Reduction in DNA-binding affinity of CYS2His2 zinc finger proteins by linker phosphorylation
Reduction in DNA-binding affinity of CYS2His2 zinc finger proteins by linker phosphorylation
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DOI：
10.1073/pnas.0402191101
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发表时间：
2004-05-18
影响因子：
11.1
通讯作者：
Berg, JM
中科院分区：
文献类型：
--
作者：
Jantz, D;Berg, JM
Cys（2）His（2）锌指蛋白是高等真核生物基因组中最大的一类转录因子。最近对lkaros转录因子的研究表明，这种锌指蛋白经历与DNA相关的细胞周期依赖性变化，这似乎是由于连接相邻锌指结构域的接头区域中的Thr或Ser残基的磷酸化。在Cys（2）His（2）超家族中，该接头序列的高度保守性表明，在这一大类转录因子中，DNA结合亲和力的细胞周期依赖性调节存在一种共同机制。通过直接比较天然化学连接产生的四种合成锌指蛋白的DNA结合特性，研究了连接子磷酸化对DNA结合亲和力的影响。这四种蛋白质，包括三个锌指结构域连接两个共识Thr-Gly-Glu-Lys-Pro连接，对应于所有四种可能的组合连接体Thr磷酸化状态。对特定DNA结合位点的基于磷酸化的DNA结合研究显示，单个连接体的磷酸化使结合亲和力降低约40倍，而两个连接体的磷酸化使结合亲和力降低130倍。纯化组分的这些结果表明，接头磷酸化确实产生DNA结合亲和力的显著降低，并支持其中单细胞周期依赖性Ser/Thr激酶可以同时抑制大量锌指转录因子的模型。
Cys(2)His(2) zinc finger proteins make up the largest class of transcription factors encoded in the genomes of higher eukaryotes. Recent studies of the lkaros transcription factor demonstrated that this zinc finger protein undergoes cell cycle-dependent changes in association with DNA that seem to be due to phosphorylation of Thr or Ser residues in the linker regions connecting adjacent zinc finger domains. The high degree of conservation of this linker sequence within the Cys(2)His(2) superfamily suggested a common mechanism for the cell cycle-dependent modulation of DNA-binding affinity throughout this large class of transcription factors. The effects of linker phosphorylation on DNA-binding affinity were investigated through a direct comparison of the DNA-binding properties of four synthetic zinc finger proteins produced by native chemical ligation. The four proteins, comprising three zinc finger domains joined by two consensus Thr-Gly-Glu-Lys-Pro linkers, correspond to all four possible combinations of linker Thr phosphorylation states. Fluorescence-based DNA-binding studies of a specific DNA-binding site revealed that phosphorylation of a single linker reduced binding affinity approximate to40-fold, whereas phosphorylation of both linkers reduced binding affinity 130-fold. These results with purified components demonstrate that linker phosphorylation does, indeed, produce a significant reduction in DNA-binding affinity and support a model wherein a single cell cycle-dependent Ser/Thr kinase could simultaneously inactivate a large number of zinc finger transcription factors.