Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo.
The anaphase-promoting complex or cyclosome (APC/C) is one of the major orchestrators of the cell division cycle in mammalian cells. The APC/C acts as a ubiquitin ligase that triggers sequential ubiquitylation of a significant number of substrates which will be eventually degraded by proteasomes during major transitions of the cell cycle. In this chapter, we present accessible methodologies to assess both in in vitro conditions and in cellular systems ubiquitylation reactions mediated by the APC/C. In addition, we also describe techniques to evidence the changes in protein stability provoked by modulation of the activity of the APC/C. Finally, specific methods to analyze interactors or posttranslational modifications of particular APC/C subunits are also discussed. Given the crucial role played by the APC/C in the regulation of the cell cycle, this review only focuses on its action and effects in actively proliferating cells.
Department of Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), Vrije Universiteit Brussel (VUB), Pleinlaan 2, Brussels, 1050, Belgium.
Cesyen
C
Cedeño
Laboratory of Pathophysiological Cell Signaling (PACS), Department of Biology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel (VUB), Pleinlaan 2, Brussels, 1050, Belgium.
Esther
E
La Monaca
Laboratory of Pathophysiological Cell Signaling (PACS), Department of Biology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel (VUB), Pleinlaan 2, Brussels, 1050, Belgium.
Mara
M
Esposito
Laboratory of Pathophysiological Cell Signaling (PACS), Department of Biology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel (VUB), Pleinlaan 2, Brussels, 1050, Belgium. ggutierr@vub.ac.be.
Gustavo J
GJ
Gutierrez
D064173:Anaphase-Promoting Complex-Cyclosome / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation / Q000235:genetics / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology*
D025801:Ubiquitin; D064173:Anaphase-Promoting Complex-Cyclosome
Anaphase-promoting complex or cyclosome (APC/C); Cdc20; Cdc27/APC3; Cdh1; Cell cycle; Phosphorylation; Ubiquitin; Ubiquitylation