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A Conserved Phosphorylation Site within the Forkhead Domain of FoxM1B Is Required for Its Activation by Cyclin-CDK1

2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês

10.1074/jbc.m109.007997

ISSN

1083-351X

Autores

Yi‐Ju Chen, Carmen Dominguez‐Brauer, Zebin Wang, John M. Asara, Robert H. Costa, Angela L. Tyner, Lester F. Lau, Pradip Raychaudhuri,

Tópico(s)

Mechanisms of cancer metastasis

Resumo

The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G1/S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G2/M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G2/M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1. The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G1/S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G2/M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G2/M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1. Transitions of the eukaryotic cell cycle are orchestrated by multiple protein kinases and by the transcriptional control of cell cycle regulators (1Nigg E.A. Nat. Rev. Mol. 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In addition, the activity of other mitotic kinases such as Polo-like kinase 1 (PLK1) 3The abbreviations used are: Plk1Polo-like kinase 1CREBcAMP-response element-binding proteinChIPchromatin immunoprecipitationCBPCREB-binding proteinMSmass spectrometryRTreverse transcriptaseWTwild typeKDkinase-deadpCMVplasmid containing cytomegalovirus promoter. must be maintained for proper mitotic progression (2Barr F.A. Silljé H.H. Nigg E.A. Nat. Rev. Mol. Cell Biol. 2004; 5: 429-440Crossref PubMed Scopus (913) Google Scholar, 5Takaki T. Trenz K. Costanzo V. Petronczki M. Curr. Opin. Cell Biol. 2008; 20: 650-660Crossref PubMed Scopus (144) Google Scholar, 6van Vugt M.A. Medema R.H. Oncogene. 2005; 24: 2844-2859Crossref PubMed Scopus (240) Google Scholar). Previous studies demonstrated that the polo-box domain in PLK1 acts as a phosphopeptide-binding domain and targets PLK1 to its substrates that have been "prime" or phosphorylated by CDK1 (7Elia A.E. Cantley L.C. Yaffe M.B. Science. 2003; 299: 1228-1231Crossref PubMed Scopus (577) Google Scholar). The polo-box domain-mediated PLK1 recruitment is responsible for both temporal and spatial regulation of PLK1 substrates (2Barr F.A. Silljé H.H. Nigg E.A. Nat. Rev. Mol. Cell Biol. 2004; 5: 429-440Crossref PubMed Scopus (913) Google Scholar, 7Elia A.E. Cantley L.C. Yaffe M.B. Science. 2003; 299: 1228-1231Crossref PubMed Scopus (577) Google Scholar). Polo-like kinase 1 cAMP-response element-binding protein chromatin immunoprecipitation CREB-binding protein mass spectrometry reverse transcriptase wild type kinase-dead plasmid containing cytomegalovirus promoter. The mammalian Forkhead box (Fox) proteins belong to a large family of transcription factors consisting of more than 50 proteins that share homology in the winged helix DNA-binding domain (8Clark K.L. Halay E.D. Lai E. Burley S.K. Nature. 1993; 364: 412-420Crossref PubMed Scopus (1098) Google Scholar, 9Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar). Within this extensive family, FoxM1 is a proliferation specific transcription factor (10Korver W. Roose J. Clevers H. Nucleic Acids Res. 1997; 25: 1715-1719Crossref PubMed Scopus (212) Google Scholar, 11Lüscher-Firzlaff J.M. Westendorf J.M. Zwicker J. Burkhardt H. Henriksson M. Müller R. Pirollet F. Lüscher B. Oncogene. 