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bioRxiv · 10.1101/2022.08.21.504716

PLK1 O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma

Yan, S.·Peng, B.·Kan, S.·Shao, G.·Xiahou, Z.·Tang, X.·Chen, Y.-x.·Dong, M.-Q.·Liu, X.·Xu, X.·Li, J.
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Abstract

The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification, whose function and substrates have entranced biologists and chemists alike. O-GlcNAcylation occurs on Ser/Thr residues and takes part in a vast array of physiological processes. OGT is essential for dividing mammalian cells, and it underscores many human diseases. Yet many of its fundamental substrates in the cell division process remains to be unveiled. Here we focus on its effect on Polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation and mitotic exit. We found that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (MS) and mutagenesis studies, the critical O-GlcNAc site is located to be Thr291. Interestingly, T291N is a uterine carcinoma mutant in the TCGA database. Biochemical assays show that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we show that PLK1-T291A and -T291N mutants display chromosome segregation defects, and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and -T291N mutants would enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, and sceHCD MS might be a new method to reveal many more O-GlcNAcylation substrates.

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Yan, S., Peng, B., Kan, S., Shao, G., Xiahou, Z., Tang, X., Chen, Y.-x., Dong, M.-Q., Liu, X., Xu, X., Li, J.. 2022-08-22. PLK1 O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma. https://doi.org/10.1101/2022.08.21.504716

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