Structural analysis of alpha-enolase - Mapping the functional domains involved in down-regulation of the c-myc protooncogene

Myc-binding protein-1 (MBP-1) is a 37-kDa protein with sequence homology to the 3' portion of the alpha-enolase gene. alpha-Enolase is a 48-kDa protei n, which plays a critical role in the glycolytic pathway. MBP-1 binds to th e c-myc P2 promoter and down-regulates c-myc expression. We have investigat ed the role of alpha-enolase in regulation of the c-myc protooncogene, RNas e protection assay shows that alpha-enolase is transcribed into a single RN A species in HeLa cells. A start codon, 400 base pairs downstream of the al pha-enolase ATG, corresponds to the MBP-1 ATG, suggesting that MBP-1 is an alternative translation initiation product of the alpha-enolase RNA. Domain mapping was performed using constructs containing truncations of the alpha -enolase gene, In vitro binding to the c-myc gene was abolished after delet ion of the N-terminal portion of alpha-enolase. In order to determine the r elationship between DNA binding activity and transcription inhibition, we p erformed co-transfection assays in HeLa cells, These studies confirmed that an N-terminal deletion of alpha-enolase is unable to downregulate c-myc pr omoter activity. Our data suggest that Lu-enolase plays an important role i n regulation of c-myc promoter activity in the form of an alternative trans lation product MBP-1, which is distinct from its role as a glycolytic enzym e.