The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.

DNA-PKcs是DNA依赖蛋白激酶（DNA-dependent protein kinase, DNA-PK）的催化亚基，其C端末端附近带有磷酸肌醇3-激酶（phosphoinositol 3-kinase, PI 3-K）结构域。源自SCID小鼠的细胞系已被用作DNA-PKcs缺陷型研究模型系统。SCID突变会导致DNA-PKcs在最远端C端发生截短，使其PI 3-K结构域保持完整。该突变蛋白在大多数SCID细胞系中表达水平较低，这使得"该突变是否会消除激酶活性"这一问题尚无定论。本研究证实，正常表达该突变蛋白的SCID细胞系，其激酶活性显著受损。我们估算，SCID成纤维细胞系中通常存在的残余激酶活性，至少较对照细胞低两个数量级。本研究结果进一步验证了"DNA-PKcs激酶活性对于DNA双链断裂（double-strand break, DSB）重接以及V(D)J重组（V(D)J recombination）不可或缺"这一结论，并证实：DNA-PKcs的远端C端区域——该区域存在于PI 3-K相关蛋白激酶中，但在真正的PI 3脂质激酶中缺失——是DNA-PKcs发挥蛋白激酶功能所必需的。我们还发现，突变型DNA-PKcs蛋白的表达会赋予细胞生长劣势，这也解释了为何大多数SCID细胞系中DNA-PKcs的表达量偏低。