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

Kazuo Kamemura, Bradley K. Hayes, Frank I. Comer, Gerald Warren Hart

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)19229-19235
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number21
DOIs
StatePublished - May 24 2002

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of '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'. Together they form a unique fingerprint.

  • Cite this