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Phosphorylation on Thr-106 and NO-modification of glyoxalase I suppress the TNF-induced transcriptional activity of NF-κB

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Abstract

Glyoxalase I (GLO1), together with glyoxalase II and the co-factor GSH, comprise the glyoxalase system, which is responsible for the detoxification of the cytotoxic glycolytic-derived metabolite methylglyoxal (MG). We, and others, have previously reported that GLO1 is subjected to several post-translational modifications, including a NO-mediated modification and phosphorylation. In this study, we demonstrate that GLO1 is a substrate for calcium, calmodulin-dependent protein kinase II (CaMKII). Site-directed mutagenesis of several serine and threonine residues revealed that CaMKII induced phosphorylation of GLO1 at a single site Thr-106. Mutagenesis of Thr-106 to Ala in GLO1 completely abolished the CaMKII-mediated phosphorylation. A phosphopeptide bracketing phosphothreonine-106 in GLO1 was used as an antigen to generate polyclonal antibodies against phosphothreonine-106. By using this phospho-specific antibody, we demonstrated that TNF induces phosphorylation of GLO1 on Thr-106. Furthermore, we investigated the role of NO-mediated modification and phosphorylation of GLO1 in the TNF-induced transcriptional activity of NF-κB. Overexpression of WT GLO1 suppressed TNF-induced NF-κB-dependent reporter gene expression. Suppression of the basal and TNF-induced NF-κB activity was significantly stronger upon expression of a GLO1 mutant that was either deficient for the NO-mediated modification or phosphorylation on Thr-106. However, upon overexpression of a GLO1 mutant that was deficient for both types of modification, the suppressive effect of GLO1 on TNF-induced NF-κB activity was completely abolished. These results suggest that NO-modification and phosphorylation of GLO1 contribute to the suppression of TNF-induced NF-κB-dependent reporter gene expression. In line with this, knock-down of GLO1 by siRNA significantly increased TNF-induced NF-κB-dependent reporter gene expression. These findings suggest that phosphorylation and NO-modification of glyoxalase I provides another control mechanism for modulating the basal and TNF-induced expression of NF-kappaB-responsive genes.

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Abbreviations

2-DE:

2-Dimensional gel electrophoresis

GLO1:

Glyoxalase I

MG:

Methylglyoxal

TNF:

Tumour necrosis factor

GSNO:

S-nitrosogluthathione

IEF:

Isoelectro focusing

CaM:

Calmodulin

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Authors and Affiliations

  1. Faculty of Medicine and Health Sciences, VIB Department of Medical Protein Research, University of Ghent, Albert Baertsoenkaai 3, 9000, Ghent, Belgium

    Virginie de Hemptinne, Dieter Rondas, Mascha Toepoel & Katia Vancompernolle

  2. Department of Biochemistry, University of Ghent, Albert Baertsoenkaai 3, 9000, Ghent, Belgium

    Katia Vancompernolle

Authors
  1. Virginie de Hemptinne
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  2. Dieter Rondas
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  3. Mascha Toepoel
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  4. Katia Vancompernolle
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Correspondence to Katia Vancompernolle.

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de Hemptinne, V., Rondas, D., Toepoel, M. et al. Phosphorylation on Thr-106 and NO-modification of glyoxalase I suppress the TNF-induced transcriptional activity of NF-κB. Mol Cell Biochem 325, 169–178 (2009). https://doi.org/10.1007/s11010-009-0031-7

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  • DOI: https://doi.org/10.1007/s11010-009-0031-7

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