M Phase-specific Phosphorylation of BRCA2 by Polo-like Kinase 1 Correlates with the Dissociation of the BRCA2-P/CAF Complex*

BRCA2 is a breast tumor susceptibility gene encoding a 390-kDa protein with functions in maintaining genomic stability and cell cycle progression. Evidence has been accumulated to support the concept that BRCA2 has a critical role in homologous recombination of DNA double-stranded breaks by interacting with RAD51. In addition, BRCA2 may have chromatin modifying activity through interaction with a histone acetyltransferase protein, p300/CBP-associated factor (P/CAF). To explore how the functions of BRCA2 may be regulated, the post-translational modifications of BRCA2 throughout the cell cycle were examined. We found that BRCA2 is hyperphosphorylated specifically in M phase and becomes dephosphorylated as cells exit M phase and enter interphase. This specific phosphorylation of BRCA2 was not observed in cells treated with DNA-damaging agents. Systematic mapping of the potential mitosis specific phosphorylation sites revealed the N-terminal 284 amino acids of BRCA2 (BR-N1) as the major region of phosphorylation and mass spectrometric analysis identified two phosphopeptides that contain “phosphorylation consensus motifs” for Polo-like kinase 1 (Plk1). Phosphorylation of BR-N1 with Plk1 recapitulated the electrophoretic mobility change as seen in BR-N1 isolated from M phase cells. Plk1 interacts with BRCA2 in vivo, and mutation of Ser193, Ser205/206, and Thr203/207 to Ala in BR-N1 abolished Plk1 phosphorylation, suggesting that BRCA2 is the substrate of Plk1. Furthermore, both the hyperphosphorylated and hypophosphorylated forms of BRCA2 bind to RAD51, whereas the M phase hyperphosphorylated form of BRCA2 no longer associates with the P/CAF, suggesting that the dissociation of P/CAF-BRCA2 complex is regulated by phosphorylation. Taken together, these results implicate a potential role of BRCA2 in modulating M phase progression.

[1]  J. Pines,et al.  Active cyclin B1–Cdk1 first appears on centrosomes in prophase , 2003, Nature Cell Biology.

[2]  Tom L. Blundell,et al.  Insights into DNA recombination from the structure of a RAD51–BRCA2 complex , 2002, Nature.

[3]  Wen-Hwa Lee,et al.  BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure. , 2002, Science.

[4]  F. Yarm.  Plk Phosphorylation Regulates the Microtubule-Stabilizing Protein TCTP , 2002, Molecular and Cellular Biology.

[5]  Kyung S. Lee,et al.  A Spindle Checkpoint Arrest and a Cytokinesis Failure by the Dominant-negative Polo-box Domain of Plk1 in U-2 OS Cells* 210 , 2002, The Journal of Biological Chemistry.

[6]  Hans Joenje,et al.  Biallelic Inactivation of BRCA2 in Fanconi Anemia , 2002, Science.

[7]  F. Uhlmann,et al.  Chromosome segregation: playing polo in prophase. , 2002, Developmental cell.

[8]  E. Nishida,et al.  Plk1 promotes nuclear translocation of human Cdc25C during prophase , 2002, EMBO reports.

[9]  M. Hung,et al.  Inhibition of cancer cell growth by BRCA2. , 2002, Cancer research.

[10]  R. Kingston,et al.  Cooperation between Complexes that Regulate Chromatin Structure and Transcription , 2002, Cell.

[11]  Y. Miki,et al.  Conserved domains in the chicken homologue of BRCA2 , 2002, Oncogene.

[12]  Ashok R Venkitaraman,et al.  Cancer Susceptibility and the Functions of BRCA1 and BRCA2 , 2002, Cell.

[13]  Yuan Yuan,et al.  Interaction with BRCA2 Suggests a Role for Filamin-1 (hsFLNa) in DNA Damage Response* , 2001, The Journal of Biological Chemistry.

[14]  A. Ashworth,et al.  Mutation in Brca2 stimulates error‐prone homology‐directed repair of DNA double‐strand breaks occurring between repeated sequences , 2001, The EMBO journal.

[15]  G. Iliakis,et al.  Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  E. Nigg,et al.  The mitotic roles of Polo-like kinase. , 2001, Journal of cell science.

[17]  D. Schild,et al.  Homologous recombinational repair of DNA ensures mammalian chromosome stability. , 2001, Mutation research.

[18]  A. Venkitaraman.  Chromosome stability, DNA recombination and the BRCA2 tumour suppressor. , 2001, Current opinion in cell biology.

[19]  D. Schild,et al.  Mutants of the Five Rad51 Paralogs Recombinational Repair in Knockout Chromosome Instability and Defective , 2022 .

[20]  W. Lee,et al.  The retinoblastoma gene: a prototypic and multifunctional tumor suppressor. , 2001, Experimental cell research.

[21]  E. Nishida,et al.  Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase , 2001, Nature.

[22]  K. Khanna,et al.  DNA double-strand breaks: signaling, repair and the cancer connection , 2001, Nature Genetics.

[23]  M. Jasin,et al.  BRCA2 is required for homology-directed repair of chromosomal breaks. , 2001, Molecular cell.

[24]  Y. Nakatani.  Histone acetylases—versatile players , 2001, Genes to cells : devoted to molecular & cellular mechanisms.

[25]  R. Shiekhattar,et al.  A Human BRCA2 Complex Containing a Structural DNA Binding Component Influences Cell Cycle Progression , 2001, Cell.

[26]  Yuan Yuan,et al.  Inhibition of breast and brain cancer cell growth by BCCIPα, an evolutionarily conserved nuclear protein that interacts with BRCA2 , 2001, Oncogene.

