Identification of O-GlcNAc sites within peptides of the Tau protein and their impact on phosphorylation

Abstract

Phosphorylation of the microtubule-associated Tau protein plays a major role in the regulation of its activity of tubulin polymerization and/or stabilization of microtubule assembly. A dysregulation of the phosphorylation/dephosphorylation balance leading to the hyperphosphorylation of Tau proteins in neurons is thought to favor their aggregation into insoluble filaments. This in turn might underlie neuronal death as encountered in many neurodegenerative disorders, including Alzheimer's disease. Another post-translational modification, the O-linked β-N-acetylglucosaminylation (O-GlcNAcylation), controls the phosphorylation state of Tau, although the precise mechanism is not known. Moreover, analytical difficulties have hampered the precise localization of the O-GlcNAc sites on Tau, except for the S400 site that was very recently identified on the basis of ETD-FT-MS. Here, we identify three O-GlcNAc sites by screening a library of small peptides sampling the proline-rich, the microtubule-associated repeats and the carboxy-terminal domains of Tau as potential substrates for the O-β-N-acetylglucosaminyltransferase (OGT). The in vitro activity of the nucleocytoplasmic OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Using phosphorylated peptides, we establish the relationship between phosphate and O-GlcNAc incorporation at these sites. Phosphorylation of neighboring residues S396 and S404 was found to decrease significantly S400 O-GlcNAcylation. Reciprocally, S400 O-GlcNAcylation reduces S404 phosphorylation by the CDK2/cyclinA3 kinase and interrupts the GSK3β-mediated sequential phosphorylation process.

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

“…Thus, we propose that 1,048 protein sequences [1,286 total (pSp/T)P(V/A/T)(S/T) sequences minus 238 (S/T)P(V/A/T)(S/T)P sequences] in the PhosphoSitePlus database may undergo phosphorylation/ O-GlcNAcylation crosstalk. This may even underrepresent the number of proteins that could undergo crosstalk at adjacent sites, as other motifs, such as (S/T)PG(S/T) in the case of Tau (33), and quite different sequences, such as res55-58 (LLPT) in the case of c-myc (11), can also participate in phosphorylation/O-GlcNAcylation crosstalk. In addition, a more global form of crosstalk also occurs whereby the enzymes involved in the crosstalk reported herein themselves contain PTMs that can alter their function (31,36).…”

“…Data that underscore our finding that Pro at P+1 prevents promiscuous O-GlcNAcylation come from experiments performed on the microtubule-associated protein Tau (33). Based on prediction software (34) and the best known O-GlcNAcylation motif (22), O-GlcNAcylation is predicted to occur on Ser202, Thr205, and Ser208 in Tau ND, no O-GlcNAcylation detected; x, two heptad repeats from RNA polymerase II; thus this 14mer peptide represents any/all of these repeats.…”

The role of protein glycosylation in Alzheimer disease

“…As O-GlcNAcylation and phosphorylation occur reciprocally, it is possible that a decrease in O-GlcNAcylation precedes the hyperphosphorylation of tau in AD brain [66,67]. Although many O-GlcNAcylation sites are believed to exist in tau, only a few have been experimentally identified [68][69][70]. For a schematic illustration, see Fig.…”

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

“…This relationship led to the hypothesis that a key role for O-GlcNAc is to interfere with phosphorylation (7). In fact, experimental evidence suggests that the interplay between these two modifications is complex, involving both negative and positive associations (8)(9)(10). Another documented function for O-GlcNAc is to disrupt binding interactions of the modified proteins, presumably by steric interference (11).…”