MOLECULAR ENDOCRINOLOGY OF PEPCK GENE REGULATION BY CAMP

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The long term objective of this research program is to determine how cAMP regulates gene expression, with particular emphasis on the mechanism of action of the cAMP regulatory element (CRE) binding protein (CREB). The CRE plays a crucial role in both basal and cAMP-stimulated transcription of the gene encoding phospho-enolpyruvate carboxykinase (PEPCK), which catalyzes the rate-limiting step in gluconeogenesis to maintain appropriate blood glucose concentrations. Protein kinase A is rapidly activated by cAMP and phosphorylates CREB on Ser 133, enhancing its ability to activate transcription. Transcription initiation involves: 1) assembly of a closed complex of general transcription factors (GTFs) and RNA polymerase II at the TATA site; 2) isomerization of the closed complex to an open complex capable of catalyzing RNA synthesis, accompanied by melting of the start site; and 3) release of the actively transcribing polymerase or promoter clearance. During the previous award, we showed that CREB contains distinct domains, a constitutive activation domain (CAD) and a kinase-inducible domain (KID), that act independently to regulate basal and hormone-induced PEPCK gene transcription. We also showed that CREB interacts with the GTFs, TFIIB and TFIID, through its CAD, and mapped three CAD subdomains that may bind to different targets in the transcription complex to enhance its assembly and stability on the promoter. Others have shown that regulatory factors are bound to the CRE and TATA sequences of the PEPCK gene in vivo, even in the absence of treatment with cAMP. Together, these data suggest the hypothesis to be tested in the current proposal: that the CAD in CREB acts at an early step to help, assemble a polymerase complex, whereas phosphorylation of KID causes a rapid change in the rate of transcription initiation by effecting the recruitment of a late factor, the isomerization/promoter melting step, or disassembly of the complex and promoter clearance by RNA polymerase II. The Specific Aims are: 1) to further define the components of the CAD that interact with the TFIIB and TFIID proteins in the initiation complex, and; 2) to determine the steps in transcription initiation that are affected by the CAD and KID, including analysis of closed and open complex formation and promoter clearance of the PEPCK gene, both in nuclear extracts with in vitro transcription assays and in hepatoma cells in vivo. These studies will help to elucidate the mechanism by which the CAD and KID in CREB maintain the PEPCK promoter in a ready state and then rapidly transmit signals to the polymerase complex in response to hormonal stimuli to maintain glucose homeostasis.