Centrosomal microtubule nucleation activity is inhibited by BRCA1-dependent ubiquitination.
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.
Sankaran
Satish
S
Department of Pathology, Brigham and Women's Hospital, NRB 630, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Starita
Lea M
LM
Groen
Aaron C
AC
Ko
Min Ji
MJ
Parvin
Jeffrey D
JD
D000782:Aneuploidy; D019313:BRCA1 Protein / Q000378:metabolism* / Q000502:physiology; D002453:Cell Cycle; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D018385:Centrosome / Q000378:metabolism / Q000648:ultrastructure*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000648:ultrastructure; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D016196:S Phase; D013997:Time Factors; D014162:Transfection; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D019313:BRCA1 Protein; D010455:Peptides; D034741:RNA, Small Interfering; D014404:Tubulin; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases; D008239:Lysine