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。尽管有许多不同的 因子可以与cAMP反应元件结合，CREB被认为是 介导对cAMP反应的主要转录因子。这个 CAMP依赖的蛋白激酶A磷酸化CREB在Ser133和 很明显，这个位点的磷酸化是允许cAMP- 调节转录的增加。最近的研究提供了 对CREB行动的实质性新见解。这些研究已经 标识了已指定为CREB绑定的协同激活剂 蛋白质(CBP)。CBP与CREB以磷酸化方式相互作用- 然而，对CBP的作用进行了明确的分析 CREB在转录反应中的作用因 缺乏CBP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 更复杂的是，存在一种密切相关的蛋白质p300，它 也被证明以磷酸化依赖的方式与CREB结合 作为依赖PKA的共激活剂发挥作用。这个 拟议的研究将利用染色质组装和体外 CREB和CBP可被操纵的转录分析 进一步评估CBP在调节转录中的作用 对CREB的回应。该系统还将用于检查 CREB诱导转录所必需的后续事件 回应。

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.