O-GlcNAcylation of tubulin inhibits its polymerization

The attachment of O-linked beta-N-acetylglucosamine (O-GlcNAc) to proteins is an abundant and reversible modification that involves many cellular processes including transcription, translation, cell proliferation, apoptosis, and signal transduction. Here, we found that the O-GlcNAc modification pattern was altered during all-trans retinoic acid (tRA)-induced neurite outgrowth in the MN9D neuronal cell line. We identified several O-GlcNAcylated proteins using mass spectrometric analysis, including alpha- and beta-tubulin. Further analysis of alpha- and beta-tubulin revealed that O-GlcNAcylated peptides mapped between residues 173 and 185 of alpha-tubulin and between residues 216 and 238 of beta-tubulin, respectively. We found that an increase in alpha-tubulin O-GlcNAcylation reduced heterodimerization and that O-GlcNAcylated tubulin did not polymerize into microtubules. Consequently, when O-GlcNAcase inhibitors were co-incubated with tRA, the extent of neurite outgrowth was decreased by 20% compared to control. Thus, our data indicate that the O-GlcNAcylation of tubulin negatively regulates microtubule formation.