TRANSCRIPTIONAL REGULATION OF N-ACETYLGLUCOSAMINYLTRANSFERASE-V BY THE SRC ONCOGENE

Transformation of baby hamster kidney fibroblasts by the Rous sarcoma virus causes a significant increase in the GlcNAc beta(1,6)Man-branche d oligosaccharides by elevating the activity and mRNA transcript level s encoding N-acetylglucosaminyltransferase V (GlcNAc-T V). Elevated ac tivity arid mRNA levels could be inhibited by blocking cell proliferat ion with herbimycin A, demonstrating that Src kinase activity can regu late GlcNAc-T V expression. 5' RACE analysis was used to identify a 3- kilobase 5'-untranslated region from GlcNAc-T V mRNA and locate a tran scriptional start site in a 25-kilobase pair GlcNAc-T V human genomic clone, A 6-kilobase pair fragment of the 5' region of the gene contain ed AP-1 and PEA3/Ets binding elements and, when co-transfected with a src expression plasmid into HepG2 cells, conferred src-stimulated tran scriptional enhancement upon a luciferase reporter gene, This stimulat ion by src could be antagonized by co-transfection with a dominant-neg ative mutant of the Raf kinase, suggesting the involvement of Ets tran scription factors in the regulation of GlcNAc-T V gene expression. The src-responsive element was localized by 5' deletion analysis to a 250 -base pair region containing two overlapping Ets sites. src stimulatio n of transcription from this region was inhibited by co-transfection w ith a dominant-negative mutant of Ets-2, demonstrating that the effect s of the src kinase on GlcNAc-T V expression are dependent on Ets.