Abstract
To investigate the mechanisms by which O-linked β-N-acetylglucosamine 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 stress-induced expression of 18 molecular chaperones, including HSP72, were reduced. GSK-3β promotes apoptosis through numerous pathways, including phosphorylation of heat shock factor 1 (HSF1) at Ser(303) (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-3β in OGT null cells recovers HSP72 expression, suggests that O-GlcNAc regulates the activity of GSK-3β. In OGT null cells, stress-induced inactivation of GSK-3β by phosphorylation at Ser(9) was ablated providing a molecular basis for these findings. Together, these data suggest that stress-induced GlcNAcylation increases HSP expression through inhibition of GSK-3β.
Highlights
O-GlcNAc is catalyzed by the uridine diphospho-N-acetylglucosamine:peptide -N-acetylglucosaminyl transferase (OGT)6 (EC 2.4.1.-) [8, 9] and a neutral O-GlcNAc-specific hexosaminidase (O-GlcNAcase; EC 3.2.1.52) (10 –13), respectively
Generation of an Inducible O-GlcNAc transferase (OGT) Knock-out Cell Line—Previously, we have shown that O-GlcNAc levels can be reduced by overexpression of Cre-recombinase in mouse embryonic fibroblasts (MEFs) carrying an Ogt exon flanked by loxP recombination sites (MEFs (OGTF/ Y))
1.51 a p value Ͻ0.01. p values derived from t test comparing chaperone expression in wild-type and OGT null cells are each time point. b p value Ͻ0.05 derived from t test comparing chaperone expression in wild-type and OGT null cells are each time point. c p value Ͻ0.1 derived from t test comparing chaperone expression in wild-type and OGT null cells are each time point. d The proteins transcribed from these genes are known as HSP72
Summary
Reagents—Reagents were purchased from Sigma, unless otherwise noted. LiCl was used at 10 mM in 0.5 M HEPES, pH 7.5, 1 h prior to heat stress. For cells treated Ϯ4HT, medium containing the drug was removed 14 h prior to the experiment, the cells were washed with phosphate-buffered saline, pH 7.4, (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to remove residual 4HT, and replaced with complete medium (DMEM 1 g/liter glucose, 10% FBS, penicillin/streptomycin). Cells were extracted with sonication (5S, Setting 3, Fisher 550 Sonic dismembrator) in buffer (20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) Nonidet P-40, pH 7.5) in the presence of protease, phosphatase, and O-GlcNAcase inhibitors. Extracts (2.5 mg) were immunoprecipitated with anti-HSF1 antibody (Assay Designs; 10 g) or control rabbit immunoglobulin (Santa Cruz Biotechnology; 10 g); which were covalently coupled to tosyl-activated Dynabeads (Invitrogen) according to the manufacturer’s instructions. Immunoprecipitates were separated on 8% BisTris gels and O-GlcNAc, HSF1, Ser(P)303 HSF1, and actin were detected by immunoblot
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