Int. J. Biol. Sci. 2008, 4 352 International Journal of Biological Sciences ISSN 1449-2288 www.biolsci.org 2008 4(6):352-361 © Ivyspring International Publisher. All rights reserved Research Paper A PP1-binding motif present in BRCA1 plays a role in its DNA repair function Young-Mi Yu1, Serena M. Pace1, Susan R. Allen1, Chu-Xia Deng2 and Lih-Ching Hsu1,3 1. Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Magee-Womens Research Institute, Pittsburgh, PA15213, USA 2. Genetics of Development and Diseases Branch, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA 3. School of Pharmacy, National Taiwan University College of Medicine, Taipei 10051, Taiwan, ROC Correspondence to: Dr. Lih-Ching Hsu, School of Pharmacy, National Taiwan University College of Medicine, 12F, No 1, Section 1, Jen-Ai Road, Taipei 10051, Taiwan. Phone: +886-2-2312-3456 ext. 88400; Fax: +886-2-2391-9098; E-mail: [email protected] Received: 2008.09.30; Accepted: 2008.10.04; Published: 2008.10.04 Protein phosphatase 1α (PP1α) regulates phosphorylation of BRCA1, which contains a PP1-binding motif 898KVTF901. Mutation of this motif greatly reduces the interaction between BRCA1 and PP1α. Here we show that mutation of the PP1-binding motif abolishes the ability of BRCA1 to enhance survival of Brca1-deficient mouse mammary tumor cells after DNA damage. The Rad51 focus formation and comet assays revealed that the DNA repair function of BRCA1 was impaired when the PP1-binding motif was mutated. Analysis of subnuclear localization of GFP-tagged BRCA1 demonstrated that mutation of the PP1-binding motif affected BRCA1 redistribution in response to DNA damage. BRCA1 is required for the formation of Rad51 subnuclear foci after DNA damage. Mutation of the PP1-binding motif in BRCA1 also affected recruitment of Rad51 to sites of DNA damage. Consistent with these findings, knockdown of PP1α in BRCA1-proficient cells by small interfering RNA also significantly reduced Rad51 focus formation induced by DNA damage. Further analysis indicated that mutation of the PP1-binding motif compromised BRCA1 activities in homologous recombination. Altogether, our data implicate that interaction with PP1α is important for BRCA1 function in DNA repair. Key words: BRCA1, protein phosphatase 1, DNA repair 1. Introduction BRCA1 is important for its function in G2/M check- point control [11] and DNA repair [12]. However, Mutations of the human breast and ovarian can- BRCA1 is primarily hypophosphorylated in mitosis cer susceptibility gene BRCA1 account for the majority and G0/G1 phases of the cell cycle [5,13], suggesting of familial breast and ovarian cancer cases. The BRCA1 that BRCA1 must undergo dephosphorylation during gene encodes a nuclear phosphoprotein of 1863 amino G2/M transition. Whether or not dephosphorylation acids (Mr ~220,000). Accumulated evidence indicates of BRCA1 is critical for its function remains unclear. that BRCA1 plays an important role in multiple cellu- Serine/threonine protein phosphatase 1 (PP1) lar functions including cell cycle checkpoint control, holoenzymes consist of catalytic subunits and regula- chromosome remodeling, transcriptional regulation, tory subunits which target the catalytic subunits to recombination, and DNA repair; therefore, is crucial distinct subcellular localizations. Together, the PP1 for maintaining genomic integrity and chromosomal complexes regulate diverse cellular functions includ- stability [1-3]. ing cell cycle progression, chromosome segregation, BRCA1 is expressed and phosphorylated in a cell cytokinesis, and cellular metabolism [14]. Recent cycle-dependent manner and is mainly phosphory- studies also indicate that PP1 is involved in check- lated in S phase [4,5]. BRCA1 is also phosphorylated point activation [15] and DNA repair [16], as well as by checkpoint kinases, including hCds1/Chk2, ATM, recovery from DNA damage checkpoint arrest [17]. and ATR, in response to DNA damage induced by We previously reported that the PP1α catalytic sub- ionizing radiation (IR), UV or chemicals. Phosphoryla- unit interacts with BRCA1 and dephosphorylates the tion of BRCA1 occurs at multiple serine residues, such protein at multiple serine residues that are phos- as S1524 and S1423 by ATM and ATR [6-9] and S988 phorylated by hCds1/Chk2, ATM and ATR [18,19]. by hCds1/Chk2 [10]. The phosphorylation state of Thus, PP1α, one of the three isoforms of the PP1 cata- Int. J. Biol. Sci. 2008, 4 353 lytic subunit (α, γ, and δ), may serve as a general USA). The mutant BRCA1 expression vectors, regulator for BRCA1 phosphorylation. pGFPBRCA1F901A and pGFPBRCA1DEL, were con- Proteins interacting with the PP1 catalytic sub- structed by replacing a 4.2 kb AflII-HindIII fragment in unit, including the majority of regulatory subunits and pGFPBRCA1 with the same fragments containing the several substrates, share a consensus PP1-binding