Cyclic AMP (cAMP) regulates the transcription of numerous genes through the protein kinase-A (PK-A) mediated phosphorylation of transcription factor CREB at Ser133. Although phosphorylation may stimulate transcriptional activators by modulating their nuclear transport or DNA- binding affinity, CREB belongs to a class of activators whose phosphorylation appears to specifically enhance their trans-activation potential. Within the CREB protein, a 60 amino acid Kinase Inducible Domain (KID) cooperates with a constitutive glutamine rich domain (Q2) in CREB to stimulate transcription of cAMP responsive genes. Current evidence suggests that the KID and Q2 domains of CREB interact with distinct proteins, both of which are critical for assembly of transcriptional initiation complex in response to cAMP. The overall objective of this proposal is to elucidate the mechanism by which cAMP stimulates transcription of target genes, focusing on the hypothesis that PK-A mediated phosphorylation of CREB stimulates protein-protein interactions which culminate in the recruitment of general transcription factors to cAMP responsive promoters. I. We will test whether interaction between a constitutive activation domain in CREB termed Q2 and a component of the general transcription factor TFIID (dTAF-II 110) is critical for PK-A inducible transcription. We will delineate regions in CREB and dTAF-II 110 which participate in complex formation, and we will monitor CREB dTAF-II 110-binding mutants for loss of PK-inducible transcription in vitro and in vivo. II. We will define regions in a recently characterized CREB binding protein (CBP) which are functionally required for cAMP responsive transcription by transient transfection assay with expression vectors encoding wild-type and mutant forms of CBP. III. We will characterize sequences which are required for phosphorylation dependent interaction between a kinase inducible domain in CREB termed KID and CBP using in vitro binding assays. IV. We will identify general transcription factors which interact functionally with CBP to stimulate transcription of cAMP responsive genes following PK-A mediated phosphorylation of CREB at Ser133. cAMP mediates a number of cellular responses to hormones and growth factors. In endocrine target organs such as thyroid and pituitary, pathologic activation of the cAMP second messenger pathway causes syndromes of endocrine neoplasia and hyperfunction. The studies proposed herein will define transcriptional intermediates which may figure importantly in the pathogenesis of these and other diseases where cAMP is inappropriately activated.
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