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Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
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Published on: February 17, 2011

BRCA1-associated protein 1 interferes with BRCA1/BARD1 RING heterodimer activity

Hiroyuki Nishikawa1, Wenwen Wu, Ayaka Koike

  • 1Division of Breast and Endocrine Surgery, Institute of Advanced Medical Science, and Department of Radiology, St. Marianna University School of Medicine, Kawasaki, Japan.

Cancer Research
|January 2, 2009
PubMed

Insights

BRCA1-associated protein 1 (BAP1) inhibits BRCA1/BARD1 E3 ligase activity, impacting DNA damage response and cell cycle progression. BAP1 plays a crucial role in regulating ubiquitination pathways critical for genomic stability.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • BRCA1, a tumor suppressor, forms a RING heterodimer E3 ligase with BARD1.
  • BRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that interacts with BRCA1.
  • The precise role of BAP1 in the E3 ligase activity of BRCA1/BARD1 remains unclear.

Purpose of the Study:

  • To elucidate the mechanism by which BAP1 influences the E3 ligase activity of the BRCA1/BARD1 complex.
  • To investigate the functional consequences of BAP1's interaction with BRCA1/BARD1 in cellular processes.
  • To determine BAP1's role in DNA damage response and cell cycle regulation.

Main Methods:

  • Protein interaction studies using domains of BAP1 (residues 182-365) and the BARD1 RING finger domain.
  • Surface plasmon resonance (BIAcore) to analyze BAP1's effect on BRCA1/BARD1 association.
  • In vitro ubiquitination assays with BRCA1/BARD1 and BAP1 (wild-type and C91S mutant) on substrates like NPM1/B23.
  • In vivo studies using short hairpin RNA (shRNA) to inhibit BAP1 expression and assess cellular responses to ionizing irradiation and cell cycle progression.

Main Results:

  • BAP1 directly interacts with BARD1, inhibiting the E3 ligase activity of the BRCA1/BARD1 complex.
  • BAP1 binding disrupts the BRCA1/BARD1 association, leading to reduced BRCA1 autoubiquitination and NPM1/B23 ubiquitination.
  • BAP1 exhibits in vitro deubiquitinating activity and also inhibits ubiquitination via a catalytically independent mechanism.
  • Depletion of BAP1 sensitizes cells to ionizing irradiation and causes S-phase progression defects.

Conclusions:

  • BAP1 acts as a negative regulator of BRCA1/BARD1 E3 ligase activity through direct interaction with BARD1.
  • BAP1 employs dual mechanisms: enzymatic deubiquitination and non-enzymatic inhibition of ubiquitination.
  • BAP1 and BRCA1/BARD1 function coordinately in regulating ubiquitination for DNA damage response and cell cycle control.

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