Dynamic interplay between O-glycosylation and O-phosphorylation of nucleocytoplasmic proteins: alternative glycosylation/phosphorylation of THR-58, a known mutational hot spot of c-Myc in lymphomas, is regulated by mitogens

• Published in: The Journal of biological chemistry Vol. 277; no. 21; pp. 19229 - 19235
• Main Authors: Kamemura, Kazuo, Hayes, Bradley K, Comer, Frank I, Hart, Gerald W
• Format: Journal Article
• Language: English
• Published: United States 24.05.2002
• Subjects: Amino Acid Sequence; Antibodies - immunology; Base Sequence; Cell Line; Cytoplasm - metabolism; DNA Primers; Glycosylation; Humans; Lymphoma - genetics; Mitogens - pharmacology; Molecular Sequence Data; Nuclear Proteins - chemistry; Nuclear Proteins - metabolism; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - immunology; Proto-Oncogene Proteins c-myc - metabolism; Threonine - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M201729200

Previously, we reported that c-Myc is glycosylated by O-linked N-acetylglucosamine at Thr-58, a known phosphorylation site and a mutational hot spot in lymphomas. In this paper, we describe the production and characterization of two Thr-58 site-specific antibodies and use them to examine the modification of Thr-58 in living cells. One antibody specifically reacts with the Thr-58-glycosylated form of c-Myc, and the other reacts only with unmodified Thr-58 in c-Myc. Using these antibodies together with a commercial anti-Thr-58-phosphorylated c-Myc antibody, we simultaneously detected three forms of c-Myc (Thr-58-unmodified, -phosphorylated, and -glycosylated). It has been reported that Thr-58 phosphorylation is dependent on a prior phosphorylation of Ser-62. Mutagenesis of Ser-62 to Ala showed a marked decrease of Thr-58 phosphorylation and a marked increase of Thr-58 glycosylation. Growth inhibition of HL60 cells by serum starvation increases Thr-58 glycosylation and correspondingly decreases its phosphorylation. Serum stimulation has the opposite effect upon the modification status of Thr-58. A candidate kinase responsible for Thr-58 phosphorylation is the glycogen synthase kinase 3 (GSK3). Lithium, a competitive inhibitor of GSK3, decreased Thr-58 phosphorylation and increased its glycosylation. Finally, we show that the Thr-58-phosphorylated form of c-Myc predominantly accumulates in the cytoplasm rather than the nucleus upon inhibition of proteasome activity. These data suggest that hierarchical phosphorylation of Ser-62 and Thr-58 and alternative glycosylation/phosphorylation of Thr-58 together regulate the myriad functions of c-Myc in cells.