1999; 18: 5620-5630Crossref PubMed Scopus (99) Google Scholar, 12Yao K.M. Sha M. Lu Z. Wong G.G. J. Biol. Chem. 1997; 272: 19827-19836Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Ye H. Kelly T.F. Samadani U. Lim L. Rubio S. Overdier D.G. Roebuck K.A. Costa R.H. Mol. Cell. Biol. 1997; 17: 1626-1641Crossref PubMed Scopus (315) Google Scholar, 14Costa R.H. Nat. Cell Biol. 2005; 7: 108-110Crossref PubMed Scopus (160) Google Scholar). Expression of Foxm1 is detected only in proliferating cells and is extinguished when cells undergo terminal differentiation and exit the cell cycle (10Korver W. Roose J. Clevers H. Nucleic Acids Res. 1997; 25: 1715-1719Crossref PubMed Scopus (212) Google Scholar, 12Yao K.M. Sha M. Lu Z. Wong G.G. J. Biol. Chem. 1997; 272: 19827-19836Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Ye H. Kelly T.F. Samadani U. Lim L. Rubio S. Overdier D.G. Roebuck K.A. Costa R.H. Mol. Cell. Biol. 1997; 17: 1626-1641Crossref PubMed Scopus (315) Google Scholar). Transcription of the Foxm1 locus results in three distinct splice variants that are almost identical in sequence but differ by the addition of two small exons: Foxm1b (HFH-11B) contains no additional exons, whereas the Foxm1c (Trident, WIN, or MPP2) and Foxm1a (HFH-11A) isoforms contain one and two additional exons, respectively (11Lüscher-Firzlaff J.M. Westendorf J.M. Zwicker J. Burkhardt H. Henriksson M. Müller R. Pirollet F. Lüscher B. Oncogene. 1999; 18: 5620-5630Crossref PubMed Scopus (99) Google Scholar, 12Yao K.M. Sha M. Lu Z. Wong G.G. J. Biol. Chem. 1997; 272: 19827-19836Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Ye H. Kelly T.F. Samadani U. Lim L. Rubio S. Overdier D.G. Roebuck K.A. Costa R.H. Mol. Cell. Biol. 1997; 17: 1626-1641Crossref PubMed Scopus (315) Google Scholar, 15Korver W. Roose J. Heinen K. Weghuis D.O. de Bruijn D. van Kessel A.G. Clevers H. Genomics. 1997; 46: 435-442Crossref PubMed Scopus (87) Google Scholar). Of these three variants, Foxm1b and Foxm1c are transcriptionally active (13Ye H. Kelly T.F. Samadani U. Lim L. Rubio S. Overdier D.G. Roebuck K.A. Costa R.H. Mol. Cell. Biol. 1997; 17: 1626-1641Crossref PubMed Scopus (315) Google Scholar, 16Wierstra I. Alves J. Biol. Chem. 2006; 387: 963-976Crossref PubMed Scopus (36) Google Scholar). Previous studies demonstrated that FoxM1 orchestrates the transcription of genes that are essential for cell cycle progression and cell proliferation. During G1/S progression, FoxM1 is required for the transcriptional activation of JNK1, KIS, SKP2, and CKS1 (17Petrovic V. Costa R.H. Lau L.F. Raychaudhuri P. Tyner A.L. J. Biol. Chem. 2008; 283: 453-460Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 18Wang I.C. Chen Y.J. Hughes D.E. Ackerson T. Major M.L. Kalinichenko V.V. Costa R.H. Raychaudhuri P. Tyner A.L. Lau L.F. J. Biol. Chem. 2008; 283: 20770-20778Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (503) Google Scholar). FoxM1 also controls transcription of a subset of G2/M-specific genes, which are essential regulators of mitosis and chromosomal segregation, such as cyclin B, Cdc25B, Aurora B, PLK1, Survivin, CENP-A, CENP-B, and CENP-F (14Costa R.H. Nat. Cell Biol. 2005; 7: 108-110Crossref PubMed Scopus (160) Google Scholar, 19Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (503) Google Scholar, 20Laoukili J. Kooistra M.R. Brás A. Kauw J. Kerkhoven R.M. Morrison A. Clevers H. Medema R.H. Nat. Cell Biol. 2005; 7: 126-136Crossref PubMed Scopus (646) Google Scholar, 21Wonsey D.R. Follettie M.T. Cancer Res. 2005; 65: 5181-5189Crossref PubMed Scopus (285) Google Scholar). Silencing of FoxM1 expression using small interfering RNA results in diminished S-phase cell population, G2/M arrest, chromosome missegregation, and polyploidization (18Wang I.C. Chen Y.J. Hughes D.E. Ackerson T. Major M.L. Kalinichenko V.V. Costa R.H. Raychaudhuri P. Tyner A.L. Lau L.F. J. Biol. Chem. 