BRCA1 Phosphorylation by Aurora-A in the Regulation of G2 to M Transition
2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês
10.1074/jbc.m311780200
ISSN1083-351X
AutoresMutsuko Ouchi, Nobuko Fujiuchi, Kaori Sasai, Hiroshi Katayama, Yoji Andrew Minamishima, Pat P. Ongusaha, Chu‐Xia Deng, Subrata Sen, Sam W. Lee, Toru Ouchi,
Tópico(s)Cancer Genomics and Diagnostics
ResumoAurora-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. Chen P.-L. von Hoff D.V. Osborne C.K. Lee W.-H. Science. 1995; 270: 789-791Crossref PubMed Scopus (309) Google Scholar, 5Okada S. Ouchi T. J. Biol. Chem. 2003; 278: 2015-2020Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Mutations in the BRCA1 locus have been found in ∼40% of familial breast cancers and most of the combined familial breast and ovarian cancers (6Alberg A.J. Helzlsouer K.J. Curr. Opin. Oncol. 1997; 9: 505-511Crossref PubMed Scopus (50) Google Scholar, 7Alberg A.J. Lam A.P. Helslsouer K.J. Curr. Opin. Oncol. 1999; 11: 435-441Crossref PubMed Scopus (37) Google Scholar). BRCA1 contains a number of structural motifs responsible for interaction with cellular proteins to regulate diverse biological functions (8Deng C. Brodie S.G. Bioessays. 2000; 22: 728-737Crossref PubMed Scopus (275) Google Scholar, 9Rosen E.M. Fan S. Pestell R.G. Goldberg I.D. J. Cell. Physiol. 2003; 196: 19-41Crossref PubMed Scopus (189) Google Scholar), transcription control (10Ouchi T. Monteiro A.N. August A. Aaronson S.A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2302-2306Crossref PubMed Scopus (333) Google Scholar, 11Ouchi T. Lee S.W. Ouchi M. Aaronson S.A. Horvath C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5208-5213Crossref PubMed Scopus (187) Google Scholar, 12Aprelikova O. Pace A.J. Fang B. Koller B.H. Liu E.T. J. Biol. Chem. 2001; 276: 25647-25650Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 13Chai Y.L. Chi J. Shao N. Shyam E. Reddy P. Rao V.N. Oncogene. 1999; 18: 263-268Crossref PubMed Scopus (158) Google Scholar, 14Chapman M.S. Verma I.M. Nature. 1996; 382: 678-679Crossref PubMed Scopus (436) Google Scholar, 15Fan S. Wang J. Yuan R. Ma Y. Meng Q. Erdos M.R. Pestell R.G. Yuan F. Auborn K.J. Goldberg I.D. Rosen E.M. Science. 1999; 284: 1354-1356Crossref PubMed Scopus (418) Google Scholar, 16Zhang H. Somasundaram K. Peng Y. Tian H. Bi D. Weber B.L. El-Deiry W.S. Oncogene. 1998; 16: 1713-1721Crossref PubMed Scopus (425) Google Scholar, 17Zheng L. Annab L.A. Afshari C.A. Lee W.-H. Boyer T.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9587-9592Crossref PubMed Scopus (193) Google Scholar), cell cycle regulation (18Xu X. Weaver Z. Linke S.P. Li C. Gotay J. Wang X.W. Harris C.C. Ried T. Deng C.X. Mol. Cell. 1999; 3: 389-395Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar, 19Yarden R.I. Pardo-Reoyo S. Sgagias M. Cowan K.H. Brody L.C. Nat. Genet. 2002; 30: 285-289Crossref PubMed Scopus (401) Google Scholar), chromatin remodeling (20Bochar D.A. Wang L. Beniya H. Kinev A. Xue Y. Lane W.S. Wang W. Kashanchi F. Shiekhattar R. Cell. 2000; 102: 257-265Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 21Hu Y.F. Hao Z.L. Li R. Genes Dev. 1999; 13: 637-642Crossref PubMed Scopus (107) Google Scholar, 22Ye Q. Hu Y.F. Zhong H. Nye A.C. Belmont A.S. Li R. J. Cell Biol. 2001; 155: 911-921Crossref PubMed Scopus (176) Google Scholar), DNA damage repair (23Gowen L.C. Avrutskaya A.V. Latour A.M. Koller B.H. Leadon S.A. Science. 1998; 281: 1009-1012Crossref PubMed Scopus (452) Google Scholar, 24Moynahan M.E. Chiu J.W. Koller B.H. Jasin M. Mol. Cell. 1999; 4: 511-518Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar, 25Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 26Zhong Q. Chen C.F. Le S. Chen Y. Wang C.C. Xiao J. Chen P.L. Sharp Z.D. Lee W.