Analysis of keratin post-translational modifications during cell division

Cell division is one of the most basic cellular processes which is required for the propagation of life. Any mistakes during cell division may lead to cell death, developmental disorders, abortion, or complex diseases like cancer. Mitotic kinases are the main drivers of mitosis and cytokinesis. It is estimated that 30% of the mitotic proteome is phosphorylated. Keratins are the members of intermediate filaments that are functionally regulated by post-translational modifications (PTM) including phosphorylation, glycosylation, sumoylation and acetylation. Keratins form highly stable polymers in epithelial cells that need to be cut through during cell division. However, it is not known how PTMs of Keratins are regulated and how the spatial organizations of Keratins are controlled during cell division.

In this study, we profiled the cell cycle-dependent phosphorylation sites on Keratins in Hela cells. We used TiO2-based phosphopeptide enrichment to isolate the phosphopeptides from mitosis, cytokinesis, and interphase cell lysates, and then analyzed them using HF Q Exactive Mass spectrometry. Our analysis revealed multiple phosphorylation sites specific to mitosis and cytokinesis, particularly in simple epithelial keratins like K8 and K18. We conducted in vitro kinase assays and identified eleven phosphorylation sites on Keratin 8 that are dependent on Aurora B kinase. One of these, Keratin 8-Ser 34, is specifically phosphorylated at the cleavage furrow and remains present at the midzone until the end of cytokinesis. Inhibiting Aurora B kinase or using a non-phosphorylatable Keratin 8 mutant resulted in residual keratin bundles at the cleavage furrow and a failure in furrow ingression.

Next, we systematically examined O-glycosylation which can compete with phosphorylation for the same residue on Keratins. For this, glycosylated peptides from mitotic and interphase lysates of Hela cells are enriched with phenyl-boronic acid-based resins. The samples were run in Orbitrap Ascend Tribrid Mass Spectrometry with EThcd based fragmentation and analyzed in MSFragger-Glyco in Fragpipe. We identified previously reported and also novel O-GlcNAc sites on Keratins, some of which are regulated in a cell cycle-dependent manner. Both co-regulated and counter-regulated glycosylation and phosphorylation sites are present when interphase and mitosis cell cycle stages are compared. Molecular analysis of these dynamic sites will provide insight into PTM crosstalks during cell division. This study offers new insights into the remodeling of Keratins through post-translational modifications in the cell division of epithelial lineages.