Ataxia Telangiectasia Mutated (ATM) Kinase and ATM and Rad3 Related Kinase Mediate Phosphorylation of Brca1 at Distinct and Overlapping Sites

Abstract

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologousrecombination, and Nbs1⅐Mre11⅐Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV)islargelyataxiatelangiectasiamutated(ATM)kinasedependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UVinduced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.Damage to DNA, which can occur from exogenous agents such as IR, 1 UV radiation, and certain chemotherapeutic drugs or from endogenously generated reactive oxygen species, poses a great threat to genomic stability. Cells respond to DNA damage by activating a complex DNA damage-response pathway that includes cell cycle arrest, transcriptional and posttranscriptional regulation of genes associated with repair, and under some circumstances, the triggering of programmed cell death. Biochemically, the DNA damage-response pathway provides a mechanism for transducing a signal from a sensor that recognizes the damage, through a transduction cascade, to a series of downstream effector molecules, which implement the appropriate response. At or close to the top of this pathway in mammalian cells are the members of the phosphoinositide kinase-related kinase (PIKK) family, which includes ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3 related). These proteins play important roles in signaling the presence of DNA damage, activating cell cycle checkpoints, and repairing DNA (1). ATM is homozygously mutated in the germline of patients with the n...

Interplay between ATM and ATR in the regulation of common fragile site stability

“…Recently it was shown that downregulation of the Chk1 kinase has a major effect on fragile site stability , hence the effect of ATM on the level of Chk1 phosphorylation is probably part of the mechanism by which ATM regulates fragile site expression. It is worth noting that in addition to Chk1, other shared targets of ATR and ATM (BRCA1, SMC1 and FANCD2) (Gatei et al, 2001;Kim et al, 2002;Ho et al, 2006) were also shown to regulate fragile site stability (reviewed by Arlt et al, 2006). Further studies are required to investigate the regulation of these proteins by ATR and ATM, under conditions that induce fragile site expression.…”

Activation and regulation of ATM kinase activity in response to DNA double-strand breaks

“…BRCA1 is also an ATM substrate and is phosphorylated on Ser1387, Ser1423 and Ser1524 after DNA damage (Cortez et al, 1999;Gatei et al, 2001). Phosphorylation of BRCA1 on these sites is closely linked to S-phase and G2/M checkpoints (Xu et al, 2001.…”

BRCA1 gene in breast cancer

“…Over the last few years, BRCA1 has been identified as a target for several upstream nuclear PI-like kinases: ATM and ATR, (Cortez et al, 1999;Chen, 2000;Tibbetts et al, 2000;Gatei et al, 2001). After DNA damage, BRCA1 is phosphorylated in vitro and in vivo on S1423 and S1524 by both ATM and ATR (see Fig.…”

“…2). However, a study utilizing a series of BRCA1 phospho-specific antibodies and cells deficient for ATM or ATR revealed that ATM mediates these phosphorylations in response to ionizing radiation (IR), while ATR mediates the phosphorylations in response to ultraviolet-C (UV) irradiation (Gatei et al, 2001). In addition, S1387 was specifically phosphorylated after IR, while S1457 was phosphorylated predominantly after UV treatment.…”