2008; 283: 20770-20778Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (503) Google Scholar, 20Laoukili J. Kooistra M.R. Brás A. Kauw J. Kerkhoven R.M. Morrison A. Clevers H. Medema R.H. Nat. Cell Biol. 2005; 7: 126-136Crossref PubMed Scopus (646) Google Scholar, 21Wonsey D.R. Follettie M.T. Cancer Res. 2005; 65: 5181-5189Crossref PubMed Scopus (285) Google Scholar). Foxm1−/−-deficient mouse embryonic fibroblasts display not only polyploidy genotype but also premature senescence, suggesting that Foxm1 is required for the maintenance of chromosomal integrity. Consistent with its important role in cell cycle progression, overexpressed Foxm1b protein has been demonstrated to promote tumor cell proliferation and progression in different mouse models (22Kalin T.V. Wang I.C. Ackerson T.J. Major M.L. Detrisac C.J. Kalinichenko V.V. Lyubimov A. Costa R.H. Cancer Res. 2006; 66: 1712-1720Crossref PubMed Scopus (244) Google Scholar, 23Kim I.M. Ackerson T. Ramakrishna S. Tretiakova M. Wang I.C. Kalin T.V. Major M.L. Gusarova G.A. Yoder H.M. Costa R.H. Kalinichenko V.V. Cancer Res. 2006; 66: 2153-2161Crossref PubMed Scopus (290) Google Scholar, 24Kalinichenko V.V. Major M.L. Wang X. Petrovic V. Kuechle J. Yoder H.M. Dennewitz M.B. Shin B. Datta A. Raychaudhuri P. Costa R.H. Genes Dev. 2004; 18: 830-850Crossref PubMed Scopus (331) Google Scholar, 25Yoshida Y. Wang I.C. Yoder H.M. Davidson N.O. Costa R.H. Gastroenterology. 2007; 132: 1420-1431Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The association between the elevated expression level of FoxM1 and tumorigenesis, as well as unfavorable prognosis, has also been observed in a number of human malignancies, including hepatocellular carcinoma, prostate cancer, lung cancer, colorectal cancer, breast cancer, and gastric cancer (13Ye H. Kelly T.F. Samadani U. Lim L. Rubio S. Overdier D.G. Roebuck K.A. Costa R.H. Mol. Cell. Biol. 1997; 17: 1626-1641Crossref PubMed Scopus (315) Google Scholar, 22Kalin T.V. Wang I.C. Ackerson T.J. 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Those observations implicated the important role of FoxM1b as a transcription factor in tumorigenesis. Thus, exploring how the function of FoxM1b is regulated in the cell might have significant impact on the design of anti-cancer therapeutics. Expression of the FoxM1b protein is barely detectable in quiescent cells. During re-entry into the cell cycle, FoxM1b is expressed at late G1/early S phase and sustained throughout G2 phase and mitosis (10Korver W. Roose J. Clevers H. Nucleic Acids Res. 1997; 25: 1715-1719Crossref PubMed Scopus (212) Google Scholar, 13Ye H. Kelly T.F. Samadani U. Lim L. Rubio S. Overdier D.G. Roebuck K.A. Costa R.H. Mol. Cell. Biol. 1997; 17: 1626-1641Crossref PubMed Scopus (315) Google Scholar). It has been shown that the transcriptional activity of FoxM1b is dependent upon the activation of the Ras-mitogen-activated protein kinase (MAPK) signaling pathway, which activates FoxM1b through cyclin-CDKs (29Major M.L. Lepe R. Costa R.H. Mol. Cell. Biol. 2004; 24: 2649-2661Crossref PubMed Scopus (221) Google Scholar). The C-terminal transcriptional activation domain of FoxM1b contains a cyclin-binding motif (LXL motif), and both CDK1 and CDK2 were shown to associate with FoxM1b. Mutation of the cyclin-binding motif eliminates the binding of both CDK1 and CDK2 (29Major M.L. Lepe R. Costa R.H. Mol. Cell. Biol. 2004; 24: 2649-2661Crossref PubMed Scopus (221) Google Scholar). Further analysis reveals that the Leu-641 residue within an LXL motif is required for the recruitment of the cyclin-CDK complex, and the Thr-596 residue is a critical CDK1 phosphorylation site within