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. 2009 Mar 3;106(9):3431-6.
doi: 10.1073/pnas.0813210106. Epub 2009 Feb 6.

Loss of p53 enhances catalytic activity of IKKbeta through O-linked beta-N-acetyl glucosamine modification

Keiko Kawauchi  1 , Keigo Araki, Kei Tobiume, Nobuyuki Tanaka
Affiliations

Loss of p53 enhances catalytic activity of IKKbeta through O-linked beta-N-acetyl glucosamine modification

Keiko Kawauchi et al. Proc Natl Acad Sci U S A. .

Abstract

The IkappaB kinase (IKK)-NF-kappaB pathway plays a critical role in oncogenesis. Recently, we have shown that p53 regulates glucose metabolism through the IKK-NF-kappaB pathway and that, in the absence of p53, the positive feedback loop between IKK-NF-kappaB and glycolysis has an integral role in oncogene-induced cell transformation. Here, we demonstrate that IKKbeta, a component of the IKK complex, was constitutively modified with O-linked beta-N-acetyl glucosamine (O-GlcNAc) in both p53-deficient mouse embryonic fibroblasts (MEFs) and transformed human fibroblasts. In p53-deficient cells, the O-GlcNAcylated IKKbeta and the activating phosphorylation of IKK were decreased by p65/NF-kappaB knockdown or glucose depletion. We also found that high glucose induced the O-GlcNAcylation of IKKbeta and sustained the TNFalpha-dependent IKKbeta activity. Moreover, the O-GlcNAcase inhibitor streptozotocin intensified O-GlcNAcylation and concomitant activating phosphorylation of IKKbeta. Mutational analysis revealed that O-GlcNAcylation of IKKbeta occurred at Ser 733 in the C-terminal domain, which was identified as an inactivating phosphorylation site, suggesting that IKKbeta O-GlcNAcylation regulates its catalytic activity. Taken together, we propose a novel mechanism for the enhancement of NF-kappaB activity by loss of p53, which evokes positive feedback regulation from enhanced glucose metabolism to IKK in oncogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
O-GlcNAcylation of IKKβ in p53-deficient cells. (A) The cell extracts from wild-type and p53−/−MEFs were subjected to immunoblot analysis with an anti-O-GlcNAc antibody (Top) and the indicated antibodies. The open arrowheads indicate the intensified bands in p53−/−MEFs. (B) The cell extracts from wild-type and p53−/−MEFs were subjected to immunoprecipitation (IP) with antibodies specific for IKKβ followed by immunoblot analysis with an anti-O-GlcNAc antibody. (C and D) MCF7 cells were infected with control- or p53-siRNA-expressing retroviruses. Glucose consumption of the indicated cells was measured. Data are means ± SD from 3 independent experiments. *P < 0.01 for the indicated comparison (t test) (C). The levels of IKKβ O-GlcNAcylation and IKKα/β phosphorylation were examined by immunoblot analysis (D).
Fig. 2.
Fig. 2.
p65-dependent IKKβ O-GlcNAcylation in p53−/−MEFs. (A) The cell extracts from p53−/−MEFs infected with control or p65 siRNA-expressing retroviruses were subjected to immunoprecipitation (IP) with an anti-IKKβ antibody, followed by immunoblot analysis with an anti-O-GlcNAc antibody. The cell extracts were subjected to immunoblot analysis with an anti-phospho-IKK (pIKK) antibody. (B) Levels of IKKβ O-GlcNAcylation and phosphorylation at the active site of IKKα/β were examined in p53−/−p65−/−MEFs infected with retroviruses encoding p65 (WT), p65 mutant (S276A) or empty vector (vec). (C) The cell extracts from p53−/−MEFs cultured in medium with or without glucose were subjected to immunoprecipitation (IP) with an anti-IKKβ antibody, followed by immunoblot analysis with an anti-O-GlcNAc antibody. The cell extracts were subjected to immunoblot analysis with an anti-phospho-IKK (pIKK) antibody.
Fig. 3.
Fig. 3.
