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 accumulated which suggests that a liver-enriched
transcription factor, likely a member of the
CCAAT/enhancer binding protein (C/EBP) family, is required along with other ubiquitously expressed
transcription factors to mediate this liver-specific response to cAMP. In this study, we examined the ability of C/EBP to participate in the cAMP-mediated activation of
phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in
hepatoma cells. Expression of a dominant repressor of C/EBP in
hepatoma cells significantly inhibited the
protein kinase A-stimulated transcription of the PEPCK promoter, suggesting that a C/EBP family member was required for maximal transcriptional activation by
protein kinase A. To provide additional support for this hypothesis, we prepared GAL4 fusion
proteins containing C/EBP domains. Both
C/EBPalpha and
C/EBPbeta 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/EBPalpha 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/EBPalpha was not required for this activity in
hepatoma cells, although in non-
hepatoma cells the basic region leucine zipper domain appeared to inhibit the ability of
C/EBPalpha to participate in mediating cAMP responsiveness. These results suggest that the liver-specific nature of the cAMP responsiveness of the PEPCK promoter involves the recruitment of
C/EBPalpha to the cAMP response unit.