FUNCTIONAL-CHARACTERIZATION OF THE L-TYPE PYRUVATE-KINASE GENE GLUCOSE RESPONSE COMPLEX

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose /insulin response element (GIRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contig uous binding site (element L3). We present in this report the identifi cation of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promot er. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The IA site binds the major late transcription factor (ML TF) in vitro and ex vivo; mutations that suppress this binding activit y also inactivated the GIRE function. Mutations transforming one or tw o noncanonical E boxes of element IA into consensus MLTF/USF binding s ites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GIRE activity: those elemen ts in which one E box has been destroyed and the other has been transf ormed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter . Consequently, the full activity of the L-PK GIRE seems to require th e cooperation between two putative MLTF/USF binding sites located in t he vicinity of an HNF4 binding site.