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Detection of Protein Ubiquitination
09:00

Detection of Protein Ubiquitination

Published on: August 19, 2009

Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase

Jessica B Casaletto1, Leta K Nutt, Qiju Wu

  • 1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

The Journal of Cell Biology
|April 13, 2005
PubMed

Insights

Xenopus nuclear factor 7 (Xnf7) inhibits the anaphase-promoting complex/cyclosome (APC), controlling mitotic exit. This novel APC inhibitor links spindle status to APC activity through direct binding to core APC components.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The anaphase-promoting complex/cyclosome (APC) is crucial for regulating cell cycle progression, particularly mitotic exit.
  • APC activity is tightly controlled by various regulatory proteins to ensure proper cell division.

Purpose of the Study:

  • To identify novel regulators of the anaphase-promoting complex/cyclosome (APC).
  • To investigate the role of Xenopus nuclear factor 7 (Xnf7) in controlling mitotic exit and APC activity.

Main Methods:

  • Immunodepletion experiments using Xenopus laevis egg extracts.
  • Assays to measure the degradation of APC substrates (cyclin B1, cyclin B2, securin).
  • Biochemical assays to determine Xnf7's interaction with APC core subunits and its ubiquitin ligase activity.

Main Results:

  • Xenopus nuclear factor 7 (Xnf7) was identified as a novel inhibitor of the APC.
  • Depletion of Xnf7 accelerated APC substrate degradation, while excess Xnf7 inhibited APC activity.
  • Xnf7 possesses intrinsic ubiquitin ligase activity essential for APC inhibition.
  • Xnf7 directly binds to APC core subunits, independent of Cdc20.
  • Xnf7 is required for spindle assembly checkpoint function in egg extracts.

Conclusions:

  • Xnf7 acts as a direct APC inhibitor, regulating the timing of mitotic exit.
  • Xnf7's ability to bind APC core components suggests a mechanism for linking spindle status to APC activity.
  • Xnf7 represents a novel link between the spindle assembly checkpoint and APC-mediated proteolysis.

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