OGT is associated with the spindle and midbody. (A and B) OGT localizes to the mitotic spindle, then to the midbody during M phase in HeLa cells as seen by immunostaining (green, OGT; red, DNA). (C) Overexpression of OGT causes polyploidy (green, OGT; red, DNA). (D) Immunostaining of purified spindles shows that O-GlcNAc–modified proteins localize to the periphery of the mitotic spindle–centrosome (labeled as 1), along the spindle (labeled as 2), and a small amount in the spindle midzone (labeled as 3) (green, α-tubulin; red, O-GlcNAc). (E) O-GlcNAc–modified proteins are enriched at the purified midbody and at the nascent nuclear envelope (green, α-tubulin; red, O-GlcNAc). (F) Immunoblot for O-GlcNAc shows enrichment in the mitotic Taxol pellet (TP) and OGT overexpression increases protein O-GlcNAcylation. (G) Mitotic phosphorylation, determined by immunoblot to proline-directed phosphorylation (MPM-2), is disrupted by OGT overexpression. (H) OGT overexpression alters the abundance of a few proteins in the Taxol pellet samples (stained with Coomassie blue dye). All blots were loaded with equal amounts of protein. (I) α-Tubulin and AURKB, markers of Taxol-stabilized spindle midbodies, are enriched in the Taxol pellet. GFP was used as a control for the effects of the overexpression procedure. Experiments shown are representatives of a minimum of three times.

OGT overexpression alters the abundance of few proteins that are associated with the spindle and midbody. (A) HeLa cells infected with GFP were cultured in SILAC medium containing no heavy isotope; cells infected with OGT were cultured in SILAC medium containing heavy isotope. After purification and mass spectral analysis, the pie chart displays the ratios of (OGT spindle and midbody/GFP spindle and midbody sample) protein abundance for the more than 700 proteins identified in the MS analysis. Eight percent of identified proteins show increased abundance of more than 200% and 1% show a decrease of more than 50% in abundance. (B)The 700 proteins at the spindle and midbody represent numerous different functional classes as categorized by UniProt. (C) Proteins with a 200% increase in abundance upon OGT overexpression fall into multiple functional classes. (D) Molecular motor proteins and adaptor proteins constitute large proportions of the proteins that decrease in abundance by greater than 50%. (E) Schematic of the chromosomal passenger protein complex that associates with OGT and OGA. (F) Immunoblot (IB) analysis of HeLa synchronized by thymidine-nocodazole from cells overexpressing either GFP or OGT shows that overexpression of OGT causes a decrease in the abundance of several proteins in the chromosomal passenger protein complex. (G) Immunoblotting confirms that the abundance of several proteins is unchanged in cells overexpressing OGT.

OGT overexpression triggers changes in mitotic phosphorylation and O-GlcNAcylation. (A) Phosphopeptides were enriched from pooled spindle-midbody extracts by purification over a titanium dioxide column. OGT overexpression decreased phosphorylation by more than 50% at 17% of the identified phosphopeptides, whereas only 7% of phosphorylation sites showed an increase of more than 200%. (B) Identified phosphorylation sites (397) occur on proteins that fall into similar functional classes. (C) Of the identified O-GlcNAcylation sites, 41% showed increased site modification of more than 200% in preparations from cells overexpressing OGT compared to cells overexpressing GFP, whereas only 4% of the O-GlcNAc sites showed decreased modification by more than 50%. (D) The GlcNAc-modified sites were predominately found on transcriptional regulators; however, RNA processing, nuclear pore, and cytoskeleton proteins also contained a large number of GlcNAcylation sites. (E) Functional classes of proteins whose O-GlcNAc site is also a reciprocal phosphorylation site. (F) Functional class of proteins that have a phosphorylation site within ± 10 amino acids proximal to an O-GlcNAcylation site.

