BRCA1 Phosphorylation by Aurora-A in the Regulation of G2 to M Transition

Abstract

Aurora-A/BTAK/STK15 localizes to the centrosome in the G2-M phase, and its kinase activity regulates the G2 to M transition of the cell cycle. Previous studies have shown that the BRCA1 breast cancer tumor suppressor also localizes to the centrosome and that BRCA1 inactivation results in loss of the G2-M checkpoint. We demonstrate here that Aurora-A physically binds to and phosphorylates BRCA1. Biochemical analysis showed that BRCA1 amino acids 1314–1863 binds to Aurora-A. Site-directed mutagenesis indicated that Ser308 of BRCA1 is phosphorylated by Aurora-A in vitro. Anti-phospho-specific antibodies against Ser308 of BRCA1 demonstrated that Ser308 is phosphorylated in vivo. Phosphorylation of Ser308 increased in the early M phase when Aurora-A activity also increases; these effects could be abolished by ionizing radiation. Consistent with these observations, acute loss of Aurora-A by small interfering RNA resulted in reduced phosphorylation of BRCA1 Ser308, and transient infection of adenovirus Aurora-A increased Ser308 phosphorylation. Mutation of a single phosphorylation site of BRCA1 (S308N), when expressed in BRCA1-deficient mouse embryo fibroblasts, decreased the number of cells in the M phase to a degree similar to that with wild type BRCA1-mediated G2 arrest induced by DNA damage. We propose that BRCA1 phosphorylation by Aurora-A plays a role in G2 to M transition of cell cycle. Aurora-A/BTAK/STK15 localizes to the centrosome in the G2-M phase, and its kinase activity regulates the G2 to M transition of the cell cycle. Previous studies have shown that the BRCA1 breast cancer tumor suppressor also localizes to the centrosome and that BRCA1 inactivation results in loss of the G2-M checkpoint. We demonstrate here that Aurora-A physically binds to and phosphorylates BRCA1. Biochemical analysis showed that BRCA1 amino acids 1314–1863 binds to Aurora-A. Site-directed mutagenesis indicated that Ser308 of BRCA1 is phosphorylated by Aurora-A in vitro. Anti-phospho-specific antibodies against Ser308 of BRCA1 demonstrated that Ser308 is phosphorylated in vivo. Phosphorylation of Ser308 increased in the early M phase when Aurora-A activity also increases; these effects could be abolished by ionizing radiation. Consistent with these observations, acute loss of Aurora-A by small interfering RNA resulted in reduced phosphorylation of BRCA1 Ser308, and transient infection of adenovirus Aurora-A increased Ser308 phosphorylation. Mutation of a single phosphorylation site of BRCA1 (S308N), when expressed in BRCA1-deficient mouse embryo fibroblasts, decreased the number of cells in the M phase to a degree similar to that with wild type BRCA1-mediated G2 arrest induced by DNA damage. We propose that BRCA1 phosphorylation by Aurora-A plays a role in G2 to M transition of cell cycle. BRCA1 phosphorylation by Aurora-A in the regulation of G2 to M transition.Journal of Biological ChemistryVol. 290Issue 36PreviewVOLUME 279 (2004) PAGES 19643–19648 Full-Text PDF Open Access The breast cancer susceptibility gene BRCA1 encodes a protein of 1863 amino acids of nuclear phosphoprotein (1Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennet L.M. Ding W. et al.Science. 1994; 266: 66-71Crossref PubMed Scopus (5289) Google Scholar, 2Thomas J.E. Smith M. Rubinfeld B. Gutowski M. Beckmann R.P. Polakis P. J. Biol. Chem. 1996; 271: 28630-28635Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 3Scully R. Chen J. Ochs R. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar, 4Chen Y. Chen C.-H. Riley D.J. Allred C. 