Structural analysis of α-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 α-enolase gene. α-Enolase is a 48-kDa protein, which plays a critical role in the glycolytic pathway. MBP-1 binds to the c- myc P2 promoter and down-regulates c-myc expression. We have investigated the role of α-enolase in regulation of the c-myc protooncogene. RNase protection assay shows that α-enolase is transcribed into a single RNA species in HeLa cells. A start codon, 400 base pairs downstream of the α-enolase ATG, corresponds to the MBP-1 ATG, suggesting that MBP-1 is an alternative translation initiation product of the α-enolase RNA. Domain mapping was performed using constructs containing truncations of the α-enolase gene. In vitro binding to the c-myc gene was abolished after deletion of the N- terminal portion of α-enolase. In order to determine the relationship between DNA binding activity and transcription inhibition, we performed co- transfection assays in HeLa cells. These studies confirmed that an N-terminal deletion of α-enolase is unable to downregulate c-myc promoter activity. Our data suggest that α-enolase plays an important role in regulation of c-myc promoter activity in the form of an alternative translation product MBP-1, which is distinct from its role as a glycolytic enzyme.