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.
Lin
Horng-Ru
HR
Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas 78245, USA.
Ting
Nicholas S Y
NS
Qin
Jun
J
Lee
Wen-Hwa
WH
D000123:Acetyltransferases / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D024682:BRCA2 Protein / Q000378:metabolism*; D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D050881:E1A-Associated p300 Protein; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D051548:Histone Acetyltransferases; D006801:Humans; D007399:Interphase; D008164:Luminescent Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008938:Mitosis; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D051135:Rad51 Recombinase; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation; D000097869:Polo-Like Kinase 1
D000123:Acetyltransferases; D024682:BRCA2 Protein; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D050881:E1A-Associated p300 Protein; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D051548:Histone Acetyltransferases; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D010744:Phosphoric Monoester Hydrolases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D051135:Rad51 Recombinase; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D000097869:Polo-Like Kinase 1; C494361:Ep300 protein, mouse; C495153:RAD51 protein, human; C495154:Rad51 protein, mouse