Inhibition by Insulin of Protein Kinase Aminduced Transcription of the Phosphoenolpyruvate Carboxykinase Gene

The minimal promoter/transcription factor requirements for induction of phosphoenolpyruvate carboxykinase (PEPCK) transcription by CAMP-activated protein kinase A (PKN and inhibition of this induction by insulin were investigated. H4 hepatoma cells were treated with or without insulin following cotramfeetion with chloramphenicol acetyltransferase reporter genes and expression vectors coding for the CAMP response element-binding protein (CREB) activation domain fused to the GAL4 DNA binding domain (CRG) and the catalytic subunit of PKA. Mutation of the PEPCK CRE to a GAL4 binding site (GCPEPCK) within the fully responsive PEPCK promoter (-600/+69) made induction by PKA dependent upon cotransfection of CRG and this induction by CRG + PKA was inhibited by insulin. Mutation of the insulin regulatory sequence (AIRS-GB-PEPCK) did not prevent induction by cAMP or inhibition by insulin. Fusion of GAL4 binding sites to the PEPCK TATA region (-40/+1, GB-PT) allowed induction by CRG + PKA and inhibition by insulin. However, hib bit ion by insulin was not observed when the CREB activation domain in CRG was replaced with the activation domain of VPl6 fG4VP16) or when the PEPCK TATA region was replaced with TATA regions from other genes. Our results indicate that the minimal requirements for induction of PEPCK by PKA and inhibition by insulin include: 1) the CREB activation domain, 2) the PEPCK TATA sequence, and 3) insulin-responsive hepatoma cells. These data suggest that specific factors interacting with both the PEPCK TATA region and the CREB activation domain are required for insulin inhibition of PKA-induced transcription.