Mitotic arrest with nocodazole induces selective changes in the level of O-linked N-acetylglucosamine and accumulation of incompletely processed N-glycans on proteins from HT29 cells.
O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant protein modification found on nuclear and cytoplasmic proteins. Several lines of evidence suggest that it is a highly dynamic modification and that the levels of this sugar on proteins may be regulated. Previous workers (Chou, C. F., and Omary, M. B. (1993) J. Biol. Chem. 268, 4465-4472) have shown that mitotic arrest with microtubule-destabilizing agents such as nocodazole causes an increase in the O-GlcNAc levels on keratins in the human colon cancer cell line HT29. We have sought to determine whether this increase in glycosylation is a general (i.e. occurring on many proteins) or a limited (i.e. occurring only on the keratins) process. A general increase would suggest that the microtubule-destabilizing agents were somehow affecting the enzymes responsible for addition and/or removal of O-GlcNAc. Our results suggest that the changes in O-GlcNAc induced by nocodazole are selective for the keratins. The levels of O-GlcNAc on other proteins, including the nuclear pore protein p62 and the transcription factor Sp1, are not significantly affected by this treatment. In agreement with these findings, nocodazole treatment caused no change in the activity of the enzymes responsible for addition or removal of O-GlcNAc as determined by direct in vitro assay. Interestingly, nocodazole treatment did cause a dramatic increase in modification of N-glycans with terminal GlcNAc residues on numerous proteins. Potential mechanisms for this and the change in glycosylation of the keratins are discussed.
Haltiwanger
R S
RS
Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-5215, USA.
Philipsberg
G A
GA
D000117:Acetylglucosamine / Q000378:metabolism*; D000581:Amidohydrolases / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D019073:HT29 Cells; D006801:Humans; D007633:Keratins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects*; D009363:Neoplasm Proteins / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology*; D028861:Nuclear Pore Complex Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism
D000970:Antineoplastic Agents; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D028861:Nuclear Pore Complex Proteins; D011134:Polysaccharides; D016329:Sp1 Transcription Factor; C049312:nuclear pore protein p62; D007633:Keratins; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D015739:Nocodazole; D000117:Acetylglucosamine