Telomeric protein Pin2/TRF1 as an important ATM target in response to double strand DNA breaks

ATM mutations are responsible for the genetic disease ataxia-telangiectasia (A-T). ATM encodes a protein kinase that is activated by ionizing radiatio n-induced double strand DNA breaks. Cells derived from A-T patients show ma ny abnormalities, including accelerated telomere loss and hypersensitivity to ionizing radiation; they enter into mitosis and apoptosis after DNA dama ge. Pin2 was originally identified as a protein involved in G(2)/M regulati on and is almost identical to TRF1, a telomeric protein that negatively reg ulates telomere elongation. Pin2 and TRF1, probably encoded by the same gen e, PIN2/TRF1, are regulated during the cell cycle. Furthermore, up-regulati on 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 un known. Here we show that Pin2/TRF1 coimmunoprecipitated with ATM, and its p hosphorylation was increased in an ATM-dependent manner by ionizing DNA dam age. Furthermore, activated ATM directly phosphorylated Pin2/TRF1 preferent ially on the conserved Ser(219)-Gln site in vitro and in vivo. The biologic al significance of this phosphorylation is substantiated by functional anal yses of the phosphorylation site mutants. Although expression of Pin2 and i ts mutants has no detectable effect on telomere length in transient transfe ction, a Pin2 mutant refractory to ATM phosphorylation on Ser(219) potently induces mitotic entry and apoptosis and increases radiation hypersensitivi ty of A-T cells. In contrast, Pin2 mutants mimicking ATM phosphorylation on Ser(219) completely fail to induce apoptosis and also reduce radiation hyp ersensitivity of A-T cells. Interestingly, the phenotype of the phosphoryla tion-mimicking mutants is the same as that which resulted from inhibition o f endogenous Pin2/TRF1 in A-T cells by its dominant-negative mutants. These results demonstrate for the first time that ATM interacts with and phospho rylates Pin2/TRF1 and suggest that Pin2/TRF1 may be involved in the cellula r response to double strand DNA breaks.