the activation domain of FoxM1b. CDK-dependent phosphorylation stimulates the FoxM1b transcriptional activity, which correlates with binding to the CREB-binding protein (CBP), the transcriptional coactivator. Mutation of Thr-596 abolishes binding to CBP and inhibits FoxM1b transcriptional activity (29Major M.L. Lepe R. Costa R.H. Mol. Cell. Biol. 2004; 24: 2649-2661Crossref PubMed Scopus (221) Google Scholar). We and others have characterized an autorepression domain in the N-terminal region between residues 1 and 232 of FoxM1b (16Wierstra I. Alves J. Biol. Chem. 2006; 387: 963-976Crossref PubMed Scopus (36) Google Scholar, 30Park H.J. Wang Z. Costa R.H. Tyner A. Lau L.F. Raychaudhuri P. Oncogene. 2008; 27: 1696-1704Crossref PubMed Scopus (64) Google Scholar, 31Laoukili J. Alvarez M. Meijer L.A. Stahl M. Mohammed S. Kleij L. Heck A.J. Medema R.H. Mol. Cell. Biol. 2008; 28: 3076-3087Crossref PubMed Scopus (115) Google Scholar). Deletion of the N-terminal repression domain renders FoxM1b constitutively active and independent of cyclin-CDK. The N-terminal repression domain inhibits FoxM1b transcriptional activity presumably by binding to the C-terminal activation domain. It is postulated that cyclin-CDK-mediated phosphorylation activates FoxM1b by disrupting the interaction of the N-terminal repression domain with the C-terminal activation domain (16Wierstra I. Alves J. Biol. Chem. 2006; 387: 963-976Crossref PubMed Scopus (36) Google Scholar, 30Park H.J. Wang Z. Costa R.H. Tyner A. Lau L.F. Raychaudhuri P. Oncogene. 2008; 27: 1696-1704Crossref PubMed Scopus (64) Google Scholar, 31Laoukili J. Alvarez M. Meijer L.A. Stahl M. Mohammed S. Kleij L. Heck A.J. Medema R.H. Mol. Cell. Biol. 2008; 28: 3076-3087Crossref PubMed Scopus (115) Google Scholar). These observations indicated a critical role of cyclin-CDK phosphorylation in the cell cycle-regulated activation of FoxM1b. However, the mechanism of temporal activation of a subset genes by FoxM1b at the G1/S and G2/M phases remains unknown. In this study, we investigated the phosphorylation pattern of the endogenous FoxM1 protein during progression through the cell cycle. We show that the endogenous FoxM1 protein maintains a hypophosphorylation state at the G1/S boundary, exhibits increased phosphorylation status from S phase to G2/M transition, and reaches its maximal phosphorylation status at mitosis. FoxM1 is dephosphorylated upon the completion of mitosis. The transcriptional activity of FoxM1b increases through the cell cycle and coincides with the phosphorylation status of FoxM1. Consistent with a recent study (32Fu Z. Malureanu L. Huang J. Wang W. Li H. van Deursen J.M. Tindal D.J. Chen J. Nat. Cell Biol. 2008; 10: 1076-1082Crossref PubMed Scopus (254) Google Scholar), we observed that PLK1 interacts with FoxM1 in M phase, phosphorylates FoxM1b on Ser-724 at the C terminus, and stimulates its transcriptional activity. Furthermore, we identified a novel, conserved phosphorylation site at Ser-251 within the forkhead box DNA binding domain of hyperphosphorylated FoxM1b using tandem mass spectrometry (liquid chromatography-MS/MS). Mutation of Ser-251 to alanine interferes with neither the subcellular localization nor the DNA binding ability of FoxM1b. However, the S251A mutant exhibits deficiency in undergoing phosphorylation by CDK1, remains predominantly hypophosphorylated and shows significantly diminished transcriptional activity. Interestingly, the transcriptional activity of the S251A mutant protein fails to be activated by CDK1, but remains responsive to PLK1 stimulation. Our results demonstrate that the transcriptional activity of FoxM1b is temporally