Biochemical analysis of the Anaphase Promoting Complex: activities of E2 enzymes and substrate competitive (pseudosubstrate) inhibitors.
The Anaphase Promoting Complex (APC) ubiquitin ligase is critical for multiple processes including cell cycle, development, meiosis, and senescence. The importance of regulation of the APC by substrate competitive (pseudosubstrate) inhibitors, such as Emi1 and BubR1, has recently been demonstrated. Substrate competitive inhibitors typically bind to enzymes via the same site as substrates, but by having any combination of increased enzyme affinity and low turnover numbers, are able to "clog" the ability of the enzyme to bind and turnover substrates. For the APC, these pseudosubstrates can both position and block the APC and have been well validated as critical regulators for the APC enzymes.We have found that the substrate competitive mechanism of inhibition is sensitive to the E2 activity driving APC catalyzed ubiquitination events. This chapter provides detailed protocols for multiple in vitro ubiquitination assays of increasing complexity and the use of pseudosubstrate inhibitors in these assays. These assays are instrumental in examining the use of E2 enzymes by the APC and the intimate relationship this has with pseudosubstrate inhibition.
Summers
Matthew K
MK
Department of Cellular Regulation, Genentech Inc., South San Francisco, CA, USA.
Jackson
Peter K
PK
D064173:Anaphase-Promoting Complex-Cyclosome; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D044783:F-Box Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013379:Substrate Specificity; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000502:physiology*; D054875:Ubiquitination / Q000502:physiology*
D018797:Cell Cycle Proteins; D044783:F-Box Proteins; C498960:FBXO5 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases