A genetic model to study GlcNAc cycling in immortalized mouse embryonic fibroblasts.

-GlcNAcylation is an abundant posttranslational protein modification in which the monosaccharide O-GlcNAc is added to Ser/Thr residues by GlcNAc transferase and removed by GlcNAcase. Analyses of GlcNAc-mediated signaling and metabolic phenomena are complicated by factors including unsatisfactory inhibitors and loss-of-function cell lines lacking identical genetic backgrounds. In this work, we generated immortalized WT, knockout, and floxed allele ( floxed) mouse embryonic fibroblast (MEF) cell lines with similar genetic backgrounds. These lines will facilitate experiments and serve as a platform to study GlcNAc cycling in mammals. As a test paradigm, we used the immortalized MEF lines to investigate how changes in GlcNAcylation affected pathological phosphorylation of the tau protein. The activity of glycogen synthase kinase 3β (GSK3β), a kinase that phosphorylates tau, decreases when expressed in knockout MEFs compared with WT cells. Phosphorylation at Thr in recombinant, tauopathy-associated tau with a proline-to-leucine mutation at position 301 (P301L) was altered when expressed in MEFs with altered GlcNAc cycling. In aggregate, our data support that GlcNAc cycling indirectly affects tau phosphorylation at Thr, but tau phosphorylation was highly variable, even in genetically stable, immortalized MEF cells. The variable nature of tau phosphorylation observed here supports the need to use cells akin to those generated here with genetically defined lesions and similar backgrounds to study complex biological processes.