A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95.
RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with HAP95.
Sugiura
Takeyuki
T
Discovery Research Laboratory, Tokyo R & D Center, Daiichi Pharmaceutical Co. Ltd., Daiichi-Sankyo group, 16-13, Kitakasai 1-Chome, Tokyo, 134-8630, Japan. sugiura.takeyuki.uu@daiichisankyo.co.jp
Yamaguchi
Aya
A
Miyamoto
Kentaro
K
D000230:Adenocarcinoma / Q000201:enzymology / Q000235:genetics; D000818:Animals; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D015513:Oncogene Proteins / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D021381:Protein Transport; D013347:Subcellular Fractions / Q000201:enzymology; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination; D015854:Up-Regulation / Q000235:genetics
C490117:AKAP8L protein, human; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D015513:Oncogene Proteins; C496945:RNF43 protein, human; D044767:Ubiquitin-Protein Ligases