Cyclic AMP induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription is mediated by multiple promoter elements

Abstract

The cis elements involved in the cAMP regulation of transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) were studied by introducing a series of block mutations (10-15 base pairs of random sequence) into eight of the protein binding domains in a region of the promoter between -490 and +73. Each mutant promoter was ligated to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected into HepG2 cells. Transcription of PEPCK-CAT was stimulated 4-fold by the addition of 8-bromo-cAMP (8-Br-cAMP), whereas overexpression of the catalytic subunit of protein kinase A in these cells increased transcription from the PEPCK promoter 30-fold. Several elements within the PEPCK promoter acted synergistically to mediate this effect. These include CRE-1 (-92 to -82) and a complex unit from -220 to -280 composed of multiple binding sites termed P3(I) (-250 to -234), P3(II) (-260 to -250), and P4 (-286 to -270). Mutation of both CRE-1 and P3(I) resulted in the complete elimination of transcriptional induction by either 8-Br-cAMP or the catalytic subunit of protein kinase A. To examine the proteins involved in this response, we replaced CRE-1, which binds both C/EBP and cAMP-responsive element binding protein (CREB), with an optimal C/EBP binding sequence which significantly decreased the binding of CREB, but maintained the affinity for C/EBP. Transcription from this modified promoter was induced by 8-Br-cAMP and the catalytic subunit of protein kinase A (PKA) to a similar extent as noted with the native PEPCK promoter. However, the results of experiments involving cotransfection of PEPCK-CAT with expression vectors for PKA and either C/EBP or CREB suggest that CREB is capable of mediating a greater responsiveness to PKA than C/EBP. Our results indicate that multiple cis elements are involved in the cAMP induction of PEPCK gene transcription and that C/EBP and CREB are potentially involved in this response.