The DNA-dependent protein kinase catalytic subunit is phosphorylated in vivo on threonine 3950, a highly conserved amino acid in the protein kinase domain
The DNA-dependent protein kinase catalytic subunit is phosphorylated in vivo on threonine 3950, a highly conserved amino acid in the protein kinase domain
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DOI：
10.1128/mcb.01962-06
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发表时间：
2007-03-01
影响因子：
5.3
通讯作者：
Meek, Katheryn
中科院分区：
文献类型：
--
作者：
Douglas, Pauline;Cui, Xiaoping;Meek, Katheryn
DNA依赖性蛋白激酶（DNA-PK）的蛋白激酶活性是通过非同源末端连接（NHEJ）过程修复DNA双链断裂（DSB）所必需的。然而，到目前为止，唯一的目标显示是功能相关的NHEJ中的DNA-PK的酶的作用是大的催化亚基DNA-PKcs本身。在体外，DNA-PKcs的自磷酸化诱导激酶失活和DNA-PKcs与DNA末端结合组分Ku 70/Ku 80的解离。磷酸化内的两个先前确定的集群的磷酸化位点不介导组装复合物的失活，仅部分调节激酶解体，这表明，额外的自磷酸化位点可能是重要的DNA-PK功能。在这里，我们表明，DNA-PKcs包含一个高度保守的氨基酸（苏氨酸3950）在一个区域类似的激活环或T-环中发现的典型的真核蛋白激酶家族的成员的蛋白激酶结构域。我们证明，苏氨酸3950是一个在体外autophosphorylation网站，这个残基，以及其他先前确定的网站在ABCDE集群，在体内被磷酸化的辐射细胞。此外，我们表明，突变的苏氨酸3950磷酸化天冬氨酸废除V（D）J重组，并导致辐射敏感性。总之，这些数据表明，苏氨酸3950是一个功能上重要的，DNA损伤诱导的磷酸化位点，该位点的磷酸化调节DNA-PKcs的活性。
The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphoryllation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphoryllation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.