SOMATOSTATIN GENE EXPRESSION--FUNCTIONAL DOMAINS OF CREB

这项建议的长期目标是分析结构 反式激活蛋白CREB的需求，使用 以生长抑素启动子为检测系统。拟议的研究旨在解决 CREB的结构/功能的三个方面。 1.CREB通过以下途径影响基础水平转录的机制 与一般转录装置相互作用。该方法 涉及基本基因所需CREB结构域的确定(S) 水平转录，使用定点定向的N-末端缺失 CREB。突变型CREB的转录活性分析 蛋白质将通过功能体外转录试验进行。 CREB蛋白突变体与普通病毒的相互作用 转录因子(S)(TFIID/TFIIB)将通过功能检测进行评估 体外转录检测，辅以重组TFIIB/TFIID。 2.CREB亚型的特异性磷酸化反应将是 即cAMP对CREB的顺序磷酸化 依赖蛋白激酶A和糖原合成酶激酶-3。这个 这种顺序的磷酸化反应的功能作用将是 通过体外和体内试验进行检测。 3.本提案的第三个方面旨在理解 CREB蛋白识别同源Cre位点并与之相互作用。 圆二色谱和2D-核磁共振研究将被用来研究 重组CREB 259-327多肽的BZIP结构域在溶液中相互作用 以CRE为主题。

The long term objective of this proposal is to analyze the structural requirements of the transactivator protein CREB, employing the somatostatin promoter as the test system. The proposed studies address three aspects of the structure/function of CREB. 1. The mechanism by which CREB effects basal level transcription, by interacting with the general transcriptional apparatus. The approach involves the determination of the domain(s) of CREB required for basal level transcription, employing site directed N-terminal deletions of CREB. Analysis of the transcriptional activity of the mutant CREB proteins will be carried out by functional in vitro transcription assays. The interactions between the CREB protein mutants and the general transcription factor(s) (TFIID/TFIIB) will be assessed by functional in vitro transcription assays, complemented with recombinant TFIIB/TFIID. 2. A specific phosphorylation reaction of the CREB isoforms will be examined; namely, the sequential phosphorylation of CREB by the cAMP dependent protein kinase A and glycogen synthase kinase-3. The functional role of this sequential phosphorylation reaction will be examined by in vitro and in vivo assays. 3. The third aspect of this proposal is designed to understand how the CREB protein recognizes and interacts with the cognate CRE site. Circular dichroism and 2D-NMR studies will be utilized to examine how the Bzip domain of a recombinant CREB 259-327 peptide interacts in solution with the CRE motif.