Centrosomal microtubule nucleation activity is inhibited by BRCA1-dependent ubiquitination

Mol Cell Biol. 2005 Oct;25(19):8656-68. doi: 10.1128/MCB.25.19.8656-8668.2005.

Abstract

In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of gamma-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of gamma-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of gamma-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aneuploidy
  • BRCA1 Protein / metabolism*
  • BRCA1 Protein / physiology
  • Cell Cycle
  • Cell Line
  • Cell Line, Tumor
  • Cell Nucleus / metabolism
  • Cells, Cultured
  • Centrosome / metabolism
  • Centrosome / ultrastructure*
  • Dose-Response Relationship, Drug
  • Humans
  • Lysine / chemistry
  • Microscopy, Fluorescence
  • Microtubules / ultrastructure
  • Mutation
  • Peptides / chemistry
  • Phenotype
  • Plasmids / metabolism
  • Protein Structure, Tertiary
  • RNA, Small Interfering / metabolism
  • S Phase
  • Time Factors
  • Transfection
  • Tubulin / chemistry
  • Tubulin / metabolism
  • Tumor Suppressor Proteins / metabolism
  • Ubiquitin / chemistry
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases / metabolism

Substances

  • BRCA1 Protein
  • Peptides
  • RNA, Small Interfering
  • Tubulin
  • Tumor Suppressor Proteins
  • Ubiquitin
  • BARD1 protein, human
  • Ubiquitin-Protein Ligases
  • Lysine