquitination-regulated cellsignaling.