The dynamic stress-induced "O-GlcNAc-ome" highlights functions for O-GlcNAc in regulating DNA damage/repair and other cellular pathways

Abstract

The modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic and essential post-translational modification of metazoans. Numerous forms of cellular injury lead to elevated levels of O-GlcNAc in both in vivo and in vitro models, and elevation of O-GlcNAc levels before, or immediately after, the induction of cellular injury is protective in models of heat stress, oxidative stress, endoplasmic reticulum (ER) stress, hypoxia, ischemia reperfusion injury, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a regulator of the cellular stress response. However, the molecular mechanism(s) by which O-GlcNAc regulates protein function leading to enhanced cell survival have not been identified. In order to determine how O-GlcNAc modulates stress tolerance in these models we have used stable isotope labeling with amino acids in cell culture to determine the identity of proteins that undergo O-GlcNAcylation in response to heat shock. Numerous proteins with diverse functions were identified, including NF-90, RuvB-like 1 (Tip49α), RuvB-like 2 (Tip49β), and several COPII vesicle transport proteins. Many of these proteins bind double-stranded DNA-dependent protein kinase (PK), or double-stranded DNA breaks, suggesting a role for O-GlcNAc in regulating DNA damage signaling or repair. Supporting this hypothesis, we have shown that DNA-PK is O-GlcNAc modified in response to numerous forms of cellular stress.

Fig. 1

SILAC strategy used in this study

Fig. 2

Samples used in the SILAC study. a 20 μg of isotope labeled extract (control, heat-stressed, PUGNAc-treated), or the combined extract, were separated by SDS–PAGE and O-GlcNAc levels were detected with CTD110.6. As a control, CTD110.6 was competed away with 100 mM GlcNAc. b Proteins eluted from the CTD110.6 immunoprecipitate were separated by SDS–PAGE and detected with G250 colloidal coomassie

Fig. 3

Numerous proteins showed elevated levels of O-GlcNAc in response to either heat stress or PUGNAc treatment. a The fold increase in response to heat stress (black bars) or PUGNAc treatment (blue bars) are indicated. The red dashed line represents a 25% increase or decrease, which represents the error in the experiment. Proteins are numbered as in Table 1. b The distribution of proteins with a 1.2−1.99 fold or 2+ fold increase in response to heat stress are shown (left panel). Whereas proteins with a 1.2−1.99 fold, 2−2.99 fold, or 3+ fold increase in response to PUGNAc treatment (right panel) are indicated

Fig. 4

Confirmation of proteins identified by the SILAC screen. Control, heat-stressed, or PUGNAc-treated cells were immunoprecipitated with either CTD110.6 or control IgM covalently coupled to cyanogen bromide activated Sepharose. Cell extract and immunoprecipitates were separated by SDS–PAGE and nuclear pore proteins (mAb414), NF-90, NF45, Carm1, Sec24, p125i, Tip49α, OGT, Actin, Tubulin, and HSP70 were detected by immunoblot

Fig. 5

Numerous proteins identified by the SILAC screen are O-GlcNAcylated dynamically in response to heat stress. Individual proteins were immunoprecipitated from control, heat-stressed, or PUGNAc-treated cells and the levels of protein, or O-GlcNAc were detected by immunoblot. An immuno-precipitation containing extraction buffer (B) was performed with the antibody of interest to confirm that bands detected by CTD110.6 did not arise from the primary antibody. As a control, CTD110.6 was competed away with 100 mM GlcNAc. In cases, such as NF-90, were numerous O-GlcNAcylated proteins are present the molecular weight of the protein of interest is indicated by an asterisk

Fig. 6

DNA-PK appears to be O-GlcNAc modified. a DNA-PK was immunoprecipitated from control, heat-stressed, or PUGNAc-treated Cos-7 cells. O-GlcNAc was detected by immunoblot. b DNA-PK immunoprecipitated from cells treated sodium chloride (100 mM, 6 h), PUGNAc (50 μM, 8 h), H₂O₂ (500 μM 6 h), bleocin (2.5 μg/ml, 6 h), and Tunicamycin (25 μg/ml, 18 h) was separated by SDS–PAGE and O-GlcNAc and DNA-PK were detected by immunoblot. As a control, O-GlcNAc was competed away with 100 mM GlcNAc. c Total cell extracts from Cos-7 cells treated with Doxorubicin (2 μM, 4 or 8 h), was separated by SDS–PAGE, and O-GlcNAc and Actin levels were detected by immunoblot