MOLECULAR ENDOCRINOLOGY OF PEPCK GENE REGULATION BY CAMP

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The long-term objective of the research program described here is to understand how cAMP regulates gene expression with particular emphasis on the mechanism of action of the cAMP response element (CRE) binding protein (CREBP) and the interplay of factors regulating expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene. The CRE plays an essential role in both basal and cAMP-stimulated transcription of the PEPCK gene. Regulated expression of PEPCK, which catalyzes the rate-limiting step in gluconeogenesis, is essential for maintaining appropriate blood glucose concentrations. Gene transcription is regulated by the interaction of trans-acting protein factors with each other and RNA polymerase, which is facilitated by binding of these factors to cis-acting DNA elements. Cyclic AMP activates protein kinase A, resulting in phosphorylation of several proteins, including the CRE binding protein. CREBP has recently been purified, cloned and shown to be directly involved in regulation of gene expression. In order to understand how CREBP interacts with other factors to regulate gene expression, it is necessary to identify the domains of CREBP that are involved in transactivation. CREBP binds to the CRE as a dimer, and two isoforms that differ by 14 amino acids and possess distinct transcriptional capacities are present in cells due to alternative splicing of the CREBP mRNA. Aim 1 is to determine the significance of the dimerization of CREBP isoforms for transactivation and to establish whether the amount or distribution of isoforms is regulated. This will be done by examining the contribution of each peptide alone compared to the interactions of the same and different isoforms in a dimer and by determining whether CREBP synthesis or splicing is regulated by hormones. Aim 2 is to identify the functional domains of CREBP involved in activation of basal and cAMP- stimulated transcription. This will be done by creating specific mutations in the CREBP cDNA and by fusing potential activation domains to the GAL4 DNA-binding domain. The transactivational capacity of mutated CREBP proteins and of CREBP/GAL4 fusion factors will be assessed in transfection and in vitro transcription assays. Aim 3 is to identify and characterize the interactions of the CRE with heterologous promoter elements, especially those involved in regulation of PEPCK. This will identify potential targets of CREBP interactions. Domains of CREBP that are required for the interplay of different transcription factors can then be identified by examining the ability of different CREBP variants and fusion factors to potentiate activation by these other factors.