IN VITRO ANALYSIS OF CREB ACTION

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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.