This is a revised application of a new proposal to study the mechanism whereby insulin inhibits activation of the PEPCK gene by cAMP in H4 hepatoma cells. The principal investigator proposes to study how transacting factors, CREB, CBP and possibly other unidentified proteins, are phosphorylated and activated by cAMP via PKA, and dephosphorylated and inhibited by presently unknown insulin mediators.
In Specific Aim 1, the principal investigator will determine which amino acids in CREB/CBP/other proteins are phosphorylated or dephosphorylated in response to cAMP and insulin, and these studies will involve phosphopeptide mapping and the use of phosphorylation-site mutants.
In Specific Aim 2, the principal investigator will test the hypothesis that a unique bridge complex formed between CREB and the PEPCK minimal promoter is disrupted during insulin action, and this will involve analysis of cis -elements in the minimal promoter and biochemical analysis of the effects of cAMP and insulin on the composition and phosphorylation state of factors associated with the bridge complex.
In Specific Aim 3, the principal investigator will determine whether an altered phosphorylation state in CREB/CBP/other proteins is due to changes in relevant nuclear kinase and phosphatase activities, and these studies will involve the use of inhibitors and purified phosphatases and kinases to alter the phosphorylation state of trans-activating factors and their activity in in vitro transcription assays.
| Quinn, P G; Yeagley, D (2005) Insulin regulation of PEPCK gene expression: a model for rapid and reversible modulation. Curr Drug Targets Immune Endocr Metabol Disord 5:423-37 |
| Yeagley, D; Agati, J M; Quinn, P G (1998) A tripartite array of transcription factor binding sites mediates cAMP induction of phosphoenolpyruvate carboxykinase gene transcription and its inhibition by insulin. J Biol Chem 273:18743-50 |
| Agati, J M; Yeagley, D; Quinn, P G (1998) Assessment of the roles of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B, and protein kinase C in insulin inhibition of cAMP-induced phosphoenolpyruvate carboxykinase gene transcription. J Biol Chem 273:18751-9 |