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Chem. 2003; 278: 2015-2020Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, L.C. Doan T.P. White R.L. Cancer Res. 2001; 61: 7713-7718PubMed Google Scholar), we BRCA1 and Aurora-A a in BRCA1 from or cells with antibodies and as indicated in The with shown in Aurora-A in the BRCA1 from We determined the of BRCA1. BRCA1 and 1314–1863 expressed as proteins in the proteins with Aurora-A protein, and by of as by with shown in interaction with Aurora-A in the 1314–1863 Interestingly, when cell with the and 1314–1863 of BRCA1 found to to Aurora-A These results BRCA1 1314–1863 is a to BRCA1 to Aurora-A through cellular in the with to that similar of proteins for These results demonstrate that BRCA1 a with Aurora-A in vivo. Aurora-A BRCA1 in BRCA1 and Aurora-A in the centrosome and a in as shown we BRCA1 is phosphorylated by Aurora-A. Aurora-A expressed in cells and with The with of and and an in kinase as a the proteins of BRCA1 from the shown in of Aurora-A by analysis of the protein with shown in phosphorylated BRCA1 the 1314–1863 the that an to the of phosphorylated the the in kinase protein in cells by BRCA1 in the shown in phosphorylation of BRCA1 is by a that binds to the We that similar of proteins as by of a phosphorylation of BRCA1 further by of a of in which to H. Zhou H. Li Q. M. S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google wild type or of Aurora-A with the expressed in cells and with as in of the with in kinase demonstrated that phosphorylated by the of by the that BRCA1 is phosphorylated by Aurora-A. These results demonstrate that BRCA1 is a of Aurora-A in vitro. Aurora-A Ser308 of studies phosphorylation of Aurora-A analysis of proteins phosphorylated by Aurora-A I.M. S. M. E.M. J.R. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). these motifs are phosphorylation by been found in and the site of BRCA1 and residues and and analysis and that phosphorylation of BRCA1 by Aurora-A is abolished in the mutant and We anti-phospho-specific which phosphorylated Ser308 of BRCA1 protein, and further in phosphorylation of the cell of cells by and with or Ser308 of the protein by an (5Okada S. Ouchi T. J. Biol. Chem. 2003; 278: 2015-2020Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Ser308 recognize the protein that the Ser308 the of BRCA1 We further explored the phosphorylation of Ser308 in transition from the G2 to the M phase of DNA damage. The cell cycle of cells at the G2 phase by a by phase cells by with and with for to arrest at the G2 of the of cells from the G2 to the M phase is G2 and cells contain a DNA and are from one by and is phosphorylated mitosis, an that the phosphorylated of be to the cells in and from the G2 cells by F. W. J. M. D. H. Z. 1998; PubMed Scopus Google Scholar, W. W. J. E. Oncogene. 2000; PubMed Scopus Google Scholar). of cells with to DNA and an that the cells in be from G2 cells in the of We to the We found that cells of cells in and that number decreased when cells at the G2 phase the of cells in increased when cells from G2 arrest these to the we further phosphorylation of Ser308 of BRCA1 in the G2 and M phosphorylation of Ser308 is by DNA damage, the cells with from G2 Cell at and the cells to analysis demonstrated that phosphorylation of Ser308 is in the M phase decreased Ser308 phosphorylation when cells to damage at the M been shown that protein and kinase activity of Aurora-A the G2 to M phase T. T. T. N. D. Y. T. S. T. N. M. Y. H. Genes 2002; PubMed Scopus Google Scholar, T. N. T. T. D. M. K. H. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Phosphorylation of of which is located in the of the kinase results in a in activity F. W. J. M. D. H. Z. 1998; PubMed Scopus Google Scholar). the cell of we Aurora-A activity is in cells Consistent with protein of Aurora-A increased in M Phosphorylation of also increased in the M phase, that kinase activity also increased in damage, phosphorylation of in protein these results demonstrate that Aurora-A phosphorylates Ser308 of BRCA1 when cells mitosis, and phosphorylation is DNA damage as a result of of Aurora-A Aurora-A by RNA in Ser308 phosphorylation of Ser308 of BRCA1 further by the protein of Aurora-A with small interfering We cell these cells are cells with RNA specific for the Aurora-A the cell as indicated in of Aurora-A protein the of BRCA1 protein, but phosphorylation of Ser308 reduced in Aurora-A These results reinforce the hypothesis that Aurora-A phosphorylates Ser308 of BRCA1 in vivo. of but the Ser308 adenovirus wild type or of Aurora-A H. Zhou H. Li Q. M. S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), and Ser308 phosphorylation of BRCA1 by infection with cells by Aurora-A and or control and cell cell with and shown in transient of wild type but the increased Ser308 phosphorylation. These results further demonstrate that Aurora-A phosphorylates Ser308 of BRCA1 BRCA1 Ser308 Phosphorylation by Aurora-A G2 to M results demonstrate that BRCA1 phosphorylated by Aurora-A when cells We explored the role of Ser308 phosphorylation in G2-M MEFs from BRCA1 exon 11-deleted mice X. W. Linke S.P. L. Li P.A. Harris C.C. Deng C.X. Nat. Genet. 