The Spen homolog Msx2-interacting nuclear target protein interacts with the E2 ubiquitin-conjugating enzyme UbcH8.
The nuclear matrix protein Msx2-interacting nuclear target protein (MINT) is a transcription factor that regulates the expression of key transcriptional effectors in diverse signaling pathways. To further understand the function and mechanism of the MINT-mediated transcription regulation, the yeast two-hybrid system was employed to screen proteins that interact with the C-terminal fragment of MINT. From a cDNA library of human lymph nodes, a cDNA encoding the ubiquitin-conjugating enzyme UbcH8 was identified. Using different truncated versions of MINT, we show that the C-terminal Spen paralog and ortholog C-terminal domain (SPOC) domain, which has been demonstrated to mediate interactions between MINT and a panel of other molecules, might be responsible for interaction between MINT and UbcH8 in yeast, as confirmed by the beta-galactosidase assay. The interaction between MINT and UbcH8 in mammalian cells was further proved by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, and co-immunoprecipitation assay. Using a reporter system, we found that MINT-mediated transcription suppression was sensitive to MG132, an inhibitor of the proteosome system. These results suggest a novel mechanism of MINT-mediated transcription regulation, and might be helpful for understanding functions of MINT.
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, Fourth Military Medical University, Changle Xi Street #17, Xian, 710032, P. R. China.
Junfeng
J
Li
Jishu
J
Wang
Xi
X
Yang
Junlin
J
Li
Hongyan
H
Qin
Xiao
X
Dong
Yangting
Y
Zhu
Liang
L
Liang
Yingmin
Y
Liang
Hua
H
Han
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D015398:Signal Transduction; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007976:Leupeptins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C476510:SPEN protein, human; D014452:Ubiquitins; C471622:UBE2L6 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde