title: A Xenopus cell-free system for analysis of the Chfr ubiquitin ligase involved in control of mitotic entry.
abstract: The checkpoint protein Chfr delays entry into mitosis in the presence of mitotic stress. We have analyzed the Chfr checkpoint pathway in the Xenopus cell-free system. We showed that Chfr is a ubiquitin ligase that targets polo-like kinase (Plk1) for degradation, leading to delayed activation of the Cdc25C phosphatase and prolonged inhibitory phosphorylation of Cdc2 at the G2/M transition. In this chapter, we will describe biochemical methods we developed to analyze the Chfr auto-ubiquitination activity and the ubiquitination of its substrate Plk1, as well as functional assays to investigate the Chfr pathway in Xenopus extracts.
authors.affiliation: Department of Biological Sciences, Stanford University, CA, USA.
authors.forename: Dongmin
authors.initials: D
authors.lastname: Kang
authors.forename: Jim
authors.initials: J
authors.lastname: Wong
authors.forename: Guowei
authors.initials: G
authors.lastname: Fang
mesh_terms: D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics*; D002460:Cell Line; D002474:Cell-Free System; D019926:Cyclin B / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000378:metabolism; D006801:Humans; D007313:Insecta; D008025:Ligases / Q000378:metabolism; D008938:Mitosis; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014981:Xenopus; D029867:Xenopus Proteins; D000097869:Polo-Like Kinase 1
chemical_list: D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins; D019926:Cyclin B; D006657:Histones; D008025:Ligases; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000097869:Polo-Like Kinase 1; C412002:CHFR protein, human; C530005:WEE1 protein, Xenopus; C499349:WEE1 protein, human