Role of the phosphorylation-dependent CREB/CBP interaction in transcriptional activation by CREB

Environmental stimuli contribute to changes in gene expression by activating intracellular signaling pathways which propagate the stimulus from the plasma membrane to the nucleus. The cAMP response element binding protein (CREB) is one of the best characterized transcription factors whose activity is regulated by phosphorylation in response to extracellular signals. A wide range of extracellular stimuli all activate CREB by inducing CREB phosphorylation at a specific residue, Ser-133. The phosphorylation of CREB at Ser-133 induces the association of CREB with a coactivator protein termed the CREB-binding protein (CBP). Specifically, phosphorylation of CREB Ser-133 allows for the association of the kinase-inducible domain (KID) of CREB with a region of CBP termed the KIX domain.; The primary goal of my Thesis work has been to more clearly understand the role of CBP in stimulus-induced CREB activation. To this end we developed a novel E. coli-based two-hybrid system that facilitates the analysis of phosphorylation-dependent protein-protein interactions. Using the bacterial two-hybrid system, we were able to examine the critical structural features within the CREB KID and the CBP KIX domain that contribute to their phosphorylation-dependent interaction.; We also adapted the E. coli two-hybrid system for selection-based assays and employed this selection-based system to identify a single point substitution in the KIX domain of CBP that increases the strength of the KID/KIX interaction. When placed in the context of full-length CBP, this substitution resulted in an increase in the ability of CBP to activate CREB-dependent transcription in mammalian cells. Likewise, a point substitution in the KIX domain that weakens the KID/KIX interaction resulted in a corresponding decrease in the ability of CBP to activate CREB-dependent transcription in mammalian cells. We conclude that the magnitude of CREB-dependent transcriptional activation reflects the strength of the interaction between CREB and CBP. In addition, we found that recruitment of CBP to CREB in the absence of extracellular stimulation is sufficient to promote CREB-dependent transcription, suggesting that the phosphorylation of Ser-133 and subsequent recruitment of CBP to CREB is the critical step at which certain signals, including those that lead to an elevation of CAMP, regulate CREB-dependent transcription.; Transcription factor complexes can also regulate transcription through modifying chromatin structure in the vicinity of the target gene. Accordingly, we investigated the importance of the intrinsic histone acetyltransferase (HAT) activity of CBP in promoting CREB-dependent transcription. We found that although the intrinsic HAT activity of CBP is not required for stimulus-induced CREB activation, this HAT activity is important for the ability of CBP to promote CREB-dependent transcription in the absence of a stimulus. In addition, we examined the role of histone deacetylases (HDACs) in the regulation of CREB-dependent gene expression. We found that pharmacologic inhibition of HDAC activity resulted in an increase in CRE-dependent transcription in several cell types. Consistent with these observations, we observed that CREB is physically associated with HDAC activity under these conditions. These findings suggest that regulation of HDAC activity may be an important mechanism for controlling the expression of CREB target genes.; Finally, we propose that the E. coli two-hybrid system that we have developed can be used to study, in an efficient and quantitative manner, any kinase-dependent protein-protein interaction. This system should prove useful both for elucidating the structural features of known signaling molecules that allow for their phosphorylation-induced interaction as well as for identifying new members within a particular signaling cascade.