IN VITRO ANALYSIS OF CREB ACTION

cAMP反应元件结合蛋白（CREB）介导 对cAMP的转录反应。 虽然有一些不同的 因子可以结合cAMP反应元件，CREB被认为是 介导对cAMP反应的主要转录因子。 的 cAMP依赖性蛋白激酶A在Ser 133磷酸化CREB， 很明显，该位点磷酸化对于允许cAMP- 介导的转录增加。 最近的研究提供了 对CREB行动的实质性新见解。 这些研究 鉴定了一种被命名为CREB结合的共激活剂 蛋白（CBP）。 CBP与CREB以磷酸化方式相互作用- 依赖的方式，但是，CBP的作用， 在CREB转录反应中的作用已经被 事实上，CBP缺陷的哺乳动物细胞尚未被鉴定。 此外，还对CBP的功能进行了分析， 由于存在一种密切相关的蛋白质p300， 也显示以磷酸化依赖性方式与CREB结合， 以PKA依赖性共激活剂的方式起作用。 的 拟议的研究将利用染色质组装和体外 其中CREB和CBP可以被操纵以 进一步评估CBP在介导转录调控中的作用， 对CREB的回应 该系统还将用于检查 随后的事件是必不可少的CREB诱导的转录 应答

The cAMP response element binding protein (CREB) mediates transcriptional responses to cAMP. Although a number of different factors can bind to cAMP responsive elements, CREB is thought to be the major transcription factor that mediates responses to cAMP. The cAMP-dependent protein kinase A phosphorylates CREB at Ser133 and it is clear that phosphorylation of this site is essential to permit cAMP- mediated increases in transcription. Recent studies have provided substantial new insight into CREB action. These studies have identified a co-activator that has been designated the CREB binding protein (CBP). CBP interacts with CREB in a phosphorylation- dependent manner, however, definitive analysis of the role that CBP plays in transcriptional responses to CREB has been complicated by the fact that mammalian cells deficient in CBP have not yet been identified. Furthermore, analysis of the function of CBP is additionally complicated by the presence of a closely related protein, p300, which has also been shown to bind to CREB in a phosphorylation-dependent manner and to function as a PKA-dependent co-activator. The proposed studies will utilize chromatin assembly and in vitro transcription assays in which CREB and CBP can be manipulated to further assess the role that CBP plays in mediating transcriptional responses to CREB. This system will also be used to examine subsequent events that are essential for CREB-induced transcriptional responses.