title: The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells.
abstract: The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G(2)/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G(2)/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G(2) phase, but not at S phase, were ∼2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G(2) phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G(2)/M transition.
authors.affiliation: Department of Biochemistry, Ajou University School of Medicine, Suwon 443-721 Korea; Graduate School of Molecular Science and Technology, Ajou University, Suwon 443-721 Korea.
authors.forename: Jo-Sun
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, Ajou University School of Medicine, Suwon 443-721 Korea; Graduate School of Molecular Science and Technology, Ajou University, Suwon 443-721 Korea.
authors.forename: Yong-Yea
authors.initials: YY
authors.lastname: Park
authors.affiliation: Department of Biochemistry, Ajou University School of Medicine, Suwon 443-721 Korea; Graduate School of Molecular Science and Technology, Ajou University, Suwon 443-721 Korea.
authors.forename: Sun-Yi
authors.initials: SY
authors.lastname: Park
authors.affiliation: B Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Hyeseon
authors.initials: H
authors.lastname: Cho
authors.affiliation: Department of Life Science, Division of Life and Pharmaceutical Sciences, and the Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, Seoul 120-750, Korea.
authors.forename: Dongmin
authors.initials: D
authors.lastname: Kang
authors.affiliation: Department of Biochemistry, Ajou University School of Medicine, Suwon 443-721 Korea; Graduate School of Molecular Science and Technology, Ajou University, Suwon 443-721 Korea. Electronic address: hscho@ajou.ac.kr.
authors.forename: Hyeseong
authors.initials: H
authors.lastname: Cho
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D056744:Cyclin B1 / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008938:Mitosis; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000737:chemistry*; D000075223:Poly-ADP-Ribose Binding Proteins; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*; D000097869:Polo-Like Kinase 1
chemical_list: D000409:Alanine; D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins; D056744:Cyclin B1; D008239:Lysine; D009363:Neoplasm Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000097869:Polo-Like Kinase 1; C412002:CHFR protein, human