2001; PubMed Scopus Google and for with or the of BRCA1 with an MEFs also with the of BRCA1. as a of BRCA1 by analysis with The of cells in the M phase by of anti-phospho-specific as in shown in MEFs showed a similar of M phase with cells of cells with resulted in in the number of M phase as a result of of G2-M which cells from of the mutant of BRCA1 in and MEFs showed of the number of M phase without DNA damage and a similar to in with the number of M phase of cells with cells the BRCA1 the of M phase compared with cells these and in these and MEFs with or the reduced of the M phase These results demonstrate that cells of BRCA1 at Ser308 from the G2 to the M We and have demonstrated BRCA1 localization in the centrosome the M phase (5Okada S. Ouchi T. J. Biol. Chem. 2003; 278: 2015-2020Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, L.C. Doan T.P. White R.L. Cancer Res. 2001; 61: 7713-7718PubMed Google Scholar). by of different types of cell with antibodies to and of the Consistent with analysis demonstrated that BRCA1 is with from cells at the phase (27Hsu L.C. Doan T.P. White R.L. Cancer Res. 2001; 61: 7713-7718PubMed Google Scholar). The biological of with have been further by exon of which contains the of BRCA1 exon in cells centrosome and in BRCA1 exon 11-deleted MEFs X. W. Linke S.P. L. Li P.A. Harris C.C. Deng C.X. Nat. Genet. 2001; PubMed Scopus Google Scholar). These results that BRCA1 plays a role in the the of BRCA1 regulation the G2-M is Recent studies have shown that Aurora-A in with protein is for T. N. T. T. D. M. K. H. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). These studies have also shown that Aurora-A in the G2 phase of the cell cycle and is for of the to the of Aurora-A at the G2-M transition when cells to at the phase T. T. T. N. D. Y. T. S. T. N. M. Y. H. Genes 2002; PubMed Scopus Google Scholar), and results also demonstrate that to an of phosphorylation of Aurora-A which is for kinase F. W. J. M. D. H. Z. 1998; PubMed Scopus Google Scholar). These studies reinforce a in which the kinase activity of Aurora-A regulates the G2 to M of the kinase in the of these observations, we BRCA1 is involved in regulation of the G2-M results of proteins demonstrate that BRCA1 and Aurora-A a in vivo. BRCA1 localizes in the centrosome in the M phase (5Okada S. Ouchi T. J. Biol. Chem. 2003; 278: 2015-2020Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, L.C. Doan T.P. White R.L. Cancer Res. 2001; 61: 7713-7718PubMed Google and Aurora-A is from the of the phase to the of the phase of the cycle Lee S.W. S. N. T. Wang Y. Deng C.X. Qin J. Ouchi T. Oncogene. 2003; 22: PubMed Scopus Google Scholar), is that from the G2 to the M Biochemical have several motifs that be phosphorylated by Aurora-A I.M. S. M. E.M. J.R. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, R. T. Nature. 2000; PubMed Scopus Google Scholar, in Scholar). are the phosphorylation by Aurora-A in and the site and in by Lee S.W. S. N. T. Wang Y. Deng C.X. Qin J. Ouchi T. Oncogene. 2003; 22: PubMed Scopus Google Scholar). We Ser308 as a phosphorylation site of the BRCA1 by which is phosphorylated by an in kinase of BRCA1 contains residues that and phosphorylation of Ser308 is determined by the amino been shown that MEFs mutant of BRCA1 contain more than two compared with normal MEFs one or centrosome from the to the M phase is for of Nat. 2002; Scopus Google Scholar). that chromosome by a of and which are the of cancer of proteins that with BRCA1 in to be to further the by which loss of BRCA1 a to We are to of the Ouchi for