A PHORBOL ESTER-INSENSITIVE AP-1 MOTIF MEDIATES THE STIMULATORY EFFECT OF INSULIN ON RAT MALIC ENZYME GENE-TRANSCRIPTION

In liver, insulin stimulates the transcription of the gene encoding th e cytosolic form of malic enzyme (ME) and modulates protein binding to two putative insulin response sequences (IRSs) in the ME promoter. On e of these IRSs resembles that identified in the phosphoenolpyruvate c arboxykinase (PEPCK) gene, whereas the other resembles that defined in the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. To assess the functional significance of these changes in protein binding, a ser ies of truncated ME-chloramphenicol acetyltransferase (CAT) fusion gen es were transiently transfected into rat H4IIE hepatoma cells. Deletio n of the PEPCK-like IRS motif had no effect on the stimulation of CAT expression by insulin. Instead, the stimulatory effect of insulin was mediated through an AP-1 motif and an Egr-1 binding site that overlaps the GAPDH-like IRS motif. Both the ME AP-1 motif and the AP-1 motif i dentified in the collagenase-1 gene promoter were able to confer a sti mulatory effect of insulin on the expression of a heterologous fusion gene, but surprisingly only the latter was able to confer a stimulator y effect of phorbol esters. Instead, the data suggest that AP-1 binds the ME AP-1 motif in an activated state such that phorbol ester treatm ent has no additional effect. The collagenase and ME AP-1 motifs were both shown to bind mainly Jun D and Fra-2, with similar affinities. Ho wever, the results of a proteolytic clipping bandshift assay suggest t hat these proteins bind the collagenase and ME AP-1 motifs in distinct conformations, which potentially explain the differences in phorbol e ster signaling through these elements.