ROLE OF PHOSPHORYLATION IN CREB STRUCTURE AND FUNCTION

通过受体依赖的核信号传导的完整性 磷酸化对维持健康至关重要。一种蛋白质， 通过环腺苷-3 '-5'- cAMP/Ca++依赖性途径是cAMP反应元件 结合蛋白（CREB）。CREB蛋白丝氨酸133位的磷酸化 已经证明对于转录激活的转录是必需的。 生长抑素基因和其它cAMP反应基因。然而，机制 磷酸化是如何改变基因转录的尚不清楚。在这 我们仔细研究了CREB可能采用的五种机制， 磷酸化可以改变受体介导的基因转录。聚焦 一个基因，人类脑啡肽原基因，和一个蛋白质， 大鼠CREB蛋白，我们将确定CREB是否通过 PKA或PKC改变了CREB的构象，改变了CREB的自身结构， CREB的结合强度（最可能是二聚化），改变了 CREB对核酸模板的结合亲和力，改变了 所述蛋白质可以改变CREB/核酸复合物的构象和/或改变CREB/核酸复合物的构象。 CREB与其他蛋白质的结合状态。我们开始分析， 利用单一形式的纯化CREB，其转录作用 很好地描述了。使用这种定义明确的材料，我们将 在体内和体外确定PKA和PKC磷酸化的位点 通过胰蛋白酶或胰蛋白酶-K消化，使用毛细管分离肽 电泳（CE）偶联质谱（MS）用于测序。 我们将测量磷酸化对CREB构象的影响， 圆二色性（CD）。CD变化将与以下分析相关： 磷酸化前后产生的CREB肽片段， 胰蛋白酶或胰蛋白酶-K消化。胰蛋白酶的大小、动力学和序列 将使用CE/MS在消化之前和之后比较肽。 构象数据将用于评估 磷酸化对CREB的自缔合常数的影响， 通过量热法。自缔合态的分子量将 通过沉降平衡进行评估。我们将测量 CREB和磷酸化CREB对核酸的常数 结合分析我们将通过荧光能量转移， 电子光谱成像和圆二色性，如果 CREB结合位点的构象可以采用替代结构 在磷酸化作用下。通过将CREB固定在柱子上，我们将检查 其与来自受体诱导的细胞提取物的其它蛋白质的缔合。 我们将使用纯化的CREB重复所有的研究，CREB被磷酸化， PKA或PKC。该定义系统的结果将用于 确定CREB磷酸化在受体介导的 基因表达的改变。这些数据将用于生成一个更 对磷酸化在细胞内作用的一般理解 发信号。

The integrity of nuclear signaling through receptor-dependent phosphorylation is critical for maintaining health. One protein that mediates transcriptional effects through cyclic-adenosine-3'-5'- monophosphate (cAMP)/Ca++-dependent pathways is the cAMP response element binding protein (CREB). Phosphorylation at serine 133 of the CREB protein has been shown to be essential for transcriptional activation of the somatostatin gene and other cAMP-responsive genes. However, the mechanism by which phosphorylation alters gene transcription is unknown. In this proposal, we carefully examine five possible mechanisms by which CREB phosphorylation can alter receptor-mediated gene transcription. Focusing on a single gene, the human proenkephalin gene, and a single protein, a rat CREB protein, we will determine if phosphorylation of CREB through either PKA or PKC alters the conformation of CREB, alters the self- association strength of CREB (most probably dimerization), alters the binding affinity of CREB for the nucleic acid template, alters the conformation of the CREB/nucleic acid complex and/or alters the association state of CREB with other proteins. We begin our analysis by utilizing a single form of purified CREB whose transcriptional effects are well characterized. Using this well-defined material, we will determine the site of PKA and PKC phosphorylation in vivo and in vitro by trypsin or tryp-K digestion, separation of peptides using capillary electrophoresis (CE) coupled to mass spectroscopy (MS) for sequencing. We will measure the effects of phosphorylation on CREB conformation by circular dichroism (CD). CD changes will be correlated with analysis of the CREB peptide fragments generated before and after phosphorylation by trypsin or tryp-K digestion. The size, kinetics and sequence of tryptic peptides will be compared before and after digestion using CE/MS. The conformational data will be used to evaluate the effect of phosphorylation on CREB's self-association constant that will be measured by calorimetry. The molecular weight of the self-associated state will be assessed by sedimentation equilibrium. We will measure the binding constant of CREB and phospho-CREB to the nucleic acid through filter binding analysis. We will examine, by fluorescence energy transfer, electron spectroscopic imaging and circular dichroism, if the conformation of the CREB binding site can adopt an alternative structure upon phosphorylation. By immobilizing CREB on columns, we will examine its association with other proteins from receptor-induced cell extracts. We will repeat all studies using purified CREB that is phosphorylated by either PKA or PKC. The results from this defined system will be used to identify the role for CREB phosphorylation in receptor-mediated alterations of gene expression. The data will be used to generate a more general understanding of the role of phosphorylation in cellular signaling.