-H. Science. 1999; 285: 747-750Crossref PubMed Scopus (525) Google Scholar), and centrosome localization and duplication (27Hsu L.C. Doan T.P. White R.L. Cancer Res. 2001; 61: 7713-7718PubMed Google Scholar, 28Xu X. Wagner K.U. Larson D. Weaver Z. Li C. Ried T. Hennighausen L. Wynshaw-Boris A. Deng C.X. Nat. Genet. 1999; 22: 37-43Crossref PubMed Scopus (624) Google Scholar). Recent studies have demonstrated that phosphorylation of specific residues of BRCA1 protein, such as Ser988, Ser1423, and Ser1524, etc., is important for regulation of survival of cells with DNA damage (29Gatei M. Zhou B.-B. Hobson K. Scott S. Young D. Khanna K.K. J. Biol. Chem. 2001; 276: 17276-17280Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 30Xu B. O'Donnell A.H. Kim S.-T. Kastan M.B. Cancer Res. 2002; 62: 4588-4591PubMed Google Scholar, 31Cortez D. Wang Y. Qin J. Elledge S.J. Science. 1999; 286: 1162-1166Crossref PubMed Scopus (869) Google Scholar, 32Tibbetts R.S. Cortez D. Brumbaugh K.M. Scully R. Livingston D. Elledge S.J. Abraham R.T. Genes Dev. 2000; 14: 2989-3002Crossref PubMed Scopus (402) Google Scholar, 33Foray N. Marot D. Randrianarison V. Venezia N.D. Picard D. Perricaudet M. Favaudon V. Jeggo P. Mol. Cell. Biol. 2002; 22: 4020-4032Crossref PubMed Scopus (70) Google Scholar). Of note, phosphorylation of BRCA1 is induced by kinases, including ATM (ataxia telangiectasia mutated), ATR, Chk2, and c-Abl, which are known to be involved in checkpoint machinery. These results indicate that the physiological function of BRCA1 is at least in part determined by phosphorylation of specific residues. The essential functions of BRCA1 have been further explored by BRCA1-null or exon 11-deleted mice (18Xu X. Weaver Z. Linke S.P. Li C. Gotay J. Wang X.W. Harris C.C. Ried T. Deng C.X. Mol. Cell. 1999; 3: 389-395Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar, 28Xu X. Wagner K.U. Larson D. Weaver Z. Li C. Ried T. Hennighausen L. Wynshaw-Boris A. Deng C.X. Nat. Genet. 1999; 22: 37-43Crossref PubMed Scopus (624) Google Scholar, 34Hakem R. de la Pompa J.L. Sirard C. Mo R. Woo M. Hakem A. Wakeham A. Potter J. Reitmair A. Billia F. Firpo E. Hui C.C. Roberts J. Rossant J. Mak T.W. Cell. 1996; 85: 1009-1023Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 35Liu C.-Y. Flesken-Nikitin A. Li S. Zeng Y. Lee W.-H. Genes Dev. 1996; 10: 1835-1843Crossref PubMed Scopus (274) Google Scholar, 36Gowen L.C. Johnson B.L. Latour A.M. Sulik K.K. Koller B.H. Nat. Genet. 1996; 12: 191-194Crossref PubMed Scopus (395) Google Scholar). Analysis of exon 11-deleted MEFs from such mutant mice indicates that compared with normal MEFs, which contain one or two centrosomes, about 25% of mutant MEFs contain more than two, leading to loss of the G2-M checkpoint and aneuploidy (18Xu X. Weaver Z. Linke S.P. Li C. Gotay J. Wang X.W. Harris C.C. Ried T. Deng C.X. Mol. Cell. 1999; 3: 389-395Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar). Interestingly, we found that without DNA damage, Ser988-phosphorylated BRCA1 resides in the centrosome and chromosome through metaphase to telophase as a result of the actions of anti-phospho-specific antibodies that recognize phosphorylated Ser988 of BRCA1 (5Okada S. Ouchi T. J. Biol. Chem. 2003; 278: 2015-2020Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). These results reinforce the working hypothesis that BRCA1 plays an important role in mitosis, but the role of phosphorylation of the protein in regulation of the M phase is largely unknown. The Aurora-A gene locus is located in the 20q13 chromosome region, which is frequently amplified in several different types of malignant tumors such as breast, colorectal, pancreatic, and bladder cancers (37Bischoff J.R. Anderson L. Zhu Y. Mossie K. Ng L. Souza B. Schryver B. Flanagan P. Clairvoyant F. Ginther C. Chan C.S. Novotny M. Slamon D.J. Plowman G.D. EMBO J. 1998; 17: 3052-3065Crossref PubMed Scopus (1109) Google Scholar, 38Zhou H. Kuang J. Zhong L. Kuo W.L. Gray J.W. Sahin A. Brinkley B.R. Sen S. Nat. Genet. 