Differential regulation of Rad18 through Rad6-dependent mono- and polyubiquitination.
Rad18 is involved in postreplication repair mainly through monoubiquitination of proliferating cell nuclear antigen (PCNA). Here we show that Rad18 protein was detected in human cells as two major bands at 75 and 85 kDa by Western blot. The bands were identified as nonubiquitinated and monoubiquitinated forms of Rad18, respectively, by mass spectrometry. Multiple ubiquitinated bands of Rad18 were detected in vitro in the presence of E1, E2 (Rad6), and methylated ubiquitin, indicating that Rad18 was monoubiquitinated at multiple sites through autoubiquitination. Rad18 self-associates, and this interaction was abolished by replacing one of the conserved cysteine residues with phenylalanine in the zinc finger domain (C207F). In the C207F mutant Rad18, monoubiquitination of Rad18 was not observed in vivo, suggesting that self-association was critical for monoubiquitination. Monoubiquitinated Rad18 was detected mainly in the cytoplasm, whereas nonubiquitinated Rad18 was detected predominantly in the nuclei. Furthermore, Rad18 was shown to be polyubiquitinated in cells treated with proteasome inhibitors. Purified Rad18 was also polyubiquitinated in an in vitro system containing E1, E2 (Rad6), and ubiquitin, and it was degraded by the addition of proteasomes. These results suggest that the amount of Rad18 in the nucleus is regulated differentially by mono- and polyubiquitination.
Miyase
Shiho
S
Institute of Molecular Embryology and Genetics, Kumamoto University Kumamoto 860-8556, Japan.
Tateishi
Satoshi
S
Watanabe
Kenji
K
Tomita
Kimio
K
Suzuki
Keiichiro
K
Inoue
Hirokazu
H
Yamaizumi
Masaru
M
D019943:Amino Acid Substitution; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D004268:DNA-Binding Proteins; D009097:Multienzyme Complexes; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases