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.
Li
Junfeng
J
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.
Wang
Jishu
J
Yang
Xi
X
Li
Junlin
J
Qin
Hongyan
H
Dong
Xiao
X
Zhu
Yangting
Y
Liang
Liang
L
Liang
Yingmin
Y
Han
Hua
H
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