O-Linked -N-acetylglucosamine (O-GlcNAc) Regulates Stress-induced Heat Shock Protein Expression in a GSK-3 -dependent Manner*

To investigate the mechanisms by which O-linked -Nacetylglucosamine modification of nucleocytoplasmic proteins (O-GlcNAc) confers stress tolerance to multiple forms of cellular injury, we explored the role(s) of O-GlcNAc in the regulation of heat shock protein (HSP) expression. Using a cell line in which deletion of the O-GlcNAc transferase (OGT; the enzyme that adds O-GlcNAc) can be induced by 4-hydroxytamoxifen, we screened the expression of 84 HSPs using quantitative reverse transcriptase PCR. In OGT null cells the stressinduced expression of 18 molecular chaperones, including HSP72, were reduced. GSK-3 promotes apoptosis through numerous pathways, including phosphorylation of heat shock factor 1 (HSF1) at Ser303 (Ser(P)303 HSF1), which inactivates HSF1 and inhibits HSP expression. In OGT null cells we observed increased Ser(P)303 HSF1; conversely, in cells in which O-GlcNAc levels had been elevated, reduced Ser(P)303 HSF1 was detected. These data, combined with those showing that inhibition of GSK-3 in OGT null cells recovers HSP72 expression, suggests that O-GlcNAc regulates the activity of GSK-3 . In OGT null cells, stress-induced inactivation of GSK-3 by phosphorylation at Ser9 was ablated providing a molecular basis for these findings. Together, these data suggest that stress-induced GlcNAcylation increases HSP expression through inhibition of GSK-3 .