C-MYC IS GLYCOSYLATED AT THREONINE-58, A KNOWN PHOSPHORYLATION SITE AND A MUTATIONAL HOT-SPOT IN LYMPHOMAS

c-Myc is a helix-loop-helix leucine zipper phosphoprotein that heterod imerizes with Max and regulates gene transcription in cell proliferati on, cell differentiation, and programmed cell death, Previously, we de monstrated that c-Myc is modified by O-linked N-acetylglucosamine (O-G lcNAc) within or nearby the N-terminal transcriptional activation doma in (Chou, T.-Y., Dang, C. V., and Hart, G. W. (1995) Proc, Natl, Acad, Sci, U.S.A. 92, 4417-4421), In this paper, we identified the O-GlcNAc attachment site(s) on c-Myc, c-Myc purified from sf9 insect cells was trypsinized, and its GlcNAc moieties were enzymically labeled with [H -3]galactose, The [H-3]galactose-labeled glycopeptides were isolated b y reverse phase high performance liquid chromatography and then subjec ted to gas-phase sequencing, manual Edman degradation, and laser desor ption/ionization mass spectrometry. These analyses show that threonine 58, an in vivo phosphorylation site in the transactivation domain, is the major O-GlcNAc glycosylation site of c-Myc. Mutation of threonine 58, frequently found in retroviral v-Myc proteins and in human Burkit t and AIDS-related lymphomas, is associated with enhanced transforming activity and tumorigenicity. The reciprocal glycosylation and phospho rylation at this biologically significant amino acid residue may play an important role in the regulation of the functions of c-Myc.