Structural Basis for Graded Inhibition of CREB:DNA Interactions by Multisite Phosphorylation.
Structural Basis for Graded Inhibition of CREB:DNA Interactions by Multisite Phosphorylation.
复制标题
通过多位点磷酸化分级抑制 CREB:DNA 相互作用的结构基础。
DOI：
10.1021/acs.biochem.8b01092
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发表时间：
2018
期刊：
影响因子：
2.9
通讯作者：
Wright,PeterE
中科院分区：
文献类型：
--
作者：
Shnitkind,Sergey;Martinez-Yamout,MariaA;Dyson,HJane;Wright,PeterE
环 AMP 反应元件结合转录因子 (CREB) 的激酶诱导结构域 (KID) 的磷酸化通过多种机制调节其功能。转录激活发生在丝氨酸 133 磷酸化后，但称为 CK 盒的邻近区域（残基 108-117）的多位点磷酸化会导致 CREB 介导的转录受到抑制。对这些相反反应机制的分子水平了解一直缺乏，部分原因是体外制备多重磷酸化 CREB 存在困难。通过取代单个残基，我们生成了工程哺乳动物 CREB，其中 CK 盒可以在体外被酪蛋白激酶磷酸化，并表征了其与环 AMP 响应元件 DNA 的相互作用。 CK 盒的磷酸化促进 KID 结构域和 DNA 结合位点（C 端碱性亮氨酸拉链 (bZip) 结构域的碱性区域）之间的分子内相互作用。磷酸化 KID 结构域和 DNA 之间对 bZip 结合的竞争导致 CREB 对 DNA 的亲和力降低。根据解离常数计算出的结合自由能与 CK 盒中磷酸基团的数量成正比，表明 DNA 结合受到类似变阻器的机制的调节。变阻器通过 Mg2+ 等阳离子浓度的变化以及替代异构体（例如缺少残基 162-272 的天然 CREB 异构体）进行调节。 CREB 的多位点磷酸化代表了一种多功能机制，通过该机制可以调整转录以满足细胞的可变需求。
Phosphorylation of the kinase inducible domain (KID) of the cyclic AMP response element binding transcription factor (CREB) regulates its function through several mechanisms. Transcriptional activation occurs following phosphorylation at serine 133, but multisite phosphorylation in a neighboring region termed the CK cassette, residues 108–117, results in inhibition of CREB-mediated transcription. A molecular-level understanding of the mechanism of these opposing reactions has been lacking, in part because of the difficulty of preparing multiply phosphorylated CREBin vitro. By substituting a single residue, we have generated an engineered mammalian CREB in which the CK cassette can be phosphorylatedin vitroby casein kinases and have characterized its interactions with cyclic AMP response element DNA. Phosphorylation of the CK cassette promotes an intramolecular interaction between the KID domain and the site of DNA binding, the basic region of the C-terminal basic leucine zipper (bZip) domain. Competition between the phosphorylated KID domain and DNA for bZip binding results in a decreased affinity of CREB for DNA. The binding free energy calculated from the dissociation constant is directly proportional to the number of phosphate groups in the CK cassette, indicating that the DNA binding is regulated by a rheostat-like mechanism. The rheostat is modulated by variation of the concentration of cations such as Mg2+and by alternative isoforms such as the natural CREB isoform that lacks residues 162–272. Multisite phosphorylation of CREB represents a versatile mechanism by which transcription can be tuned to meet the variable needs of the cell.