THE ALPHA-ISOFORM OF THE CCAAT ENHANCER-BINDING PROTEIN IS REQUIRED FOR MEDIATING CAMP RESPONSIVENESS OF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE PROMOTER IN HEPATOMA-CELLS/

The gene coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1. 32) is expressed in all gluconeogenic tissues, but stimulation of its rate of transcription by cAMP is robust only in liver, Evidence has ac cumulated which suggests that a liver-enriched transcription factor, l ikely a member of the CCAAT/enhancer binding protein (C/EBP) family, i s required along with other ubiquitously expressed transcription facto rs to mediate this liver-specific response to cAMP. In this study, we examined the ability of C/EBP to participate in the cAMP-mediated acti vation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in hepatoma cells, Expression of a dominant repressor of C/EBP in hep atoma cells significantly inhibited the protein kinase A-stimulated tr anscription of the PEPCK promoter, suggesting that a C/EBP family memb er was required for maximal transcriptional activation by protein kina se A. To provide additional support for this hypothesis, we prepared G AL4 fusion proteins containing C/EBP domains. Both C/EBP alpha and C/E BP beta GAL4 fusion proteins were capable of stimulating transcription from promoters containing binding sites for the DNA-binding domain of GAL4, However, only the GAL4-C/EBP alpha fusion protein demonstrated the ability to synergize with the other transcription factors bound to the PEPCK promoter which are required to mediate cAMP responsiveness, The DNA-binding domain of C/EBP alpha was not required for this activ ity in hepatoma cells, although in non-hepatoma cells the basic region leucine zipper domain appeared to inhibit the ability of C/EBP alpha to participate in mediating cAMP responsiveness, These results suggest that the liver-specific nature of the cAMP responsiveness of the PEPC K promoter involves the recruitment of C/EBP alpha to the cAMP respons e unit.