Identification of a gamma-tubulin-binding domain in BRCA1.
We previously reported (L-C. Hsu and R. L. White, Proc. Natl. Acad. Sci. USA, 95: 12983-12988, 1998) that hypophosphorylated BRCA1 is associated with mitotic centrosomes in vivo, perhaps through its interaction with gamma-tubulin. In vitro evidence presented here indicates that full-length BRCA1 protein generated by in vitro translation interacts with gamma-tubulin. A specific domain of BRCA1 protein, BRCA1 fragment no. 3 (BF3; amino acids 504-803), is both necessary and sufficient to bind gamma-tubulin. BF3 and gamma-tubulin coimmunoprecipitated when coexpressed in cells. In addition, expression of BF3 interfered with the interaction between BRCA1 and gamma-tubulin. Stable transformants of COS-7 cells that overexpressed BF3 showed a reduced growth rate partly because of increased apoptosis. Furthermore, overexpression of BF3 in COS-7 cells results in the accumulation of mitotic cells with multiple centrosomes and abnormal spindles. Okadaic acid, an inhibitor of protein phosphatases types 1 and 2A, induces hyperphosphorylation of BRCA1, a reduction of both BRCA1 and gamma-tubulin associated with mitotic centrosomes, and an accumulation of abnormal spindle formation. Thus, attenuating the interaction between BRCA1 and gamma-tubulin, and their association with mitotic centrosomes, may induce an increase of aneuploid cell population and contribute to tumorigenesis.
Hsu
L C
LC
Department of Oncological Sciences, University of Utah, Salt Lake City, Utah 84112, USA. lhsu@genetics.utah.edu
Doan
T P
TP
White
R L
RL
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019556:COS Cells; D002455:Cell Division / Q000502:physiology; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005091:Exons; D006801:Humans; D019319:Okadaic Acid / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D014162:Transfection; D014404:Tubulin / Q000378:metabolism*
D019313:BRCA1 Protein; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D014404:Tubulin; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases