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 OGlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G 1 /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 OGlcNAc). 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.

The sweet side of the cell cycle

“…Disrupting O -GlcNAc cycling via OGT or OGA overexpression severely alters M-phase progression causing a prolonged M-phase [29]. Additionally, OGA knockdown causes defects in M-phase progression and a higher incidence of delayed M-phase exit [40]. Taken together, these data point to critical roles for O-GlcNAcylation in the control of mitotic progression [29].…”

“…Furthermore, O -GlcNAc regulates histone mitotic phosphorylation. OGT/OGA overexpression and OGA knockdown reduce Ser10 Histone H3 phosphorylation by Aurora kinase B (AURKB), leading to distortions of the spindle architecture and impaired formation [40,42]. The loss of spindle fidelity increases the number of multipolar spindle cells [40,42].…”

“…OGT/OGA overexpression and OGA knockdown reduce Ser10 Histone H3 phosphorylation by Aurora kinase B (AURKB), leading to distortions of the spindle architecture and impaired formation [40,42]. The loss of spindle fidelity increases the number of multipolar spindle cells [40,42]. Intriguingly, spindle fidelity can be rescued after inhibiting O -GlcNAc turnover in OGT/OGA overexpressing cells [42].…”

O-GlcNAc glycosylation stoichiometry of the FET protein family: only EWS is glycosylated with a high stoichiometry

“…O-GlcNAc glycosylation is a reversible and dynamic post-translational modification of the Ser/Thr residues of nuclear, cytosolic, and mitochondrial proteins found in multicellular organisms, and is an important modulator of a wide range of cellular functions throughout life. 12,13) We and others have reported that EWS is an O-GlcNAc glycosylated protein [14][15][16][17][18] but there are no reports on the glycosylation status of either FUS or TAF15. In the present study, the first comparative analysis of the glycosylation stoichiometry of FET proteins was performed.…”

O-GlcNAc: a novel regulator of immunometabolism

“…Finally, O-GlcNAcylation is essential for T cell malignancy, since T cell specific OGT knockout prevented malignant transformation of PTEN-knockout thymocytes (Swamy et al 2016). However, this is likely due more to O-GlcNAc’s essential role in organizing the mitotic spindle and regulating cellular division than in regulating T cell cancers specifically (Tan et al 2013; Slawson et al 2005; Lanza et al 2016). Many unanswered questions remain in our understanding of how O-GlcNAc regulates CD4 + T cell differentiation and how aberrant O-GlcNAcylation may promote inflammatory diseases.…”