Validation of O-GlcNAcylation site mapping. (A, C, and E)FETDMS/MS spectra recorded on (A) [M + 4H]⁺⁴ ions (m/z 815.6) for the modified peptide, gSTEANVLPPSgSIGFTFSVPVAK, from NUP153; (C) [M + 3H]⁺³ ions (m/z 531.9) for the modified peptide, TITVPVgSGSPK, from EMSY; (E) [M + 3H]⁺³ ions (m/z 499.2) for the modified peptide, SAPAgSQASLR, from NUMA. Abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; and V, Val. Predicted m/z values for product ions of type c′ and type z′•(monoisotopic and average masses for singly and doubly charged ions, respectively) are listed above and below the peptide sequence. Observed product ions are underlined and also labeled in the spectrum. Ions in the precursor isolation window are labeled with a triangle (▼). Brackets enclose ions corresponding to charge-reduced species, as well as fragments derived from these charge-reduced species. Product ions resulting from loss of an aminomethyl triazole radical are labeled with a circle (●). Insert displays the isotopic distribution, calculated monoisotopic mass, and experimental monoisotopic mass of precursor ions corresponding to the labeled and unlabeled forms of the indicated peptide. (B, D, and F) Nup153, NuMA1, or EMSY were immunopurified from lysates of thymidine-nocodazole synchronized cells overexpressing either GFP or OGT. Precipitates were washed, separated on SDS-PAGE, and transferred to membranes for blotting. Membranes were probed for O-GlcNAc. For Nup153 and NuMA1, the increase in O-GlcNAc in the samples from the OGT-overexpressing cells is evident. For EMSY, the increase is not readily apparent, which may be due to only one of seven O-GlcNAcylation sites exhibiting increased modification.

NuMA1 localization is disrupted in cells exhibiting aneuploidy. HeLa cells overexpressing OGT were released for 12 hours from double thymidine block, then fixed and stained for α-tubulin (green) or NuMA1 (red). In cells with normal number of mitotic spindles, NuMA1 is at the centromeres (top panel). However, as the number of spindles increase (middle and bottom panel), a subset of NuMA1 appears to localize to the membrane (arrows) of cells and less NuMA1 is associated with the centromeres.

OGT overexpression disrupts the CDK1 activation circuit. (A and B) CAD MS/MS spectra of phosphopeptides from CDK1 containing pThr¹⁴ (A) or pTyr¹⁵ (B). Insets show the relative abundances of the precursor ions originating from the two samples. (C) Immunoblotting of thymidine-nocodazole lysates with a phosphorylation-specific antibody to Thr¹⁴ on CDK1 (pT14-CDK1) reveals increased phosphorylation at this site in OGT-overexpressing cells. The total amount of CDK1 was slightly lower in OGT-expressing cells compared to GFP-expressing controls. All blots were performed a minimum of three times with different lysates. (D) Immunoblotting with an antibody that recognizes proteins phosphorylated on Ser residue within a CDK1 consensus phosphorylation motif shows that CDK1 activity is decreased in OGT-overexpressing cells. (E) Immunoblotting with an antibody that recognizes nucleophosmin (NPM) phosphorylated on Thr²³⁴ and Thr²³⁷ shows that phosphorylation of this protein is decreased in OGT-overexpressing cells. (F) Immunoblotting of phosphorylated (pThr⁴⁹⁵) and total MYT1 shows that phosphorylation of this protein is decreased and its abundance is increased in OGT-overexpressing cells. (G) Immunoblotting of phosphorylated (pThr²¹⁰) and total PLK1 shows that the abundance of PLK1 is decreased in OGT-overexpressing cells. (H) Relative mRNA amounts at M phase of CDK1, CDC25, PLK1, and MYT1 in cells overexpressing OGT. All transcripts were normalized to actin.

Model illustrating how OGT overexpression affects cell cycle progression. OGT overexpression causes a decrease in the protein and mRNA abundance of PLK1, although the amount of PLK1's activating phosphorylation is not changed. MYT1, a target of PLK1, displays an increase in protein amount but a reduction in phosphorylation, whereas the other PLK1 target, CDC25C, shows a decrease in mRNA amount. MYT1 phosphorylates CDK1, inhibiting CDK1 activity, and leading to a decrease in CDK1 mitotic phosphorylation on numerous substrates. Thus, the increase in CDK1 inhibitory phosphorylation leads to a reduction in the phosphorylation of CDK1 substrates.