title: Identification of ubiquitin target proteins using cell-based arrays.
abstract: 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 RhoGDI2, 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.
authors: Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing 100850, China.
authors: Tao
authors: T
authors: Zhou
authors: Bing
authors: B
authors: Liang
authors: Gui-Ying
authors: GY
authors: Su
authors: Wei-Li
authors: WL
authors: Gong
authors: Hui-Yan
authors: HY
authors: Li
authors: Li-Feng
authors: LF
authors: Tian
authors: Kun
authors: K
authors: He
authors: Jie
authors: J
authors: Zhao
authors: Jiang-Hong
authors: JH
authors: Man
authors: Tao
authors: T
authors: Li
authors: Wei-Hua
authors: WH
authors: Li
authors: Zhi-Yi
authors: ZY
authors: Zhang
authors: Chen-Hui
authors: CH
authors: Wang
authors: Ai-Ling
authors: AL
authors: Li
authors: Hui
authors: H
authors: Liu
authors: Xin
authors: X
authors: Pan
authors: Pei-Jing
authors: PJ
authors: Zhang
authors: Bao-Feng
authors: BF
authors: Jin
authors: Xue-Min
authors: XM
authors: Zhang
mesh_terms: D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D061988:Proteasome Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry*; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
chemical_list: C569658:ARHGDIB protein, human; D018076:DNA, Complementary; D020730:Guanine Nucleotide Dissociation Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D061988:Proteasome Inhibitors; D011506:Proteins; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; C098416:MAP-kinase-activated kinase 3; C470551:NLK protein, human; D017346:Protein Serine-Threonine Kinases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde