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.
