The Negative Cross-Talk between SAG/RBX2/ROC2 and APC/C E3 Ligases in Regulation of Cell Cycle Progression and Drug

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Shizhen Zhang1, Yanwen Shen1, Hua Li2

  • 1Cancer Institute of the Second Affiliated Hospital and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou 310029, China.

Cell Reports
|September 9, 2020
PubMed

Insights

The E3 ligase SAG inhibits the anaphase-promoting complex/cyclosome (APC/C) to regulate cell cycle progression. This cross-talk ensures accurate cell division and response to anti-microtubule drugs.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The anaphase-promoting complex/cyclosome (APC/C) is a crucial E3 ubiquitin ligase regulating cell cycle progression.
  • APC/C utilizes UBE2C and UBE2S E2 enzymes for K11-linked substrate ubiquitylation.
  • SAG (RBX2/ROC2), a component of Cullin-RING E3 ligases, also interacts with UBE2C/UBE2S for K11-linked polyubiquitylation.

Purpose of the Study:

  • To investigate the potential cross-talk between the APC/C and SAG E3 ligases.
  • To elucidate the role of SAG in regulating APC/C activity and cell cycle progression.
  • To understand the feedback mechanism involving APC/C-mediated degradation of SAG.

Main Methods:

  • Co-immunoprecipitation assays to study protein interactions.
  • Western blotting to assess protein levels and ubiquitylation.
  • Cell cycle analysis (e.g., flow cytometry) to monitor progression.
  • RNA interference (RNAi) for gene knockdown.
  • Site-directed mutagenesis to create degradation-resistant SAG variants.

Main Results:

  • SAG competes with APC2 for binding to UBE2C/UBE2S, acting as an endogenous inhibitor of APC/C.
  • SAG knockdown leads to premature APC/C activation, causing mitotic slippage and resistance to anti-microtubule agents.
  • SAG is a substrate of APC/CCDH1, targeted for degradation during the G1 phase.
  • A degradation-resistant SAG mutant (SAG-R98A/L101A) accelerates G1-to-S phase progression.

Conclusions:

  • A negative cross-talk exists between SAG and APC/C, where SAG inhibits APC/C activity and is subsequently degraded by APC/C.
  • This regulatory loop involving SAG and APC/C is critical for ensuring the fidelity of cell cycle progression.
  • The interplay between SAG and APC/C impacts cellular responses to anti-microtubule drugs and cell cycle checkpoint control.
Keywords:
APC/CAPC2SAGUBE2CUBE2Scell cyclemitotic slippageubiquitylation

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