Mitotic arrest-associated enhancement of O-linked glycosylation and phosphorylation of human keratins 8 and 18.
Arrest of the human colonic cell line HT29 at the G2/M phase of the cell cycle resulted in changes in keratin assembly that were coupled with a significant increase in the O-linked glycosylation and serine phosphorylation of keratin polypeptides 8 and 18 (K8/18). With mitotic arrest, enhanced keratin phosphorylation occurred preferentially on K8, whereas K18 showed a higher glycosylation level than K8. Removal of the arresting agent allowed cells to proceed through the cell cycle with a concomitant decrease in K8/18 glycosylation. In contrast, keratins isolated from S phase-enriched cells, obtained after synchronization with aphidicolin, did not show enhanced glycosylation. Tryptic peptide analysis of keratins in G2/M-arrested cells showed changes in the glycopeptide pattern of K8 and in the phosphopeptide patterns of K8 and K18. Labeling of K8/18 immunoprecipitates, isolated from G2/M-arrested cells, with [3H]galactose followed by beta-elimination showed that K8/18 glycosylation consisted of single N-acetylglucosamine residues. Threonine was identified as the site of glycosylation after comparing acid hydrolysis products of beta-eliminated and non-beta-eliminated K8 and K18. Specific cleavage at tryptophan residues indicated that K18 glycosylation and phosphorylation were restricted to the head and proximal rod domains, whereas K8 did not show the same restriction. Our results show a unique association of the single O-linked N-acetylglucosamine type of modification of keratins with mitotic arrest in HT29 cells. There was no reciprocal relationship between K8/18 glycosylation and phosphorylation, and each keratin showed a preferential G2/M cell cycle-associated increase in either serine phosphorylation or threonine glycosylation.
Chou
C F
CF
Palo Alto Veterans Administration Medical Center, California.
Omary
M B
MB
D016590:Aphidicolin / Q000494:pharmacology; D002453:Cell Cycle; D002460:Cell Line; D003703:Demecolcine / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D007633:Keratins / Q000378:metabolism*; D008938:Mitosis; D010449:Peptide Mapping; D010766:Phosphorylation
D016590:Aphidicolin; D007633:Keratins; D003703:Demecolcine