regulated through phosphorylation modifications by multiple protein kinases during progression of the cell cycle. Moreover, we show that phosphorylation of Ser-251 in the forkhead box domain of FoxM1b is a critical event for activation of its transcriptional activity at the G2/M phases by CDK1. Human osteosarcoma U2OS cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone Laboratories Inc.) and 100 units of penicillin/streptomycin at 37 °C with 5% CO2. Cells were transfected with plasmid DNA using LipofectamineTM 2000 (Invitrogen) in serum-free tissue culture medium following the manufacturer's protocol. Four hours after transfection, cells were fed with complete Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cell cycle synchronization was performed by double thymidine block. U2OS cells were arrested at G1/S transition for 17 h with 2.5 mm thymidine (Sigma) with a 7-h release interval. Arrested cells were released into fresh medium to follow cell cycle progression. To analyze cell cycle progression, flow cytometry analyses were carried out using a Beckman Coulter EPICS Elite ESP apparatus, and data were analyzed by FCSPress software (Ray Hicks, FCSPress, Cambridge, UK). To isolate mitotic cells, we used the mitotic shake-off method. Briefly, U2OS cells were arrested by 300 ng/ml of nocodazole (Sigma) treatment for 16 h. The partially detached nocodazole-arrested cells were shaken-off and washed three times with phosphate-buffered saline before being plated to allow re-entry into the cell cycle. The pCMV T7-FoxM1b expression vector, 6×FoxM1 TATA luciferase reporter plasmid, and −749 bp Aurora B promoter-luciferase construct are described previously (19Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (503) Google Scholar, 29Major M.L. Lepe R. Costa R.H. Mol. Cell. Biol. 2004; 24: 2649-2661Crossref PubMed Scopus (221) Google Scholar). Human wild type and K82R kinase-dead PLK1 plasmids were kindly provided by David V. Hansen (Stanford University). Site-directed mutagenesis was performed by using the QuikChange® site-directed mutagenesis kit (Stratagene), and the FoxM1b point mutations were verified by sequencing (University of Illinois, Chicago, DNA Sequencing Facility). The following antibodies were used for immunoblotting and immunoprecipitation: mouse monoclonal T7 tag (Novagen, Madison, WI); mouse monoclonal Plk1 (F8), mouse monoclonal CBP (C1), normal mouse IgG, and normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal Plk1 (pT210), mouse monoclonal cyclin B1 (GNS-11) (BD Biosciences); mouse monoclonal MPM2, rabbit anti-phosphohistone H3 (Ser-10) (Upstate, Lake Placid, NY); rabbit phospho-Rb (Ser-795) (Cell Signaling Technology, Danvers, MA); mouse anti-α-tubulin (T6074), mouse anti-β-actin (A5441) (Sigma); and rabbit polyclonal anti-FoxM1 antibody was generated as described previously (19Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (503) Google Scholar). All procedures including lyses, clarification, and immunoprecipitation were performed at 4 °C. Whole cell extracts were prepared using the Nonidet P-40 lysis buffer as described (29Major M.L. Lepe R. Costa R.H. Mol. Cell. Biol. 2004; 24: 2649-2661Crossref PubMed Scopus (221) Google Scholar). For lysis, cells were incubated for 20 min in ice-cold Nonidet P-40 lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 5 mm EDTA, 5 mm EGTA, 1% Nonidet P-40, 5% glycerol, 10 mm β-glycerolphosphate, and 1 mm Na3VO4). Lysates were clarified by centrifugation at 13,000 × g for 20 min, and supernatants were collected. Immunoprecipitations were carried out with 500 μg of total protein extract from each sample. Cell lysates were first pre-cleared