1998; 20: 189-193Crossref PubMed Scopus (1141) Google Scholar). In particular, the 20q11–q13 region is amplified in 40% of breast cancer cell lines as well as in 12–18% of primary tumors. The encoded protein, Aurora-A, is a member of the Ser/Thr kinase family, and recent studies have demonstrated that Aurora-A is involved in the G2-M checkpoint and mitosis commitment (39Marumoto T. Hirota T. Morisaki T. Kunitoku N. Zhang D. Ichikawa Y. Sasayama T. Kuninaka S. Mimori T. Tamaki N. Kimura M. Okano Y. Saya H. Genes Cells. 2002; 7: 1173-1182Crossref PubMed Scopus (186) Google Scholar, 40Meraldi P. Honda R. Nigg E. EMBO J. 2002; 21: 483-492Crossref PubMed Scopus (573) Google Scholar, 41Anand S. Penrhyn-Lowe S. Venkitaraman A.R. Cancer Cell. 2003; 3: 51-62Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 42Hirota T. Kunitoku N. Sasayama T. Marumoto T. Zhang D. Nitta M. Hatakeyama K. Saya H. Cell. 2003; 114: 585-598Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). In Drosophila, mutations in the Aurora-A gene result in incomplete centrosome positioning, leading to abnormal formation of spindle poles and astral microtubules (43Giet R. McLean D. Descamps S. Lee M.J. Raff J.W. Prigent C. Glover D.M. J. Cell Biol. 2002; 156: 437-451Crossref PubMed Scopus (288) Google Scholar, 44Glover D.M. Leibovitz M.H. McLean D.A. Parry H. Cell. 1995; 81: 95-105Abstract Full Text PDF PubMed Scopus (698) Google Scholar). In Xenopus egg extracts, Eg2, a homologue of Aurora-A, has been shown to be involved in mitotic spindle assembly (45Roghi C. Giet R. Uzbekov R. Morin N. Chartrain I. Le Guellec R. Couturier A. Doree M. Philippe M. Prigent C. J. Cell Sci. 1998; 111: 557-572Crossref PubMed Google Scholar). Furthermore, recent studies have demonstrated that Aurora-A is inactivated by DNA damage at the end of the G2 phase, and overexpression of Aurora-A abrogates the G2 checkpoint in higher eukaryotes (39Marumoto T. Hirota T. Morisaki T. Kunitoku N. Zhang D. Ichikawa Y. Sasayama T. Kuninaka S. Mimori T. Tamaki N. Kimura M. Okano Y. Saya H. Genes Cells. 2002; 7: 1173-1182Crossref PubMed Scopus (186) Google Scholar). Significantly, Aurora-A is recruited to the centrosome during early G2, and it becomes phosphorylated and activated in centrosomes late in the G2 phase (42Hirota T. Kunitoku N. Sasayama T. Marumoto T. Zhang D. Nitta M. Hatakeyama K. Saya H. Cell. 2003; 114: 585-598Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Moreover, small interfering RNA-mediated depletion of Aurora-A results in the failure to enter mitosis, suggesting that Aurora-A-dependent signaling in the centrosome is crucial for mitosis commitment. On the basis of these observations, we explored whether BRCA1 regulates mitosis entry through functional interaction with Aurora-A in the G2 to M phases. Our results demonstrate that Aurora-A binds to BRCA1 and phosphorylates Ser308 of BRCA1. A phospho-specific antibody recognizing phosphorylated Ser308 showed that this phosphorylation increases in early M phase but is inhibited by IR damage. A biological assay by means of BRCA1 (–/–) MEFs revealed that BRCA1 phosphorylation by Aurora-A might regulate mitotic entry. Given that both Aurora-A and BRCA1 are closely associated with breast carcinogenesis, our results suggest a model in which derailed regulation of G2-M transition by these proteins predisposes to cancer development. Cell Culture, Synchronization, and Fluorescence-activated Cell Sorter Analysis—MCF7, U2OS cells, and BRCA1 (–/–) MEFs (46Xu X. Qiao W. Linke S.P. Cao L. Li W.