The BRCA1 C-terminal (BRCT) domain has recently been implicated as BV-6

The BRCA1 C-terminal (BRCT) domain has recently been implicated as BV-6 a phospho-protein binding domain. data not only implicate BV-6 CtIP as a critical player in cell cycle checkpoint control but also provide molecular mechanisms by which BRCA1 controls multiple cell cycle transitions after DNA damage. DNA damage occurs frequently in the cell. It can be generated during normal DNA replication UV light and X-ray exposure oxidative base damages or after various chemical treatments. To maintain genomic integrity cells have developed elaborate DNA repair systems and various cell cycle checkpoints that ensure the repair of DNA lesions before cell cycle progression resumes. Two large protein kinases ATM and ATR are the critical upstream kinases that control the DNA damage-induced cell cycle checkpoints (1 20 Many nuclear proteins including BRCA1 are phosphorylated by ATM and ATR and participate BV-6 in these cell cycle checkpoint pathways (29). The breast cancer tumor suppressor gene BRCA1 encodes a 1 863 nuclear protein with C-terminal tandem BRCA1 C-terminal (BRCT) motifs. The BRCT domains are essential for the tumor suppressor function of BRCA1. The majority of clinically relevant BRCA1 mutations lead to truncated gene products that lack one or both BRCT domains. Missense mutations that disrupt the secondary structure of BRCA1 BRCT domains were also identified in early-onset breast and ovarian cancer patients. Genetically removing BRCA1 BRCT domains in mice results in increased tumor incidence (17). The molecular mechanism underlying the tumor suppression function of BRCA1 BRCT domains may be linked with DNA damage-induced cell cycle checkpoint controls (10 22 26 38 Recent biochemical studies demonstrated that the BRCA1 BRCT domains are a phospho-protein binding motif (18 19 38 These observations were recently confirmed by the structural analyses of BRCA1 BRCT domains in BV-6 complexes with phospho-peptides (2 6 23 30 However since the BRCA1 BRCT domains participate in multiple cell cycle checkpoints (10 22 33 it is still puzzling how BRCA1 controls these distinct cellular activities. CtIP was a protein originally identified as a binding partner of transcriptional suppressor CtBP (21). Subsequently CtIP was also shown to interact with BRCA1 BRCT domains by two-hybrid screening (14 31 39 Although CtIP is phosphorylated after DNA damage it is controversial whether or not DNA damage regulates the physical interaction between BRCA1 and CtIP (32). Moreover the role of CtIP in BRCA1-dependent cell cycle checkpoint control has not been studied. Here we show that CtIP is a phosphorylation-dependent binding partner of the BRCA1 BRCT domains. The cell cycle-regulated BRCA1/CtIP complex is required for the G2/M transition and Chk1 activation after DNA damage. MATERIALS AND METHODS Cell culture and antibodies. All cell lines were maintained in RPMI 1640 medium with 10% fetal calf serum at 37°C in 5% (vol/vol) CO2. To arrest cells at the G1/S boundary cells were treated with 2 μM thymidine for 19 h and then released for 10 h followed by Mouse monoclonal antibody to Keratin 7. The protein encoded by this gene is a member of the keratin gene family. The type IIcytokeratins consist of basic or neutral proteins which are arranged in pairs of heterotypic keratinchains coexpressed during differentiation of simple and stratified epithelial tissues. This type IIcytokeratin is specifically expressed in the simple epithelia lining the cavities of the internalorgans and in the gland ducts and blood vessels. The genes encoding the type II cytokeratinsare clustered in a region of chromosome 12q12-q13. Alternative splicing may result in severaltranscript variants; however, not all variants have been fully described. a second thymidine (2 μM) block for 18 h. Arrested cells were released into cell cycle by removal of drug and the addition of fresh cell culture medium. Cells were harvested at the indicated time points. Rabbit anti-BRCA1 (C23) antibody was a generous gift from David Livingston. Polyclonal anti-CtIP antibody was generated by immunizing a rabbit with glutathione is fivefold higher than that between BRCT and BACH1 suggesting that BRCA1/CtIP complex is less stable (unpublished data). This probably explains that BRCA1/CtIP complex is only transiently formed in G2 cells when CtIP protein level is also accumulated. On the contrary BRCA1/BACH1 complex is much more stable and exists in S-to-M-phase cells. The Ser327 site of CtIP is phosphorylated mainly in G2 phase which is distinct from the phosphorylation of BACH1 at Ser990. The phosphorylation of Ser327 is cell cycle regulated and also appears to correlate with the expression level of CtIP. Since the Ser327 site is a Ser-Pro site a preferred phosphorylation site by cyclin-dependent kinases it is possible that CtIP is phosphorylated by one or more of BV-6 the cyclin-dependent kinases in G2 phase. Although the Ser327 phosphorylation of CtIP is required for the G2/M transition checkpoint this phosphorylation event is not induced by DNA damage (data not shown). One hypothesis is that the phosphorylation of CtIP at Ser327 site may be required for its.