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 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 alpha-enolase in regulation of the c-myc protooncogene. RNase protection assay shows that alpha-enolase is transcribed into a single RNA species in HeLa cells. A start codon, 400 base pairs downstream of the alpha-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 deletion of the N-terminal portion of alpha-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 alpha-enolase is unable to down-regulate c-myc promoter activity. Our data suggest that alpha-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.
Subramanian
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Comprehensive Cancer Center, Department of Biochemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294-3300, USA.
Miller
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D014408:Biomarkers, Tumor; D015152:Blotting, Northern; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D017353:Gene Deletion; D016259:Genes, myc / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000502:physiology*; D010957:Plasmids; D014176:Protein Biosynthesis; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014162:Transfection; D025521:Tumor Suppressor Proteins
D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D008156:Luciferases; D012260:Ribonucleases; C095157:ENO1 protein, human; D010751:Phosphopyruvate Hydratase