The SUMO-targeted ubiquitin ligase RNF4 localizes to etoposide-exposed mitotic chromosomes: implication for a novel DNA damage response during mitosis.
RNF4, a SUMO-targeted ubiquitin ligase (STUbL), localizes to the nucleus and functions in the DNA damage response during interphase of the cell cycle. RNF4 also exists in cells undergoing mitosis, where its regulation and function remain poorly understood. Here we showed that administration of etoposide, an anticancer DNA topoisomerase II poison, to mitotic human cervical cancer HeLa cells induced SUMO-2/3-dependent localization of RNF4 to chromosomes. The FK2 antibody signals, indicative of poly/multi-ubiquitin assembly, were detected on etoposide-exposed mitotic chromosomes, whereas the signals were negligible in cells depleted for RNF4 by RNA interference. This suggests that RNF4 functions as a STUbL in the etoposide-induced damage response during mitosis. Indeed, RNF4-depletion sensitized mitotic HeLa cells to etoposide and increased cells with micronuclei. These results indicate the importance of the RNF4-mediated STUbL pathway during mitosis for the maintenance of chromosome integrity and further implicate RNF4 as a target for topo II poison-based therapy for cancer patients.
Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan.
Masayuki
M
Saito
Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan.
Yuka
Y
Fujimitsu
Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan.
Takeshi
T
Sasano
Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan.
Yushi
Y
Yoshikai
Department of Biochemistry, Shimane University School of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan.
Reiko
R
Ban-Ishihara
Department of Biochemistry, Shimane University School of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan.
Yuko
Y
Nariai
Department of Biochemistry, Shimane University School of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan. Electronic address: turano@med.shimane-u.ac.jp.
Takeshi
T
Urano
Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan; Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan. Electronic address: hisa@kumamoto-u.ac.jp.
Hisato
H
Saitoh
D000818:Animals; D002877:Chromosomes, Human / Q000378:metabolism*; D004260:DNA Repair; D004250:DNA Topoisomerases, Type II; D005047:Etoposide / Q000494:pharmacology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced; D008938:Mitosis; D009687:Nuclear Proteins / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000276:immunology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000276:immunology; D014157:Transcription Factors / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*
D009687:Nuclear Proteins; C114070:RNF4 protein, human; D025842:SUMO-1 Protein; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; D005047:Etoposide; D004250:DNA Topoisomerases, Type II
Chromosome; DNA topoisomerase II; Etoposide (VP-16); Mitosis; Ring finger protein 4 (RNF4); Small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase