CARBOHYDRATE REGULATION OF THE RAT L-TYPE PYRUVATE-KINASE GENE REQUIRES 2 NUCLEAR FACTORS - LF-A1 AND A MEMBER OF THE C-MYC FAMILY

Transcription of the L-type pyruvate kinase (L-PK) gene is induced in response to increased carbohydrate metabolism in the liver. We have de monstrated previously that a segment of the 5'-flanking region of the L-PK gene between -183 and -96 is necessary and sufficient for the glu cose response in primary hepatocytes. To explore the protein factors t hat are involved in carbohydrate regulation, we have performed mutatio nal analyses and in vitro binding studies of this segment. Sequences c ritical for the glucose response were mapped from -171 to -124. This s egment contains the consensus binding sites for two nuclear transcript ion factors: LF-A1 and MLTF. Both factors are capable of binding to th e corresponding L-PK sites in vitro. Mutational and functional analyse s indicated that LF-A1 is indeed involved in glucose induction of the L-PK gene. The PK MLTF-like site consists of two imperfect CACGTG moti fs, the core binding site for a family of transcription factors relate d to c-myc. Unexpectedly, mutations in either motif that resulted in d efective glucose stimulation retained in vitro binding to MLTF. Furthe rmore, an authentic MLTF binding site from the adenovirus major late p romoter was not functionally interchangeable with the natural sequence . These data indicate that binding of MLTF, in presence of LF-A1, is n ot capable of supporting the glucose response. Conversion of either im perfect motif to CACGTG within the context of the mutations disrupting the opposite site restored the response to elevated glucose. Thus, th e factor that recognizes the PK MLTF-like site and participates in med iating the carbohydrate response of the L-PK gene appears to be a memb er of the c-myc family distinct from MLTF.