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15-deoxy-Δ12,14-prostaglandin J2 inhibition of NF-κB-DNA binding through covalent modification of the p50 subunit

AuthorsCernuda-Morollón, Eva; Pineda-Molina, Estela; Cañada, F. Javier ; Pérez-Sala, Dolores
Issue Date2001
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJournal of Biological Chemistry 276(38): 35530- 35536 (2001)
AbstractCyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-ΚB. Here we explore the possibility that the NF-ΚB subunit p50 may be a target for the cyclopentenone 15-deoxy-Δ 12,14-prostaglandin J2 (15d-PGJ2). This prostaglandin inhibited the DNA binding ability of recombinant p50 in a dose-dependent manner. The inhibition required the cyclopentenone moiety and could be prevented but not reverted by glutathione and dithiothreitol. Moreover, a p50 mutant with a C62S mutation was resistant to inhibition, indicating that the effect of 15d-PGJ2 was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ2 was demonstrated by reverse-phase high pressure liquid chromatography and mass spectrometry analysis that showed an increase in retention time and in the molecular mass of 15d-PGJ2-treated p50, respectively. The interaction between p50 and 15d-PGJ2 was relevant in intact cells. 15d-PGJ2 effectively inhibited cytokine-elicited NF-κB activity in HeLa without reducing IκBα degradation or nuclear translocation of NF-κB subunits. 15d-PGJ2 reduced NF-κB DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF-κB proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ2 resulted in the formation of a 15d-PGJ 2-p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ2 that results in inhibition of DNA binding.
Publisher version (URL)http://dx.doi.org/10.1074/jbc.M104518200
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