PROTEIN/PROTEIN INTERACTIONS OF CREB/ATF PROTEINS

信号转导途径最终会聚在 转录激活产生特定的基因表达模式 对环境刺激的反应。 转录起始 由这些信号传导途径介导的是由协调调节 特异性转录因子的表达和/或激活，所述转录因子结合 基因的控制区域。 对机制的具体见解 潜在的转录激活最近出现的研究 这些转录因子的结构和功能。 的 CREB/ATF家族的转录反式激活蛋白只有 最近被发现，似乎提供了一个链接之间的 调节基因表达的细胞激活剂 信号通路和病毒基因表达的调节 反式激活蛋白 了解的性质和重要性 真核基因调控机制中的蛋白质/蛋白质相互作用 表达对于理解正常和异常调节是必不可少的， 基因转录。 我们最近开发了一种快速灵敏的 一种技术，允许检测这些相互作用之间 哺乳动物转录因子。 利用125 I标记的重组CREB 和ATF-2肽来探测HeLa细胞核提取物的蛋白质印迹， 确定了多个独立的核因子， 蛋白质/蛋白质相互作用与这些含亮氨酸拉链 转录调节蛋白。 在本申请中，我们建议 利用这种技术分离表达蛋白质的cDNA克隆， 与CREB和/或ATF-2的特异性蛋白/蛋白相互作用。 此外，本发明还提供了一种方法， 我们将对DNA结合和转录进行详细的分析， 几种CREB/ATF蛋白的特征。 蛋白质/蛋白质含量测定 我们已经开发的互动将允许系统的方法， 鉴定和克隆参与真核生物调控的蛋白质， 转录调控 的鉴定和表征。 真核转录复合物的组成部分将允许研究， 解决正常和异常控制细胞的分子机制， 基因表达。

Signal transduction pathways converge ultimately at the level of transcriptional activation to produce specific patterns of gene expression in response to environmental stimuli. The initiation of transcription mediated by these signalling pathways is regulated by the coordinate expression and/ or activation of specific transcription factors that bind to the control regions of genes. Specific insights into the mechanisms underlying transcriptional activation have recently arisen from the studies of the structure and functions of these transcription factors. The CREB/ATF family of transcriptional transactivating proteins has only recently been discovered and appears to provide a link between the regulation of gene expression in response to activators of cellular signalling pathways and the regulation of gene expression by viral transactivating proteins. Understanding the nature and importance of protein/protein interactions in the mechanisms of eukaryotic gene expression is essential to understanding the normal and aberrant regulation of gene transcription. We have recently developed a rapid and sensitive technique which allows the detection of these interactions between mammalian transcription factors. Utilizing 125I-labelled recombinant CREB and ATF-2 peptides to probe western blots of HeLa nuclear extracts, we have identified multiple separate nuclear factors that form specific protein/protein interactions with these leucine zipper-containing transcriptional regulatory proteins. In this application, we propose to utilize this technique to isolate cDNA clones expressing proteins that form specific protein/protein interactions with CREB and/or ATF-2. In addition, we will perform detailed analyses of the DNA-binding and transcriptional characteristics of several CREB/ATF proteins. The assay of protein/protein interactions we have developed will allow a systematic approach to identifying and cloning proteins involved in the control of eukaryotic transcriptional regulation. The identification and characterization of the components of eukaryotic transcription complexes will allow studies that address the molecular mechanisms of normal and aberrant control of cellular gene expression.