Identification of ubiquitin target proteins using cell-based arrays.
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.
Zhou
Tao
T
Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing 100850, China.
Liang
Bing
B
Su
Gui-Ying
GY
Gong
Wei-Li
WL
Li
Hui-Yan
HY
Tian
Li-Feng
LF
He
Kun
K
Zhao
Jie
J
Man
Jiang-Hong
JH
Li
Tao
T
Li
Wei-Hua
WH
Zhang
Zhi-Yi
ZY
Wang
Chen-Hui
CH
Li
Ai-Ling
AL
Liu
Hui
H
Pan
Xin
X
Zhang
Pei-Jing
PJ
Jin
Bao-Feng
BF
Zhang
Xue-Min
XM
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
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