Telomeric Protein Pin2/TRF1 as an Important ATM Target in Response to Double Strand DNA Breaks

Abstract

ATM mutations are responsible for the genetic disease ataxia-telangiectasia (A-T). ATM encodes a protein kinase that is activated by ionizing radiation-induced double strand DNA breaks. Cells derived from A-T patients show many abnormalities, including accelerated telomere loss and hypersensitivity to ionizing radiation; they enter into mitosis and apoptosis after DNA damage. Pin2 was originally identified as a protein involved in G 2 /M regulation and is almost identical to TRF1, a telomeric protein that negatively regulates telomere elongation. Pin2 and TRF1, probably encoded by the same gene, PIN2/TRF1, are regulated during the cell cycle. Furthermore, up-regulation of Pin2 or TRF1 induces mitotic entry and apoptosis, a phenotype similar to that of A-T cells after DNA damage. These results suggest that ATM may regulate the function of Pin2/TRF1, but their exact relationship remains unknown. Here we show that Pin2/TRF1 coimmunoprecipitated with ATM, and its phosphorylation was increased in an ATM-dependent manner by ionizing DNA damage. Furthermore, activated ATM directly phosphorylated Pin2/TRF1 preferentially on the conserved Ser 219 -Gln site in vitro and in vivo. The biological significance of this phosphorylation is substantiated by functional analyses of the phosphorylation site mutants. Although expression of Pin2 and its mutants has no detectable effect on telomere length in transient transfection, a Pin2 mutant refractory to ATM phosphorylation on Ser 219 potently induces mitotic entry and apoptosis and increases radiation hypersensitivity of A-T cells. In contrast, Pin2 mutants mimicking ATM phosphorylation on Ser 219 completely fail to induce apoptosis and also reduce radiation hypersensitivity of A-T cells. Interestingly, the phenotype of the phosphorylation-mimicking mutants is the same as that which resulted from inhibition of endogenous Pin2/ TRF1 in A-T cells by its dominant-negative mutants. These results demonstrate for the first time that ATM interacts with and phosphorylates Pin2/TRF1 and suggest that Pin2/TRF1 may be involved in the cellular response to double strand DNA breaks.Mutations in the ATM gene are responsible for the rare autosomal human recessive disorder ataxia-telangiectasia (A-T), 1 characterized by progressive neurological degeneration, telangiectasia, growth retardation, premature aging, immunodeficiency, mitotic checkpoint defect, hypersensitivity to ionizing radiation, gonadal atrophy, genomic instability, and predisposition to cancer (1). ATM encodes a protein kinase that is activated by ionizing DNA damage and is critical for maintaining genome stability, telomere maintenance, and induction of cell cycle checkpoints by double strand DNA breaks (2-4). ATM has been shown to bind and/or phosphorylate many key regulators, including p53, ␤-adaptin, c-Abl, Chk1-2, Brca1, and Nijmegen breakage syndrome protein (5-15). Identification of these ATM target proteins opens new avenues for understanding the physiological function of ATM as well as for explaining the pleiotropic pheno...

ATM function and telomere stability

“…The striking correlation between the appearance of retained ATM and of histone H2AX (?-H2AX), and the rapid association of a fraction of ATM with ?-H2AX foci, are consistent with a major role for ATM in the early detection of DSBs and subsequent induction of cellular responses. It has been reported that ATM protein interacts with TRF1 (Kishi et al, 2001) and lack of TRF2 results in apoptosis in ATM function dependent (Karlseder et al, 1999). These reports further propose the role of ATM in telomere maintenance.…”

“…How the absence of ATM in¯uences the association of telomeres with the nuclear matrix is not presently clear. One assumption is that ATM regulates the function of Pin2/TRF1 as activated ATM directly phosphorylated Pin2/TRF1 preferentially on the conserved Ser(219)-Gln site in vitro and in vivo (Kishi et al, 2001). Expression of Pin2 and its mutants has no detectable e ect on telomere length in transient transfection assays, however, a Pin2 mutant refractory to ATM phosphorylation on Ser(219) enhances cell killing by IR in A-T cells.…”

ATM's leucine-rich domain and adjacent sequences are essential for ATM to regulate the DNA damage response

“…ATM phosphorylates multiple proteins involved in the DNA damage response, including p95/nibrin (Lim et al, 2000;Zhao et al, 2000), Brca1 (Cortez et al, 1999), the p53 tumor-suppressor gene (Banin et al, 1998;Canman et al, 1998), the checkpoint kinase chk2 (Blasina et al, 1999;Matsuoka et al, 2000), SMC1 (Banin et al, 1998;Kim et al, 2002;Yazdi et al, 2002), BLM (Beamish et al, 2002), FANCD2 (Taniguchi et al, 2002), and Pin2/Trf1 (Kishi et al, 2001). The phosphorylation of these proteins by ATM can regulate their functional activity.…”

DNA damage transiently increases TRF2 mRNA expression and telomerase activity

“…20 Following DNA damage, ATM phosphorylates TRF1 and suppresses its ability to induce abortive mitosis and apoptosis. 21,22 On the other hand, TRF2 overexpression also negatively regulates telomere length, probably by an active degradation process. 19,23 TRF2 protects telomeres from end-toend fusion, 24 presumably because it mediates the proper folding of the telomere.…”