Role for O-glycosylation of RFP in the interaction with enhancer of polycomb.
We recently demonstrated that RFP, which belongs to the large B-box RING finger protein family, interacts with Enhancer of Polycomb 1 (EPC1) and functions as a transcriptional repressor in human cultured cells. In this study, we examined the expression of RFP and EPC1 in mouse tissues by immunoblotting as well as their interaction by a pull-down assay. Both RFP and EPC1 proteins are expressed in several mouse tissues including testis, spleen, thymus, adrenal gland, cerebrum, and cerebellum. In addition, they were coprecipitated from the lysate of mouse testis. Pull-down assays using glutathione S-transferase (GST)-fused EPC1 proteins revealed that RFP is associated with the EPcA, EPcB, and carboxy-terminal (CT) regions of EPC1. Although RFP is highly expressed as 58- and 68-kDa proteins in mouse testis, the EPC1 CT region more strongly interacted with the 68-kDa form than the EPcA or EPcB region. Interaction of the 58-kDa form of RFP with each region was weak compared with that of the 68-kDa form with the EPC1 CT region. Because the 68-kDa form of RFP was almost completely digested with O-glycosidase but not with N-glycosidase, this suggested that O-glycosylation of RFP plays a role in its interaction with the EPC1 CT region that may be responsible for transcriptional repression. In addition, the luciferase reporter gene assay showed that expression of the EPcA region strongly impairs the transcriptional repressive activity of RFP.
Tezel
Gaye
G
Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Shimono
Yohei
Y
Murakumo
Yoshiki
Y
Kawai
Kumi
K
Fukuda
Toshifumi
T
Iwahashi
Naoko
N
Takahashi
Masahide
M
D000311:Adrenal Glands / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002531:Cerebellum / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012097:Repressor Proteins; D013154:Spleen / Q000378:metabolism; D013687:Telencephalon / Q000378:metabolism; D013737:Testis / Q000378:metabolism; D013950:Thymus Gland / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic; D000097573:Red Fluorescent Protein
D002868:Chromosomal Proteins, Non-Histone; D018076:DNA, Complementary; D005982:Glutathione Transferase; D006026:Glycoside Hydrolases; D008156:Luciferases; D008164:Luminescent Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D000097573:Red Fluorescent Protein; C485469:EPC1 protein, human