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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK049600-02
Application #
2430233
Study Section
Endocrinology Study Section (END)
Program Officer
Sato, Sheryl M
Project Start
1996-06-01
Project End
1999-05-31
Budget Start
1997-06-01
Budget End
1998-05-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Physiology
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033