Phosphorylation on Thr-106 and NO-modification of glyoxalase I suppress the TNF-induced transcriptional activity of NF-kappaB.
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-kappaB. Overexpression of WT GLO1 suppressed TNF-induced NF-kappaB-dependent reporter gene expression. Suppression of the basal and TNF-induced NF-kappaB 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-kappaB activity was completely abolished. These results suggest that NO-modification and phosphorylation of GLO1 contribute to the suppression of TNF-induced NF-kappaB-dependent reporter gene expression. In line with this, knock-down of GLO1 by siRNA significantly increased TNF-induced NF-kappaB-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.
de Hemptinne
Virginie
V
Faculty of Medicine and Health Sciences, VIB Department of Medical Protein Research, University of Ghent, Albert Baertsoenkaai 3, 9000, Ghent, Belgium.
Rondas
Dieter
D
Toepoel
Mascha
M
Vancompernolle
Katia
K
D001483:Base Sequence; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D017931:DNA Primers; D017930:Genes, Reporter; D006801:Humans; D007791:Lactoylglutathione Lyase / Q000737:chemistry / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000737:chemistry*; D010766:Phosphorylation; D013912:Threonine / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D017931:DNA Primers; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D013912:Threonine; D009569:Nitric Oxide; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D007791:Lactoylglutathione Lyase