Chan D W, Ye R, Veillette C J, Lees-Miller S P
Department of Biological Sciences, University of Calgary, Canada.
Biochemistry. 1999 Feb 9;38(6):1819-28. doi: 10.1021/bi982584b.
Ku antigen is composed of 70 and 82 kDa subunits (Ku70 and Ku80, respectively) that together bind with high affinity to ends of double-stranded DNA and other DNA structures in vitro. When bound to DNA, the Ku 70/80 heterodimer enhances the kinase activity of the catalytic subunit of the DNA-dependent protein kinase, DNA-PKcs. Ku and DNA-PKcs are required for V(D)J recombination and DNA double-strand break repair in vivo and may also play a role in regulation of transcription. Ku is phosphorylated by DNA-PKcs in vitro, and cells that lack DNA-PKcs are deficient in Ku phosphorylation in vitro, suggesting that Ku may be a physiological target for DNA-PK. Here we have identified the sites of DNA-PK phosphorylation in human Ku protein. We find that Ku70 is phosphorylated at a single serine residue, serine 6, located in the putative transcriptional activation domain, and Ku80 is phosphorylated at serines 577 and 580 and at threonine 715. Interestingly, none of the phosphorylation sites identified in Ku correspond to the serine-glutamine consensus for DNA-PK phosphorylation, consistent with previous reports that DNA-PK can recognize additional phosphorylation motifs.
Ku抗原由70 kDa和82 kDa的亚基（分别为Ku70和Ku80）组成，二者在体外可共同与双链DNA末端及其他DNA结构高亲和力结合。与DNA结合时，Ku 70/80异二聚体可增强DNA依赖性蛋白激酶催化亚基DNA-PKcs的激酶活性。在体内，V(D)J重组和DNA双链断裂修复需要Ku和DNA-PKcs，它们可能还在转录调控中发挥作用。在体外，Ku可被DNA-PKcs磷酸化，缺乏DNA-PKcs的细胞在体外Ku磷酸化存在缺陷，这表明Ku可能是DNA-PK的生理靶点。在此，我们确定了人Ku蛋白中DNA-PK磷酸化的位点。我们发现，Ku70在位于假定转录激活域的单个丝氨酸残基丝氨酸6处被磷酸化，Ku80在丝氨酸577、丝氨酸580和苏氨酸715处被磷酸化。有趣的是，在Ku中鉴定出的磷酸化位点均不符合DNA-PK磷酸化的丝氨酸-谷氨酰胺共有序列，这与之前关于DNA-PK可识别其他磷酸化基序的报道一致。