TNFα-induced NF-κB activation is prolonged by exposure to high glucose. (A) HepG2 cells were incubated in normal medium (5.6 mM) or high glucose medium (30 mM) for 3 h. The cell extracts were subjected to immunoprecipitation with an anti-IKKβ antibody, followed by immunoblot analysis with an anti-O-GlcNAc antibody. (B) HepG2 cells were treated with STZ (5 mM) for 3 h. Levels of IKKβ O-GlcNAcylation and the phosphorylation at the active-site of IKKα/β (pIKK) were examined (Top). The nuclear extracts from the cells were subjected to EMSA with a radiolabeled probe containing the NF-κB consensus sequence (Bottom). The open arrowhead indicates the band for NF-κB with the p65 component. The solid arrowhead indicates a nonspecific band. (C) HepG2 cells were transfected with a NF-κB Luc reporter plasmid and phRL-TK as an internal control. After 24 h, the transfected cells were preincubated in normal medium (5.6 mM) or high glucose medium (30 mM) for 3 h and then stimulated with TNFα (100 ng/ml) for 12 h before harvesting. The luciferase activity is shown normalized to Renilla luciferase activity. Data are means ± SD from 3 independent experiments. (D and E) HepG2 cells were incubated in normal medium (5.6 mM) or high glucose medium (30 mM) for 3 h and stimulated with TNFα (100 ng/ml) for the indicated periods. The nuclear extracts were subjected to EMSA using a radiolabeled-κB oligonucleotide probe. The upper and lower panels are results from same experiment at different time points (D). The catalytic activity of IKKβ was estimated by an in vitro kinase assay (IVK) (E; Top). The cell extracts from the treated cells were subjected to immunoprecipitation with an anti-IKKβ antibody, followed by immunoblot analysis with an anti-O-GlcNAc antibody (E; Middle).
Fig. 4.
Fig. 4.
O-GlcNAcylation of IKKβ at Ser 733 regulates its catalytic activity. (A) NIH 3T3 cells infected with mouse IKKβ siRNA-expressing retrovirus were superinfected with HA-tagged human IKKβ wild type (WT). The cell extracts from the infected cells were subjected to immunoblot analysis with the indicated antibodies. Tubulin was used as a loading control. (B) NIH 3T3 cells infected with mouse IKKβ siRNA-expressing retrovirus were superinfected with retroviruses encoding HA-tagged human IKKβ wild type (WT), S733E, S740E, or S750E mutants. The cell extracts were subjected to immunoprecipitation with an anti-HA antibody followed by immunoblot analysis with an anti-O-GlcNAc antibody. (C) NIH 3T3 cells infected with mouse IKKβ siRNA-expressing retrovirus were superinfected with retroviruses encoding HA-tagged human IKKβ WT, S733E, or S733A mutants. The level of IKKβ O-GlcNAcylation was examined in cells infected with each retrovirus. (D) National Institutes of Health 3T3 cells infected with mouse IKKβ siRNA-expressing retrovirus were superinfected with retroviruses encoding HA-tagged human IKKβ WT, S750E, or S750A mutants. The level of IKKβ O-GlcNAcylation was examined in the cells infected with each retrovirus. (E and F) p53−/−MEFs infected with mouse IKKβ siRNA-expressing retrovirus were superinfected with HA-tagged human IKKβ wild-type (WT), S733E, or S733A mutants. The cell extracts from the infected cells were subjected to immunoprecipitation with an anti-HA antibody followed by immunoblot analysis with the indicated antibodies to examine the levels of IKKβ O-GlcNAcylation and phosphorylation at the active site of IKKα/β (E). Glucose consumption was estimated from the concentration of glucose in the culture media. Data are means ± SD. from 3 independent experiments *P < 0.01 for the indicated comparison (t test) (F). (G) A model of IKKβ activation regulated by phosphorylation and O-GlcNAcylation at the C-terminal domain. In the basal state, IKKβ is reciprocally modified with O-GlcNAc at Ser 733. Stimulation, such as by TNFα, induces activating phosphorylation in its kinase domain of IKKβ (i) and subsequently autophosphorylates the inactivating phosphorylation sites at its C-terminal domain, including the Ser 733 residue (ii). For IKKβ modified with O-GlcNAc, the TNFα-induced kinase activity is sustained by blockade of inactivating phosphorylation (iii).
Fig. 5.
Fig. 5.
IKKβ O-GlcNAcylation is enhanced in transformed human fibroblasts. (A and B) Tig3 cells were transformed by serial infection with retroviruses encoding the SV40 T antigen, Myc, and Ha-Ras (SMR cells). The glucose consumption of control Tig3 cells and transformed Tig3 cells (SMR) was estimated from the concentration of glucose in the culture media. Data are means ± SD. from 3 independent experiments. *P < 0.01 for the indicated comparison (t test) (A). Levels of IKKβ O-GlcNAcylation and the active-site of IKKα/β phosphorylation were examined in control Tig3 cells and transformed Tig3 cells (SMR) (B). (C) A model for the proposed link between p53, IKK, NF-κB, and glycolysis. p53 regulates an activation loop between IKK, NF-κB, and glucose metabolism.
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