DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key member of the phosphatidylinositol-3 kinase-like (PIKK) family of protein kinases with critical roles in DNA-double strand break repair, transcription, metastasis, mitosis, RNA processing, and innate and adaptive immunity. The absence of DNA-PKcs from many model organisms has led to the assumption that DNA-PKcs is a vertebrate-specific PIKK. Here, we find that DNA-PKcs is widely distributed in invertebrates, fungi, plants, and protists, and that threonines 2609, 2638, and 2647 of the ABCDE cluster of phosphorylation sites are highly conserved amongst most Eukaryotes. Furthermore, we identify highly conserved amino acid sequence motifs and domains that are characteristic of DNA-PKcs relative to other PIKKs. These include residues in the Forehead domain and a novel motif we have termed YRPD, located in an α helix C-terminal to the ABCDE phosphorylation site loop. Combining sequence with biochemistry plus structural data on human DNA-PKcs unveils conserved sequence and conformational features with functional insights and implications. The defined generally progressive DNA-PKcs sequence diversification uncovers conserved functionality supported by Evolutionary Trace analysis, suggesting that for many organisms both functional sites and evolutionary pressures remain identical due to fundamental cell biology. The mining of cancer genomic data and germline mutations causing human inherited disease reveal that robust DNA-PKcs activity in tumors is detrimental to patient survival, whereas germline mutations compromising function are linked to severe immunodeficiency and neuronal degeneration. We anticipate that these collective results will enable ongoing DNA-PKcs functional analyses with biological and medical implications.

DNA 依赖性蛋白激酶催化亚基 (DNA-PKcs) 是磷脂酰肌醇-3 激酶样 (PIKK) 蛋白激酶家族的关键成员，在 DNA 双链断裂修复、转录、转移、有丝分裂、RNA 加工中起关键作用，以及先天性和适应性免疫。许多模式生物缺乏 DNA-PKcs 导致假设 DNA-PKcs 是脊椎动物特异性 PIKK。在这里，我们发现 DNA-PKcs 广泛分布于无脊椎动物、真菌、植物和原生生物中，并且 ABCDE 磷酸化位点簇的苏氨酸 2609、2638 和 2647 在大多数真核生物中高度保守。此外，我们确定了高度保守的氨基酸序列基序和结构域，这些基序和结构域是 DNA-PKcs 相对于其他 PIKKs 的特征。这些包括前额结构域中的残基和我们称为 YRPD 的新基序，位于 ABCDE 磷酸化位点环的 α 螺旋 C 末端。将序列与生物化学以及人类 DNA-PKcs 的结构数据相结合，揭示了具有功能洞察力和含义的保守序列和构象特征。定义的普遍渐进的 DNA-PKcs 序列多样化揭示了进化痕迹分析支持的保守功能，这表明对于许多生物体而言，由于基本的细胞生物学，功能位点和进化压力保持相同。对导致人类遗传病的癌症基因组数据和种系突变的挖掘表明，肿瘤中强大的 DNA-PKcs 活性不利于患者的生存，而损害功能的种系突变与严重的免疫缺陷和神经元变性有关。我们预计这些集体结果将使正在进行的具有生物学和医学意义的 DNA-PKcs 功能分析成为可能。