[27]  Wen-Hwa Lee,et al.  Lessons learned from BRCA1 and BRCA2 , 2000, Oncogene.

[28]  S. Elledge,et al.  Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress. , 2000, Genes & development.

[29]  T. Kouzarides,et al.  The BRCA2 activation domain associates with and is phosphorylated by a cellular protein kinase , 2000, Oncogene.

[30]  S. West,et al.  Gross chromosomal rearrangements and genetic exchange between nonhomologous chromosomes following BRCA2 inactivation. , 2000, Genes & development.

[31]  D. J. Clarke,et al.  Checkpoints controlling mitosis , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[32]  T. Katagiri,et al.  Potential role of BRCA2 in a mitotic checkpoint after phosphorylation by hBUBR1. , 2000, Cancer research.

[33]  Jong-Soo Lee,et al.  hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response , 2000, Nature.

[34]  S. Kim,et al.  Constitutive activation of cyclin B1-associated cdc2 kinase overrides p53-mediated G2-M arrest. , 2000, Cancer research.

[35]  Phang-lang Chen,et al.  Expression of BRC Repeats in Breast Cancer Cells Disrupts the BRCA2-Rad51 Complex and Leads to Radiation Hypersensitivity and Loss of G2/M Checkpoint Control* , 1999, The Journal of Biological Chemistry.

[36]  S. Elledge,et al.  Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. , 1999, Science.

[37]  A. Ashworth,et al.  Absence of Brca2 causes genome instability by chromosome breakage and loss associated with centrosome amplification , 1999, Current Biology.

[38]  C. Wang,et al.  Association of BRCA1 with the hRad50-hMre11-p95 complex and the DNA damage response. , 1999, Science.

[39]  A. Ashworth,et al.  Interaction between the Product of the Breast Cancer Susceptibility Gene BRCA2 and DSS1, a Protein Functionally Conserved from Yeast to Mammals , 1999, Molecular and Cellular Biology.

[40]  B. Ponder,et al.  Mitotic checkpoint inactivation fosters transformation in cells lacking the breast cancer susceptibility gene, Brca2. , 1999, Molecular cell.

[41]  D. Glover,et al.  Polo-like kinases: a team that plays throughout mitosis. , 1998, Genes & development.

[42]  E. Nigg.  Polo-like kinases: positive regulators of cell division from start to finish. , 1998, Current opinion in cell biology.

[43]  T. Kouzarides,et al.  BRCA2 associates with acetyltransferase activity when bound to P/CAF , 1998, Oncogene.

[44]  J. Lamerdin,et al.  XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages. , 1998, Molecular cell.

[45]  Y. Chen,et al.  The BRC repeats in BRCA2 are critical for RAD51 binding and resistance to methyl methanesulfonate treatment. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[46]  A. Hinnebusch,et al.  Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis. , 1998, Analytical chemistry.

[47]  A. Wallgren,et al.  A large deletion disrupts the exon 3 transcription activation domain of the BRCA2 gene in a breast/ovarian cancer family. , 1998, Cancer research.

[48]  B. Ponder,et al.  Involvement of Brca2 in DNA repair. , 1998, Molecular cell.

[49]  P. Bartel,et al.  RAD51 Interacts with the Evolutionarily Conserved BRC Motifs in the Human Breast Cancer Susceptibility Gene brca2 * , 1997, The Journal of Biological Chemistry.

[50]  A. Ashworth,et al.  Nuclear location and cell cycle regulation of the BRCA2 protein. , 1997, Cancer research.

[51]  A. Ashworth,et al.  Tumorigenesis and a DNA repair defect in mice with a truncating Brca2 mutation , 1997, Nature Genetics.

[52]  T. Mak,et al.  Brca2 is required for embryonic cellular proliferation in the mouse. , 1997, Genes & development.

[53]  T. Ludwig,et al.  Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos. , 1997, Genes & development.

[54]  Luke Hughes-Davies,et al.  Transcriptional activation functions in BRCA2 , 1997, Nature.

[55]  G. Eichele,et al.  Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2 , 1997, Nature.

[56]  A. Berchuck,et al.  Cell cycle control of BRCA2. , 1996, Cancer research.

[57]  M. Solomon,et al.  A Predictive Scale for Evaluating Cyclin-dependent Kinase Substrates , 1996, The Journal of Biological Chemistry.

[58]  B. Howard,et al.  A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A , 1996, Nature.

[59]  J. Rommens,et al.  The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds , 1996, Nature Genetics.

[60]  D. Bentley,et al.  Identification of the breast cancer susceptibility gene BRCA2 , 1995, Nature.

[61]  R. Erikson,et al.  Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1 , 1995, Molecular and cellular biology.

[62]  J. Gudmundsson,et al.  Different tumor types from BRCA2 carriers show wild-type chromosome deletions on 13q12-q13. , 1995, Cancer research.

[63]  D. Easton,et al.  Consistent loss of the wild type allele in breast cancers from a family linked to the BRCA2 gene on chromosome 13q12-13. , 1995, Oncogene.

[64]  P. Sung.  Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. , 1994, Science.

[65]  E. Nigg,et al.  Targets of cyclin-dependent protein kinases. , 1993, Current opinion in cell biology.

[66]  J. Labbé,et al.  In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase , 1990, Cell.

[67]  Phang-lang Chen,et al.  Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation , 1989, Cell.

[68]  P. Chambon,et al.  The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer , 1988, Cell.

[69]  Erich A. Nigg,et al.  Cell division: Mitotic kinases as regulators of cell division and its checkpoints , 2001, Nature Reviews Molecular Cell Biology.