by incubation with 20 μl of protein A-Sepharose beads (50% slurry) (Amersham Biosciences) for 1 h. The supernatants were then incubated with 2 μg of antibodies and protein A-Sepharose beads overnight with gentle rocking at 4 °C. The beads were washed three times with lysis buffer, and bound proteins were eluted from the beads in SDS sample buffer. Coimmunoprecipitated proteins were then detected by Western blot analysis. For the dephosphorylation assay using λ-phosphatase, FoxM1 immunoprecipitates were washed twice in lysis buffer in the absence of phosphatase inhibitors and resuspended in phosphatase buffer (50 mm Tris, pH 7.5, 100 mm NaCl, 0.1 mm EGTA, 2 mm dithiothreitol, and 0.01% Brij 35). Parallel samples were incubated with or without 100 units of λ-phosphatase (New England Biolabs, Ipswich, MA) at 30 °C for 30 min. U2OS cells were plated in a 24-well plate at a density of 105 cells/well. Cells were transfected using 1 μl of Lipofectamine 2000 with 0.1 μg of either pCMV FoxM1b expression construct or pCMV empty vector, 0.2 μg of either 6×FoxM1 TATA luciferase reporter or the −749 bp Aurora B promoter luciferase reporter, and 1 ng of pCMV-Renilla luciferase, which serve as an internal control. Twenty-six hours after transfection, cells were harvested and extracts were prepared for dual luciferase assay (Promega, Madison, WI) as described previously. The luciferase levels were normalized to Renilla luciferase activity (19Wang I.C. Chen Y.J. Hughes D. Petrovic V. Major M.L. Park H.J. Tan Y. Ackerson T. Costa R.H. Mol. Cell. Biol. 2005; 25: 10875-10894Crossref PubMed Scopus (503) Google Scholar, 29Major M.L. Lepe R. Costa R.H. Mol. Cell. Biol. 2004; 24: 2649-2661Crossref PubMed Scopus (221) Google Scholar). All experiments were performed in triplicate, and repeated twice. A vector expressing T7-FoxM1b was coexpressed with either wild type or kinase-dead PLK1 plasmid in U2OS cells in five 15-cm dishes. T7-FoxM1b was immunoprecipitated by T7 tag antibody-conjugated beads (Novagen). The immunoprecipitated proteins were resolved in 7% SDS-PAGE. The hyper- and hypophosphorylated forms of the FoxM1b protein were visualized by Coomassie Blue staining. The gel bands were excised, washed, reduced with 10 mm dithiothreitol (Sigma), and alkylated with 55 mm iodoacetamide (Sigma) followed by digestion with modified sequencing grade trypsin (Promega). The extracted tryptic peptides were analyzed by data-dependent reversed-phase microcapillary liquid chromatography-tandem mass spectrometry using a LTQ linear ion trap mass spectrometer (ThermoFisher Scientific). MS/MS spectra were searched against the concatenated target and decoy (reversed) Swiss-Prot protein data base using Sequest (Proteomics Browser, ThermoFisher Scientific) with differential modifications for Ser/Thr/Tyr phosphorylation (+79.97) and the sample processing artifacts for Met oxidation (+15.99) and Cys alkylation (+57.02). Phosphorylated and unphosphorylated peptide sequences were identified if they initially passed the following Sequest scoring thresholds: 1+ ions, Xcorr ≥ 2.0, Sf ≥ 0.4, p ≥ 5; 2+ ions, Xcorr ≥ 2.0, Sf ≥ 0.4, p ≥ 5; 3+ ions, Xcorr ≥ 2.60, Sf ≥ 0.4, p ≥ 5 against the target protein data base. Passing MS/MS spectra that represented the phosphorylated forms in the FoxM1b sequence were manually inspected to be sure that all b- and y-fragment ions aligned with the assigned sequence and modification sites. Determination of the exact sites of phosphorylation was aided using GraphMod software (Proteomics Browser). For the in vitro kinase assays, 500 μg of protein lysates from U2OS cells transfected with T7-FoxM1b wild type or S251A mutant