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.
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.
Kamemura
Kazuo
K
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
Hayes
Bradley K
BK
Comer
Frank I
FI
Hart
Gerald W
GW
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D008223:Lymphoma / Q000235:genetics*; D008934:Mitogens / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
D000906:Antibodies; D017931:DNA Primers; D008934:Mitogens; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; D013912:Threonine