Analysis of the structural properties of cAMP-responsive element-binding protein (CREB) and phosphorylated CREB
Analysis of the structural properties of cAMP-responsive element-binding protein (CREB) and phosphorylated CREB
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DOI：
10.1074/jbc.271.23.13716
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发表时间：
1996-06-07
影响因子：
4.8
通讯作者：
Brennan, RG
中科院分区：
文献类型：
--
作者：
Richards, JP;Bachinger, HP;Brennan, RG
转录因子CREB （cAMP响应元件结合蛋白）是由蛋白激酶A （PKA）磷酸化单个丝氨酸残基激活的。为了研究通过磷酸化调控CREB的可能机制，我们对全长野生型CREB蛋白进行了结构和生物物理表征，这是一个改变的CREB蛋白（CREB/SER），其中dna结合域的三个半胱氨酸残基被丝氨酸残基和一个包含CREB整个激活域的截断蛋白（ACT265）所取代。圆二色性（CD）表明CREB和CREB/SER具有相同的二级结构，包含约20%的α -螺旋结构，9%的β -链结构，34%的β -转结构和37%的随机螺旋结构。PKA磷酸化不会改变CREB或与DNA结合的CREB的CD谱，因此不会改变其二级结构。蛋白酶裂解模式表明PKA磷酸化不会引起CREB的全局构象变化。此外，通过荧光各向异性DNA结合测定，PKA磷酸化不会改变CREB对典型或非典型CRE序列的DNA结合亲和力。由于PKA磷酸化CREB导致其与转录共激活因子CREB结合蛋白和p300的特异性结合，我们认为PKA激活CREB是通过与这些蛋白产生特异性的互补相互作用而发生的，而不是通过先前提出的磷酸化依赖的构象变化或增加DNA结合亲和力的机制。
The transcription factor CREB (cAMP responsive element binding protein) is activated by protein kinase A (PKA) phosphorylation of a single serine residue. To investigate possible mechanisms of CREB regulation by phosphorylation, we initiated a structural and biophysical characterization of the full-length, wild-type CREB protein, an altered CREB protein (CREB/SER) in which the three cysteine residues in the DNA-binding domain were replaced with serine residues and a truncated protein (ACT265) which encompasses the entire activation domain of CREB. Circular dichroism (CD) reveals that CREB and CREB/SER have identical secondary structures and contain approximately 20% alpha-helix, 9% beta-strand, 34% beta-turn, and 37% random coil structures. PKA phosphorylation does not alter the CD spectra, and therefore the secondary structure, of CREB or of CREB bound to DNA. Protease cleavage patterns indicate that PKA phosphorylation does not induce a global conformational change in CREB. Furthermore, PKA phosphorylation does not change the DNA binding affinity of CREB for either canonical or non-canonical CRE sequences as measured by a fluorescence anisotropy DNA binding assay. Since PKA phosphorylation of CREB results in its specific binding to the transcriptional coactivators CREB-binding protein and p300, we suggest that the PKA activation of CREB occurs by the production of specific, complementary interactions with these proteins, rather than through the previously proposed mechanisms of a phosphorylation-dependent conformational change or increased DNA binding affinity.