-M. Furth P.A. Harris C.C. Deng C.X. Nat. Genet. 2001; 28: 266-271Crossref PubMed Scopus (297) Google Scholar) were maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum. For synchronization of the cell cycle at the G1/S boundary, a double-thymidine block and release was performed (47Spector D.L. Goldman R.D. Leinwand L.A. Cells: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1998: 14.5-14.7Google Scholar). Briefly, MCF7 cells were treated with 2.5 mm thymidine for 16 h and then released by washing them with phosphate-buffered saline three times followed by placing them in fresh medium containing Dulbecco's modified Eagle's medium, 10% fetal bovine serum. After 8 h, the cells were retreated with thymidine for 16 h. The cells were washed with phosphate-buffered saline again and maintained in fresh Dulbecco's modified Eagle's medium, 10% fetal bovine serum medium containing 20 nm roscovitine (Sigma) for 12 h to synchronize them at the G2 phase. The cells were washed with phosphate-buffered saline to release them into mitosis, and M phase cells were collected after 30 min. For each time point, the cells were stained with propidium iodide and fluorescein isothiocyanate-conjugated anti-phospho-histone H3 antibody (Cell Signaling) as described previously (48Xu B. Kim S.-T. Kastan M.B. Mol. Cell. Biol. 2001; 21: 3445-3450Crossref PubMed Scopus (472) Google Scholar). IR 1The abbreviations used are: IR, ionizing radiation; MEF, mouse embryo fibroblast; aa, amino acid; GST, glutathione S-transferase; KD, kinase-deficient; WT, wild type. was administered using a MARK2 IRRADIATOR (J. L. Shepherd & Associates, San Fernando, CA). Plasmid Construction and Adenovirus Production—Wild type and kinase-defective forms of Aurora-A have been described previously (49Katayama H. Zhou H. Li Q. Tatsuka M. Sen S. J. Biol. Chem. 2001; 276: 46219-46224Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). BRCA1 S308N was generated using QuikChange (Stratagene) with primers of 5′-TGTAATAAAAACAAACAGCCT-3′ and its complement sequence. FLAG-tagged BRCA1 cDNA was subcloned into pBabepuro, and retrovirus was generated as described (50Aglipay J.A. Lee S.W. Okada S. Fujiuchi N. Phtsuka T. Wang Y. Johnstone R.W. Deng C.X. Qin J. Ouchi T. Oncogene. 2003; 22: 8931-8938Crossref PubMed Scopus (106) Google Scholar). Both cDNAs were subcloned into a pAdTRACK-CMV vector, and recombination was performed in a BJ5180 bacterial strain as reported previously (11Ouchi T. Lee S.W. Ouchi M. Aaronson S.A. Horvath C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5208-5213Crossref PubMed Scopus (187) Google Scholar). The mammalian GST tag vector, pEBG, was obtained from Bruce Mayer at the University of Connecticut Health Center. Transfection, Immunoprecipitation, and Immunoblot Analysis—For small interfering RNA analysis of Aurora-A, double-stranded RNA (5′-AAAUGCCCUGUCUUACUGUCA-3′) was synthesized (Dharmacon) and transfected with OligofectAMINE (Invitrogen). Transfection was performed with FuGENE (Roche Applied Science) or LipofectAMINE (Invitrogen) according to the manufacturer's protocol. Rabbit polyclonal phospho-Ser-specific antibody recognizing phosphorylated Ser308 of BRCA1 was generated by Research Genetics, Inc. against keyhole limpet hemocyanin-conjugated synthetic peptides; EFCNKSpKQPGLAR. The following antibodies were purchased for Western blot analysis: antibodies for BRCA1 (C-20, Santa Cruz; Ab-1, Calbiochem), Aurora-A (3092, Cell Signaling), Aurora-A/T288-P (3091, Cell Signaling), GST (Z-5, Santa Cruz), actin (H196, Santa Cruz), and FLAG (M2, Sigma). The