Danska J S, Holland D P, Mariathasan S, Williams K M, Guidos C J
Division of Surgical Research, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.
Mol Cell Biol. 1996 Oct;16(10):5507-17. doi: 10.1128/MCB.16.10.5507.
The scid gene product has been identified as the 460-kDa catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs p460), a member of the phosphatidylinositol 3-kinase family. DNA-PK activity is undetectable in scid cells, but the molecular basis for this defect has not been identified. Here we report that expression of p460 in scid lymphocyte precursors is detectable but is reduced at least 10-fold relative to that in wild-type lymphocytes. In addition, we show that the scid mutation disturbs p460 nuclear association, presumably affecting its role in DNA repair pathways. To examine the molecular basis for our observations, we used a degenerate PCR strategy to clone the C-terminal p460 kinase domain from wild-type and scid thymocytes. Northern (RNA) analysis with these probes revealed normal steady-state p460 mRNA levels in scid cells, suggesting that the reduced abundance of p460 protein is due to a posttranscriptional defect. Sequence comparisons identified a single-base-pair alteration in the scid C-terminal p460 kinase domain, resulting in a premature stop codon. This mutation is predicted to truncate p460 by approximately 8 kDa, but it preserves the conserved motifs required for kinase activity in members of the phosphoinositidyl 3-kinase family. Despite a computed molecular weight alteration of less than 2%, we were able to visualize this difference by Western blot (immunoblot) analysis of wild-type and scid p460. These data demonstrate that the scid DNA-PKes mutation is not a null allele and suggest a molecular rationale for the well-described leakiness of the scid phenotype.
scid基因产物已被鉴定为DNA依赖性蛋白激酶（DNA-PKcs p460）的460 kDa催化亚基，它是磷脂酰肌醇3激酶家族的成员。在scid细胞中检测不到DNA-PK活性，但这种缺陷的分子基础尚未明确。在此我们报告，在scid淋巴细胞前体中可检测到p460的表达，但相对于野生型淋巴细胞，其表达量至少降低了10倍。此外，我们发现scid突变扰乱了p460与细胞核的结合，推测这影响了其在DNA修复途径中的作用。为了研究我们观察结果的分子基础，我们使用简并PCR策略从野生型和scid胸腺细胞中克隆C端p460激酶结构域。用这些探针进行的Northern（RNA）分析显示scid细胞中p460 mRNA的稳态水平正常，这表明p460蛋白丰度降低是由于转录后缺陷所致。序列比较确定scid C端p460激酶结构域中有一个单碱基对改变，导致提前出现终止密码子。预计该突变会使p460截短约8 kDa，但它保留了磷脂酰肌醇3激酶家族成员激酶活性所需的保守基序。尽管计算得出的分子量变化小于2%，但我们通过对野生型和scid p460进行蛋白质印迹（免疫印迹）分析能够观察到这种差异。这些数据表明scid DNA-PKes突变不是无效等位基因，并为scid表型中描述详尽的渗漏现象提供了分子解释。
