Phosphorylation of the cAMP response element binding protein CREB by cAMP-dependent protein kinase A and glycogen synthase kinase-3 alters DNA-binding affinity, conformation, and increases net charge
Phosphorylation of the cAMP response element binding protein CREB by cAMP-dependent protein kinase A and glycogen synthase kinase-3 alters DNA-binding affinity, conformation, and increases net charge
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DOI：
10.1021/bi970982t
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发表时间：
1998-03-17
期刊：
影响因子：
2.9
通讯作者：
Habener, JF
中科院分区：
文献类型：
--
作者：
Bullock, BP;Habener, JF
cAMP 反应元件结合蛋白 CREB 响应 cAMP 依赖性蛋白激酶 A (PKA) 和其他蛋白激酶的磷酸化，激活基因转录。磷酸化 CREB 通过招募 CREB 结合蛋白等转录共激活因子来激活转录。在这里，我们描述了分析磷酸化对 CREB DNA 结合亲和力的影响以及溶液中 CREB/DNA 复合物结构特征的实验。对 CREB 缺失突变体的分析表明，反式激活结构域内的氨基酸序列促进 CREB 与含有 cAMP 响应元件的荧光标记寡核苷酸的高亲和力结合。体外实验表明，磷酸化在作为初始激酶的 PKA 和糖原合酶激酶 3 (GSK-3) 之间进行，但在作为辅助激酶的酪蛋白激酶 II 之间不进行。荧光电泳迁移率变动分析表明，PKA 磷酸化导致 CREB 与对称生长抑素 CRE (SMS-CRE) 和不对称生长抑素上游元件 (SMS-UE) 的结合亲和力增加 3-5 倍。 GSK-3 对 CREB 的持续磷酸化减弱了响应 PKA 的增强的 DNA 结合，因此充当 PKA 诱导的结合的抑制剂。 Ferguson 图分析表明，PKA 和 GSK-3 对 CREB 的磷酸化导致 CREB/DNA 复合物的球形尺寸和净正表面电荷增加。此外，这些分析还揭示了一个意想不到的发现，即在存在和不存在 DNA 的情况下，CREB 都会以四聚体的形式结合。这些发现提出了一个模型，通过该模型，CREB 的磷酸化改变了 CREB/DNA 复合物的二级结构和电荷特征，从而导致结合亲和力的改变。
The cAMP response element binding protein CREB activates the transcription of genes in response to phosphorylation by cAMP-dependent protein kinase A (PKA) and other protein kinases, Phosphorylated CREB activates transcription by recruiting transcriptional co-activators such as the CREB binding protein. Here, we describe experiments that analyze the effects of phosphorylation on the DNA binding affinity of CREB and the structural characteristics of the CREB/DNA complex in solution, Analysis of deletion mutants of CREB indicate that amino acid sequences within the transactivation domain promote high-affinity binding of CREB to fluorescently labeled oligonucleotides containing cAMP response elements. In vitro experiments indicate that phosphorylation is processive between PKA as the initial kinase and glycogen synthase kinase-3 (GSK-3) but not casein kinase II as the secondary kinase. Fluorescent electrophoretic mobility shift assays show that phosphorylation by PKA results in a 3-5-fold increase in the binding affinity of CREB to both the symmetrical somatostatin CRE (SMS-CRE) and the asymmetric somatostatin upstream element (SMS-UE). Processive phosphorylation of CREB by GSK-3 attenuates the enhanced DNA binding in response to PKA thus acts as an inhibitor of PKA-induced binding. Ferguson plot analyses demonstrate that phosphorylation of CREB by PKA and GSK-3 result in an increase in the spherical size and the net positive surface charge of the CREB/DNA complex. Moreover, these analyses uncovered the unexpected finding that CREB associates as a tetramer both in the presence and absence of DNA, These findings suggest a model by which phosphorylation of CREB alters the secondary structure and charge characteristics of the CREB/DNA complex resulting in an alteration in binding affinity.