mo- mutations from modified pBRCA1GFPF901A and tif (R/K)(V/I)xF, which binds to a hydrophobic pBRCA1GFPDEL with the BRCA1 stop codon. All groove on the surface of the PP1 catalytic subunit and plasmid clones were sequence-verified. serves as an anchor for the initial binding of these Cell culture and transfection proteins to PP1 [14]. Mutation of this consensus motif by substitution of phenylalanine with alanine or dele- A mouse mammary tumor cell line 780 (Brca1 tion of the motif greatly reduces the binding between exon 11-deleted), 293T and COS-7 cells were cultured these proteins and the PP1 catalytic subunit. We re- in DMEM supplemented with 2 mM L-glutamine and cently identified a PP1-binding motif 898KVTF901 in 10% FBS, and ES-2 cells were cultured in McCoy’s 5A human BRCA1. Mutation of this motif substantially medium supplemented with 1.5 mM L-glutamine and 10% FBS. Transfection was performed using Lipofec- reduces the interaction between BRCA1 and PP1α tamine 2000 (Invitrogen, Carlsbad, CA, USA) accord- [19]. ing to the manufacturer’s instructions. Cells were BRCA1 is implicated in the repair of DNA dou- harvested for analysis 24-48 h after transfection. Tar- ble-strand breaks (DSBs) by either homologous re- get-specific 20-25 nucleotide small interfering RNAs combination (HR) or non-homologous end-joining α (NHEJ) [1,2,20]. It has been reported that BRCA1 also (siRNAs) for human PP1 and control siRNA were participates in nucleotide-excision repair [21,22]. purchased from Santa Cruz (CA, USA) and siRNA Rad51, a homologue of bacterial RecA protein, is in- transfection was also performed using Lipofectamine volved in the repair of DSBs by HR, which is an er- 2000. ror-free DNA repair process. Expression of Rad51 is Analysis of cell cycle distribution by flow cytometry also cell cycle-dependent [23] and the protein forms Cells were harvested by trypsinization, fixed in nuclear foci in S phase [24]. BRCA2 has been shown to ice-cold methanol, and subjected to propidium iodide interact with Rad51 and regulate the assembly of staining and flow cytometric analysis as described Rad51 filaments at the sites of DNA repair. BRCA1 previously [32]. forms a complex with both Rad51 [25] and BRCA2 [26]. BRCA1 associates with Rad51 at subnuclear foci Western analysis [25]. After DNA damage, BRCA1 is phosphorylated, Cells were harvested by trypsinization and lysed relocalizes along with Rad51 to the damaged regions, in RIPA buffer supplemented with protease inhibitors. and displays large nuclear foci [27,28]. BRCA1 also Cell lysates containing ~100 μg of protein were sub- associates with the MRE11/RAD50/NBS1 complex, jected to 4-12% SDS-PAGE, followed by Western which participates in HR, NHEJ, and the DNA dam- analysis. Antibodies used were human BRCA1 anti- age response [28]. It has been demonstrated that body Ab-1 (MS110) (Calbiochem, San Diego, CA, hCds1/Chk2 phosphorylation of BRCA1 regulates the USA), mouse Brca1 M-20, PP1α C-19, Rad51 H-92 an- repair of DSBs by promoting error-free HR and sup- tibodies (Santa Cruz), and γ-tubulin antibody GTU-88 pressing error-prone NHEJ [12]. BRCA1 may also par- (Sigma, St Louis, MO, USA). Quantitative analysis was ticipate in an error-free subtype of NHEJ [29,30]. Thus, performed using Quantity One software (BioRad, BRCA1 is required for accurate DNA repair and Hercules, CA, USA). BRCA1-deficient cells are hypersensitive to DNA Cell survival assay damaging agents which induce DSBs or crosslinks of DNA strands. Here, we sought to test whether inter- Cell survival was measured by colony formation. action with PP1 was involved in the DNA repair func- One day after transfection, 500 or 1000 cells were tion of BRCA1 using Brca1∆11/∆11 mouse mammary seeded in T-25 flasks. The next day, cells were either 137 tumor cells [31]. left untreated (C) or exposed to 5 Gy of IR from a Cs source (Sheperd Mark I 30-1 Irradiator) at a dose rate 2. Materials and Methods of ~110 cGy/min. Two weeks after IR treatment, colo- Vector construction nies were fixed in cold methanol and stained with Giemsa. Colonies with at least 50 cells were then To construct pGFPBRCA1, a ~5.6 kb XhoI-HindIII scored. Relative cell survival was calculated as the BRCA1 cDNA fragment containing the human BRCA1 ratio of colony numbers in irradiated and untreated cDNA sequence (containing the stop codon) was sub- groups (IR/C). cloned into pGFP-C3 (Clontech, Mountain View, CA, Int. J. Biol. Sci. 2008, 4 354 Immunofluorescence staining for three weeks. Colonies were fixed and stained with Cells were seeded into 4-well or 8-well chamber Giemsa. XHATM-resistant colonies (at least 50 cells slides. The next day, cells were either left untreated or per colony) were quantified. The HR frequency was treated with 100 μM of 5-fluorodeoxyuridine (5-FUdR) calculated as the number of resistant colonies divided 5 or irradiated with 5 Gy of IR.