cell extracts were prepared in EBC buffer (50 mm HEPES, pH 7.6, 250 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA, pH 8.0, with mixed protease inhibitor; Sigma). Total cell lysates (1 mg) were used for immunoprecipitation with the indicated antibodies and protein G-agarose (Sigma). The samples were washed with EBC buffer four times and subjected to SDS-PAGE. The secondary antibodies (Jackson Immuno-laboratory) were peroxidase-conjugated anti-mouse IgG (H+L) or anti-rabbit IgG (H+L). Film was developed by ECL. GST Pull-down Assay—Purification of GST-BRCA1 constructs was described previously (25Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). Purified Aurora-A was produced by cleavage of GST-Aurora-A by thrombin according to the manufacturer's protocol (Amersham Biosciences). Briefly, 1 μg of purified Aurora-A was incubated with 1 μg of GST-BRCA1 fusion proteins for 1 h at 4 °C. Glutathione beads were added to each sample, and the samples were further rotated for 1 h at 4 °C. After extensive washing with NET-N buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 100 mm NaF, 200 μm sodium orthovanadate) followed by NET-N buffer containing 500 mm of NaCl, the samples were loaded in 7.5% SDS-PAGE and blotted with anti-Aurora-A antibody. GST pull-down assay using total cell lysates of MCF7 cells were described previously (10Ouchi T. Monteiro A.N. August A. Aaronson S.A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2302-2306Crossref PubMed Scopus (333) Google Scholar, 11Ouchi T. Lee S.W. Ouchi M. Aaronson S.A. Horvath C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5208-5213Crossref PubMed Scopus (187) Google Scholar). Briefly, 500 μg of lysates were incubated with about 1 μg of GST fusion proteins for 1 h at 4 °C. After glutathione beads were added to each sample, the samples were further rotated for 1h at 4 °C. After extensive washing with NET-N buffer, the samples were loaded in 7.5% SDS-PAGE and blotted with anti-Aurora-A antibody. In Vitro Kinase Assay—The immunoprecipitates were washed with NET-N buffer containing 500 mm of NaCl followed by kinase buffer (50 mm Tris-HCl, pH 7.5, 15 mm MgCl2, 1 mm dithiothreitol). Each sample was incubated with 20 μl of kinase buffer containing 5 μCi of [γ-32P]ATP and 1 μg of GST-BRCA1 proteins for 10 min at 30 °C and then separated in SDS-PAGE. The gels were dried and autoradiographed. BRCA1 Forms a Complex with Aurora-A—Aurora-A/BTAK/STK15 is a Ser/Thr kinase that is localized in the centrosome and frequently amplified in human cancer (38Zhou H. Kuang J. Zhong L. Kuo W.L. Gray J.W. Sahin A. Brinkley B.R. Sen S. Nat. Genet. 1998; 20: 189-193Crossref PubMed Scopus (1141) Google Scholar, 39Marumoto T. Hirota T. Morisaki T. Kunitoku N. Zhang D. Ichikawa Y. Sasayama T. Kuninaka S. Mimori T. Tamaki N. Kimura M. Okano Y. Saya H. Genes Cells. 2002; 7: 1173-1182Crossref PubMed Scopus (186) Google Scholar, 40Meraldi P. Honda R. Nigg E. EMBO J. 2002; 21: 483-492Crossref PubMed Scopus (573) Google Scholar, 41Anand S. Penrhyn-Lowe S. Venkitaraman A.R. Cancer Cell. 2003; 3: 51-62Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 42Hirota T. Kunitoku N. Sasayama T. Marumoto T. Zhang D. Nitta M. Hatakeyama K. Saya H. Cell. 2003; 114: 585-598Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Recent studies have shown that Aurora-A plays a crucial role in mitotic entry and that Aurora-A activation in the G2-M transition is inhibited by DNA damage (39Marumoto T. Hirota T. Morisaki T. Kunitoku N. Zhang D. Ichikawa Y. Sasayama T. Kuninaka S. Mimori T. Tamaki N. Kimura M. Okano Y. Saya H. Genes Cells. 