NEUROENDOCRINE REGULATION OF CREB BY CAMP AND CALCIUM

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The cAMP and Ca2+ signaling pathways are used throughout the neuroendocrine system to regulate secretion and gene expression. It has recently been shown that cAMP and Ca2+ can converge to regulate the activity of a single transcription factor, CREB. This convergence likely permits CREB to integrate multiple extracellular signals in the regulation of transcription. Presumably the effects of Ca2+ on CREB activation are mediated by Ca2+/calmodulin-dependent protein kinases (CaM Kinases). Recent studies from this laboratory have demonstrated that specific CaM Kinases can have very different effects on the activation of CREB. In particular, we found that CaM Kinase II can inhibit activation of CREB while CaM Kinase IV can activate CREB. The proposed studies seek to further characterize the ability of specific CaM Kinases to regulate activation of the CREB/ATF transcription factor family. The specific aims include: i) Test the hypothesis that phosphorylation of Ser142 of CREB is physiologically regulated. This will involve preparation of an antibody which detects phosphorylation of Ser142. 2) Determine if the inhibitory effects of CaMKII are also observed for ATF1, a related leucine-zipper transcription factor, which has also been shown to respond to changes in cAMP and Ca2+. The region surrounding the negative regulatory site of CREB at Ser142 is conserved, but not identical in ATF1. 3) Examine the ability of CaMKI to activate CREB. It is possible that CaM Kinases other than CaMKIV can activate CREB. We will use transfection assays to determine if CaMKI can activate CREB. 4) Test the hypothesis that the inhibitory effects of CaMKII on activation of CREB are due to the effects of phosphorylation at Ser142 to block interaction between CREB and the co- activator protein, CREB binding protein (CBP). 5) Map the portions of CREB and Cl3P which are required for high affinity, PKA-dependent and CaM Kinase-induced interactions. 6) Develop an episomal, cAMP-responsive reporter gene to examine effects of cAMP and Ca2+ on the interaction of CREB with chromatin structure. These studies should provide new insight into the mechanisms which permit cAMP and Ca2+ to regulate members of the CREB/ATF family.