Sequence-specific DNA binding and transcription factor phosphorylation by Ku autoantigen/DNA-dependent protein kinase
Sequence-specific DNA binding and transcription factor phosphorylation by Ku autoantigen/DNA-dependent protein kinase
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DOI：
10.1074/jbc.272.9.5647
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发表时间：
1997-02-28
影响因子：
4.8
通讯作者：
Hache, RJG
中科院分区：
文献类型：
--
作者：
Giffin, W;KwastWelfeld, J;Hache, RJG
NRE1是一个DNA序列元件，Ku抗原/DNA依赖蛋白激酶(DNA-PK)催化亚基通过它抑制糖皮质激素诱导的小鼠乳腺肿瘤病毒转录。虽然Ru是DNA末端的亲和力结合者，并具有沿DNA移位的能力，但我们报告了直接序列特异性Ku结合比DNA末端结合具有更高的亲和力(K-d=0.84+/-0.24 nM)。Ku结合与几个Ku可以积累的序列的比较揭示了两类序列。与NRE1相似的序列有效地竞争NRE1结合。相反，与NRE1缺乏相似性的序列竞争Ku很差，并且在没有DNA末端的情况下不被识别。DNA-PK对糖皮质激素受体(GR)融合蛋白的磷酸化反映了Ku的DNA结合偏好，并表明GR与DNA-PK在DNA上的共定位是有效磷酸化的关键。DNA-PK对GR融合蛋白的磷酸化定位于Ser-527。这个位点位于GR的DNA和配体结合域之间的GR核定位序列附近，因此，如果得到证实，它的磷酸化可能会影响体内受体的功能。
NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ru is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (K-d = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.