2002; 7: 1173-1182Crossref PubMed Scopus (186) Google Scholar). Because we and others have found that BRCA1 is also localized in the centrosome (5Okada S. Ouchi T. J. Biol. Chem. 2003; 278: 2015-2020Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 27Hsu L.C. Doan T.P. White R.L. Cancer Res. 2001; 61: 7713-7718PubMed Google Scholar), we examined whether BRCA1 and Aurora-A form a complex in normally growing cells. BRCA1 was immunoprecipitated from unsynchronized BRCA1-mutated HCC1937 or MCF7 cells with anti-BRCA1 antibodies C-20 and Ab-1 as indicated in Fig. 1A. The samples were immunoblotted with anti-Aurora-A antibody. As shown in Fig. 1A, Aurora-A was detected in the BRCA1 immunoprecipitates from MCF7 cells. We next determined the Aurora-A-binding region of BRCA1. BRCA1 aa 1–324, 260–553, 502–802, 758–1064, 1005–1313, and 1314–1863 were expressed as N-terminal GST fusion proteins in bacteria. After purification, the GST fusion proteins were incubated with purified Aurora-A protein, and samples were extensively washed by NET-N buffer containing 500 mm of NaCl as described under "Experimental Procedures" followed by immunoblotted with anti-Aurora-A antibody. As shown in Fig. 1B (top panel), interaction with Aurora-A was detected in the aa 1314–1863 region. Interestingly, when cell lysates were incubated with six GST-BRCA1 fragments above, both the aa 758–1064 and 1314–1863 segments of BRCA1 were found to bind to Aurora-A (Fig. 1B, bottom panel). These results suggest that, although BRCA1 aa 1314–1863 is a primary binding region to Aurora-A, BRCA1 aa 758–1064 may indirectly bind to Aurora-A through cellular protein(s). GST-BRCA1 fragments were immunoblotted in the separate SDS-PAGE with anti-GST antibody to confirm that similar amounts of GST fusion proteins were used for this assay (Fig. 1C). These results demonstrate that BRCA1 forms a complex with Aurora-A in vivo. Aurora-A Phosphorylates BRCA1 in Vitro—Because both BRCA1 and Aurora-A co-exist in the centrosome and form a complex in vivo as shown above, we investigated whether BRCA1 is phosphorylated by Aurora-A. GST-tagged Aurora-A was expressed in 293T cells and affinity-precipitated with GSH beads. The samples were extensively washed with NET-N buffer containing 500 mm of NaCl and divided into six aliquots, and an in vitro kinase assay was performed using as a substrate the purified GST fusion proteins of BRCA1 from the experiment shown in Fig. 1C. Expression of GST-tagged Aurora-A was confirmed by anti-GST immunoblot analysis after GST pull-down of the protein with GSH beads (Fig. 2A). As shown in Fig. 2B, GST-Aurora-A strongly phosphorylated BRCA1 aa 260–553 and, less efficiently, the aa 1314–1863 segments. To rule out the possibility that an unknown kinase(s) binding to the GST region of GST-Aurora-A phosphorylated the substrates, the same in vitro kinase assay was performed expressing GST protein alone in 293T cells followed by affinity purification. Because precipitated GST did not phosphorylate BRCA1 segments used in the experiment shown in Fig. 2B, phosphorylation of BRCA1 aa 260–553 is not carried out by a kinase(s) that binds to the GST tag (data not shown). We confirmed that similar levels of GST-BRCA1 proteins were used as substrates by Coomassie Blue staining of a gel (Fig. 2C). Aurora-A-dependent phosphorylation of BRCA1 was further confirmed by means of a kinase-deficient (KD) form of Aurora-A, in which Lys162 was mutated to arginine (49Katayama H. Zhou H. Li Q. Tatsuka M. Sen S. J. Biol. Chem. 2001; 276: 46219-46224Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar); wild type (WT) or KD forms of Aurora-A with the N-terminal GST tag were expressed in 293T cells and affinity-precipitated with GSH-beads as described in Fig. 2A. After extensive washing of the samples with NET-N buffer containing high salt concentration, in vitro kinase assay demonstrated that purified GST-BRCA1 (260–553) was phosphorylated by the WT of Aurora-A, not by the KD form, showing that BRCA1 (260–553) is phosphorylated by Aurora-A. These results demonstrate that BRCA1 is a substrate of Aurora-A in vitro. Aurora-A Phosphorylates Ser308 of BRCA1—Previous studies identified putative consensus phosphorylation sites of Aurora-A using mass spectrometry analysis of 28 kinetochore proteins phosphorylated by Aurora-A (51Cheeseman I.M. Anderson S. Jwa M. Green E.M. Kang J.