SOMATOSTATIN GENE EXPRESSION--FUNCTIONAL DOMAINS OF CREB

本提案的长期目标是分析 反式激活蛋白CREB的要求，采用 生长抑素启动子作为测试系统。 拟议的研究涉及 CREB的结构/功能的三个方面。 1. CREB影响基础水平转录的机制， 与一般的转录装置相互作用。的方法 涉及确定基础免疫应答所需的CREB结构域。 水平转录，采用定点N-末端缺失， CREB。 突变型CREB的转录活性分析 通过功能性体外转录测定来进行蛋白质。 CREB蛋白突变体和一般蛋白之间的相互作用 转录因子（TFIID/TFIIB）将通过功能评估， 体外转录测定，与重组TFIIB/TFIID互补。 2. CREB同种型的特异性磷酸化反应将是 检查;即，CREB通过cAMP的顺序磷酸化 依赖性蛋白激酶A和糖原合成酶激酶-3。 的 这种连续磷酸化反应的功能作用将是 通过体外和体内测定进行检查。 3.本提案的第三个方面旨在了解 CREB蛋白识别同源CRE位点并与其相互作用。 圆二色性和2D-NMR研究将被用来检查如何 重组CREB 259-327肽的Bzip结构域在溶液中相互作用 CRE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.