Mitotic arrest with anti-microtubule agents or okadaic acid is associated with increased glycoprotein terminal GlcNAc's

Abstract

The two major intermediate filament glycoproteins in human simple epithelia are keratins 8 and 18 (K8/18). A dramatic increase in terminal N-acetylglucosamine (GlcNAc) residues in K8/18 was previously noted after arresting cells in G2/M using anti-microtubule agents. Here we use in vitro galactosylation to show that increased terminal GlcNAc's is a general phenomenon that occurs in glycoproteins isolated from nuclear and plasma membrane fractions after cells are arrested in mitosis using colcemid, nocodazole, or okadaic acid. All three agents also resulted in a hyperphosphorylated form of K8 as determined by phosphatase treatment and tryptic phosphopeptide mapping. The altered glycosylation was found to be independent of microtubule disassembly, and was not directly related to the G2/M phase of the cell cycle after aphidicolin synchronization. Staurosporine (1 microM) inhibited K8/18 phosphorylation in okadaic acid- or nocodazole-treated cells, and inhibited the increase in K8/18 glycosylation without inhibiting the increase in terminal GlcNAc's of membrane-associated glycoproteins. In contrast, brefeldin A resulted in a dramatic increase in terminal GlcNAc's of membrane-associated but not intermediate filament proteins. Golgi complex-related staining using anti-beta-COP antibody showed significant fragmentation under conditions associated with altered membrane protein glycosylation. Our results suggest that Golgi disruption may be involved in the observed increase in terminal GlcNAc's of membrane but not intermediate filament glycoproteins. The mechanism of increased glycoprotein terminal GlcNAc's in association with mitotic arrest appears to be distinct for intermediate filaments and membrane-associated proteins, and in the case of intermediate filament proteins, phosphorylation may play an important role. Some of the effects of agents that induce mitotic arrest may be mediated by glycosylation changes.