-S. Yates III, J.R. Chan C.S.M. Drubin D.G. Barnes G. Cell. 2002; 111: 163-172Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Among these motifs are K(S/T) sequences whose phosphorylation by lpl1p/Aurora-A has been found in Dam1, Spc34, and the autophosphorylation site of lpl1p/Aurora. In BRCA1 aa 260–553, five Ser and Thr residues fit this motif (Ser308, Ser444, Ser451, Thr464, and Thr528), and mutagenic analysis (S308N, S444A, S451A, T464A, and T528A) revealed that phosphorylation of BRCA1 aa 260–553 by Aurora-A is abolished only in the S308N mutant (Fig. 3A and data not shown). We generated rabbit anti-phospho-specific antibody, S308P, which recognizes phosphorylated Ser308 of BRCA1 protein, and further studied in vivo phosphorylation of the protein. To characterize S308P antibody, total cell lysates of MCF7 cells were treated by λ-phosphatase and immunoblotted with anti-BRCA1 or Ser308 antibodies. After treatment, shift-down of the protein was detected by an anti-BRCA1 antibody (MAb21A8) (5Okada S. Ouchi T. J. Biol. Chem. 2003; 278: 2015-2020Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Ser308 did not recognize the protein after phosphatase treatment, indicating that the Ser308 antibody specifically detects the Ser308-phosphorylated form of BRCA1 (Fig. 3B). We further explored the phosphorylation of Ser308 in transition from the G2 to the M phase under conditions of DNA damage. The cell cycle of MCF7 cells was synchronized at the G2 phase by a double-thymidine block followed by roscovitine treatment (see "Experimental Procedures"). Briefly, S phase cells synchronized by double-thymidine block treatment were washed with phosphate-buffered saline and incubated with fresh medium containing 20 nm roscovitine for 12 h to arrest at the G2 phase. Rapid assessment of the progression of cells from the G2 to the M phase is difficult because both G2 and cells undergoing mitosis contain a 4 n DNA content and thus are not distinguishable from one another by standard propidium iodide staining and flow cytometry. Because histone H3 is phosphorylated exclusively during mitosis, an antibody that specifically recognizes the phosphorylated form of histone H3 can be used to identify the cells in mitosis and thus distinguish them from the G2 cells by flow cytometry (52Juan G. Traganos F. James W. Ray J. Roberge M. Sauve D. Anderson H. Darzynkiewicz Z. Cytometry. 1998; 32: 71-77Crossref PubMed Scopus (220) Google Scholar, 53Walter A.O. Seghezzi W. Korver W. Sheung J. Lees E. Oncogene. 2000; 19: 4906-4916Crossref PubMed Scopus (230) Google Scholar). Co-staining of cells with propidium iodide to assess DNA content and an anti-phosphohistone H3 antibody demonstrates that the cells in mitosis can be distinguished from G2 cells in the 4 n population of cells. We used anti-phospho-histone H3 antibody to quantify the mitosis population (Fig. 3C). We found that unsynchronized MCF7 cells contained 1.64% of cells in mitosis and that this number was decreased when cells were arrested at the G2 phase (0.24%). As predicted, the numbers of cells in mitosis increased when cells were released from G2 arrest (2.99%). Using these protocols to s
Referência(s)