plasmids were coimmunoprecipitated overnight with T7 tag antibody and protein A-Sepharose beads. The immunoprecipitates were washed three times with lysis buffer and twice with kinase buffer. To start the kinase reaction, 20 μl of kinase buffer supplemented with 50 μm ATP, 5 μCi of [γ-32P]ATP, and 5 μg of recombinant cyclin B1-CDK1 proteins was added to the beads, and samples were incubated at 30 °C for 30 min. Reactions were terminated by adding an equal volume of 2× Laemmli sample buffer (Bio-Rad). The samples were separated on SDS-PAGE and analyzed by Coomassie Blue staining and then dried prior to autoradiography. T7-FoxM1b wild type and S251A mutant constructs were subcloned from pCMV T7-FoxM1b and pCMV T7-FoxM1bS251A vectors into a pLPCX-puro vector, respectively. Retrovirus stocks were prepared by transfecting 24 μg of the vectors into the Amphotropic 293 cells in a 15-cm plate using the calcium phosphate method. Viral supernatants were collected at 48 and 72 h post-transfection, filtered, concentrated, and stored at −80 °C until use (pLPCX-puro vector and Amphotropic 293 cells were kindly provided by Chia-Chen Chen, University of Illinois, Chicago). U2OS cells were transduced by concentrated viral supernatant in Dulbecco's modified Eagle's medium and 10 μg/ml Polybrene. Forty-eight hours after transduction, cells were selected with 1 μg/ml puromycin. A modified McKay assay was performed with the double-stranded FoxM1 binding site oligonucleotide (from 6×FoxM1 TATA-luciferase reporter gene) (29Major M.L. Lepe R. Costa R.H. Mol. Cell. Biol. 2004; 24: 2649-2661Crossref PubMed Scopus (221) Google Scholar, 33Hoffman W.H. Biade S. Zilfou J.T. Chen J. Murphy M. J. Biol. Chem. 2002; 277: 3247-3257Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 34Lin T. Chao C. Saito S. Mazur S.J. Murphy M.E. Appella E. Xu Y. Nat. Cell Biol. 2005; 7: 165-171Crossref PubMed Scopus (705) Google Scholar). Briefly, U2OS cells, transfected with a vector expressing either T7-FoxM1b wild type or S251A mutant protein, were lysed in 1 ml of McKay binding buffer (10% glycerol, 5 mm EDTA, 20 mm Tris, pH 7.2, 100 mm NaCl, 0.1% Nonidet P-40 and protease inhibitors). Cell debris was removed from the supernatant by spinning down at 14,000 × g for 10 min at 4 °C, and protein concentrations were determined by the Bradford method with the Bio-Rad protein assay reagent (Bio-Rad). Three hundred micrograms of cell extract was pre-cleared by incubation with 20 μl of protein A-Sepharose beads (50% slurry) (Amersham Biosciences) for 40 min and then incubated with 500,000 cpm of 32P-labeled FoxM1 binding site oligonucleotide, 35 μl of T7 tag antibody-conjugated beads (Novagen), and 1 μg/μl of poly(dI/dC) overnight with gentle rocking at 4 °C. The beads were washed three times with the McKay washing buffer (2% glycerol, 5 mm EDTA, 20 mm Tris, pH 7.2, 100 mm NaCl, 0.1% Nonidet P-40 and protease inhibitors), and resuspended in 100 μl of TE buffer (10 mm Tris, pH 8.0, 1 mm EDTA, pH 8.0). Five micrograms of proteinase K were then added and samples were incubated for 30 min at 37 °C. The 32P-labeled FoxM1 binding site oligonucleotides were purified using the QIAquick Nucleotide Removal kit (Qiagen), eluted with 20 μl of ddH2O and resolved on 5% non-denaturing acrylamide gel. For competition experiments, a 100-fold excess of cold probe was preincubated with extract for 10 min prior to the binding reaction. After electrophoresis, the gel was dried and exposed to x-ray film. U2OS, U2OS-WT, and U2OS-251A cell lines were used to perform chromatin immunoprecipitation (ChIP) assay as described (39Hunter

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