Abstract
Centrosome stability is required for successful mitosis in mammalian cells. Amplification of the centrosome leads to chromosomal missegregation and generation of aneuploidy, which are closely associated with cell transformation and tumorigenesis (Doxsey, S. J. (2001) Nat. Cell Biol. 3, E105-E108; Hinchcliffe, E. H., and Sluder, G. (2001) Genes Dev. 15, 1167-1181; Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., and Doxsey, S. J. (2001) Cancer Res. 61, 2212-2219). However, there are currently limited insights into mechanism(s) for this critical biological event. Here we show that Gadd45a, a DNA damage-inducible protein that is regulated by tumor suppressors p53 and BRCA1, participates in the maintenance of centrosome stability. Mouse embryonic fibroblasts derived from gadd45a knock-out mice exhibit centrosome amplification (designated as increased centrosome numbers). Introduction of exogenous Gadd45a into mouse embryonic fibroblasts isolated from gadd45a-null mice substantially restored the normal centrosome profile. In contrast to p21(waf1/cip1), which ensures coordinated initiation of centrosome, Gadd45a had no significant effect on centrosome duplication in S phase. Interestingly Gadd45a was found to physically associate with Aurora-A protein kinase, whose deregulated expression results in centrosome abnormality. Furthermore Gadd45a was demonstrated to strongly inhibit Aurora-A kinase activity and to antagonize Aurora-A-induced centrosome amplification. These findings identify a novel mechanism for Gadd45a in the maintenance of centrosome stability and broaden understandings of p53- and BRCA1-regulated signaling pathways in maintaining genomic fidelity.
Highlights
Requires the assembly of a strictly bipolar mitotic apparatus that will ensure that all daughter chromosomes are segregated to opposite sides of the cell before the completion of mitosis
We examined whether overexpression of Gadd45a protein in gadd45a-deficient cells restores normal centrosome status. pMSCV-green fluorescent proteins (GFPs)-Gadd45a, a retroviral expression vector that produces GFP-Gadd45a fusion protein, was transfected into gadd45aϪ/Ϫ Mouse embryonic fibroblasts (MEFs) cells. 96 h later, cells were stained with anti␥-tubulin antibody, and centrosomes were examined in 400 GFP-positive cells
MEFs derived from gadd45a-null mice display aneuploidy, chromosomal aberrations, abnormal cytokinesis, and gene amplification [11]
Summary
Plasmid Clones and Antibodies—Myc-tagged Gadd45a expression vectors were described previously [29, 30]. pCMV45 and pCMV-p21 are the expression vectors for Gadd45a. GST-Aurora-A, GST-Cdc, and GST-Cyclin B1 fusion proteins were incubated with cellular lysates isolated from HeLa Gadd45a-inducible cells, which were collected at 24 h after withdrawal of tetracycline and which expressed substantial amounts of Gadd45a protein, and followed by GST pulldown assays. To determine whether Gadd45a protein inhibits Aurora-A activity, Myc-tagged Aurora-A expression vector was transfected into HeLa cells. These observations demonstrate that Gadd45a antagonizes induction of centrosome amplification by Aurora-A
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