title: Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects.
abstract: Alterations in O-GlcNAc cycling, the addition and removal of O-GlcNAc, lead to mitotic defects and increased aneuploidy. Herein, we generated stable O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G1/S, the OGA knockdown cells progressed normally through S phase but demonstrated mitotic exit defects. Cyclin A was increased in the knockdown cells while Cyclin B and D expression was reduced. Retinoblastoma protein (RB) phosphorylation was also increased in the knockdown compared to control. At M phase, the knockdown cells showed more compact spindle chromatids than control cells and had a greater percentage of cells with multipolar spindles. Furthermore, the timing of the inhibitory tyrosine phosphorylation of Cyclin Dependent Kinase 1 (CDK1) was altered in the OGA knockdown cells. Although expression and localization of the chromosomal passenger protein complex (CPC) was unchanged, histone H3 threonine 3 phosphorylation was decreased in one of the OGA knockdown cell lines. The Ewing Sarcoma Breakpoint Region 1 Protein (EWS) participates in organizing the CPC at the spindle and is a known substrate for O-GlcNAc transferase (OGT, the enzyme that adds O-GlcNAc). EWS O-GlcNAcylation was significantly increased in the OGA knockdown cells promoting uneven localization of the mitotic midzone. Our data suggests that O-GlcNAc cycling is an essential mechanism for proper mitotic signaling and spindle formation, and alterations in the rate of O-GlcNAc cycling produces aberrant spindles and promotes aneuploidy.
authors.affiliation: a Department of Biochemistry and Molecular Biology , University of Kansas Medical Center , Kansas City , KS , USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Lanza
authors.affiliation: a Department of Biochemistry and Molecular Biology , University of Kansas Medical Center , Kansas City , KS , USA.
authors.forename: Ee Phie
authors.initials: EP
authors.lastname: Tan
authors.affiliation: a Department of Biochemistry and Molecular Biology , University of Kansas Medical Center , Kansas City , KS , USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: a Department of Biochemistry and Molecular Biology , University of Kansas Medical Center , Kansas City , KS , USA.
authors.forename: Miranda
authors.initials: M
authors.lastname: Machacek
authors.affiliation: b Department of Cancer Biology , University of Kansas Medical Center , Kansas City , KS , USA.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Brinker
authors.affiliation: c KU Cancer Center, University of Kansas Medical Center , Kansas City , KS , USA.|d Department of Molecular Biosciences , University of Kansas , Lawrence , KS , USA.
authors.forename: Mizuki
authors.initials: M
authors.lastname: Azuma
authors.affiliation: a Department of Biochemistry and Molecular Biology , University of Kansas Medical Center , Kansas City , KS , USA.|c KU Cancer Center, University of Kansas Medical Center , Kansas City , KS , USA.|e Institute for Reproductive Health and Regenerative Medicine, University of Kansas Medical Center , Kansas City , KS , USA.|f KU Alzheimer Disease Center, University of Kansas Medical Center , Kansas City , KS , USA.
authors.forename: Chad
authors.initials: C
authors.lastname: Slawson
mesh_terms: D055785:Gene Knockdown Techniques / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000502:physiology*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D008941:Spindle Apparatus / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism*
chemical_list: D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases
keywords: AurB; EWS; M Phase; O-GlcNAc; O-GlcNAcylation; OGA; OGT; cell cycle; spindle