Ting Nicholas S Y, Pohorelic Brant, Yu Yaping, Lees-Miller Susan P, Beattie Tara L
Department of Biochemistry and Molecular Biology and Department of Oncology, Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive N.W. Calgary, AB T2N 4N1, Canada.
Nucleic Acids Res. 2009 Oct;37(18):6105-15. doi: 10.1093/nar/gkp636. Epub 2009 Aug 5.
Telomere integrity in human cells is maintained by the dynamic interplay between telomerase, telomere associated proteins, and DNA repair proteins. These interactions are vital to suppress DNA damage responses and unfavorable changes in chromosome dynamics. The DNA-dependent protein kinase (DNA-PK) is critical for this process. Cells deficient for functional DNA-PKcs show increased rates of telomere loss, accompanied by chromosomal fusions and translocations. Treatment of cells with specific DNA-PK kinase inhibitors leads to similar phenotypes. These observations indicate that the kinase activity of DNA-PK is required for its function at telomeres possibly through phosphorylation of essential proteins needed for telomere length maintenance. Here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a direct substrate for DNA-PK in vitro. Phosphorylation of hnRNP A1 is stimulated not only by the presence of DNA but also by the telomerase RNA component, hTR. Furthermore, we show that hnRNP A1 is phosphorylated in vivo in a DNA-PK-dependent manner and that this phosphorylation is greatly reduced in cell lines which lack hTR. These data are the first to report that hTR stimulates the kinase activity of DNA-PK toward a known telomere-associated protein, and may provide further insights into the function of DNA-PK at telomeres.
人类细胞中的端粒完整性是通过端粒酶、端粒相关蛋白和DNA修复蛋白之间的动态相互作用来维持的。这些相互作用对于抑制DNA损伤反应和染色体动态变化中的不利改变至关重要。DNA依赖性蛋白激酶（DNA-PK）对这一过程至关重要。缺乏功能性DNA-PKcs的细胞显示出端粒丢失率增加，伴有染色体融合和易位。用特异性DNA-PK激酶抑制剂处理细胞会导致类似的表型。这些观察结果表明，DNA-PK的激酶活性可能通过磷酸化维持端粒长度所需的必需蛋白，从而在端粒发挥其功能。在此，我们表明异质性核核糖核蛋白A1（hnRNP A1）在体外是DNA-PK的直接底物。hnRNP A1的磷酸化不仅受到DNA的刺激，还受到端粒酶RNA组分hTR的刺激。此外，我们表明hnRNP A1在体内以DNA-PK依赖的方式被磷酸化，并且在缺乏hTR的细胞系中这种磷酸化大大减少。这些数据首次报道hTR刺激DNA-PK对已知端粒相关蛋白的激酶活性，并可能为DNA-PK在端粒的功能提供进一步的见解。
