Hydrogen Peroxide Activates NF-κB through Tyrosine Phosphorylation of IκBα and Serine Phosphorylation of p65: EVIDENCE FOR THE INVOLVEMENT OF IκBα KINASE AND Syk PROTEIN-TYROSINE KINASE

Yasunari Takada,Asok Mukhopadhyay,Gopal C Kundu,Ganapati H Mahabeleshwar,Sujay Singh,Bharat B Aggarwal

doi:10.1074/jbc.m212389200

Abstract

Although it is well established that reactive oxygen intermediates mediate the NF-kappaB activation induced by most agents, how H2O2 activates this transcription factor is not well understood. We found that treatment of human myeloid KBM-5 cells with H2O2 activated NF-kappaB in a dose- and time-dependent manner much as tumor necrosis factor (TNF) did but unlike TNF, H2O2 had no effect on IkappaBalpha degradation. Unexpectedly, however, like TNF-induced activation, H2O2-induced NF-kappaB activation was blocked by the calpain inhibitor N-Ac-Leu-Leu-norleucinal, suggesting that a proteosomal pathway was involved. Although H2O2 activated IkappaBalpha kinase, it did not induce the serine phosphorylation of IkappaBalpha. Like TNF, H2O2 induced the serine phosphorylation of the p65 subunit of NF-kappaB, leading to its nuclear translocation. We found that H2O2 induced the tyrosine phosphorylation of IkappaBalpha, which is needed for NF-kappaB activation. We present several lines of evidence to suggest that the Syk protein-tyrosine kinase is involved in H2O2-induced NF-kappaB activation. First, H2O2 activated Syk in KBM-5 cells; second, H2O2 failed to activate NF-kappaB in cells that do not express Syk protein; third, overexpression of Syk increased H2O2-induced NF-kappaB activation; and fourth, reduction of Syk transcription using small interfering RNA inhibited H2O2-induced NF-kappaB activation. We also showed that Syk induced the tyrosine phosphorylation of IkappaBalpha, which caused the dissociation, phosphorylation, and nuclear translocation of p65. Thus, overall, our results demonstrate that H2O2 induces NF-kappaB activation, not through serine phosphorylation or degradation of IkappaBalpha, but through Syk-mediated tyrosine phosphorylation of IkappaBalpha

Highlights

It is well established that reactive oxygen intermediates mediate the Nuclear factor-␬B (NF-␬B) activation induced by most agents, how H2O2 activates this transcription factor is not well understood
We found that treatment of human myeloid KBM-5 cells with H2O2 activated NF-␬B in a dose- and time-dependent manner much as tumor necrosis factor (TNF) did but unlike TNF, H2O2 had no effect on I␬B␣ degradation
In this report we investigated the effect of H2O2 on NF-␬B activation, I␬B␣ phosphorylation, I␬B␣ degradation, p65 phosphorylation, and nuclear translocation, and the role of proteintyrosine kinase Syk in H2O2-induced NF-␬B activation

Summary

Introduction

It is well established that reactive oxygen intermediates mediate the NF-␬B activation induced by most agents, how H2O2 activates this transcription factor is not well understood. Overall, our results demonstrate that H2O2 induces NF-␬B activation, not through serine phosphorylation or degradation of I␬B␣, but through Syk-mediated tyrosine phosphorylation of I␬B␣. On activation by agents such as TNF, I␬B␣ is phosphorylated at serine residues 32 and 36, ubiquitinated at lysine residues 21 and 22, and degraded through the proteosomal pathway, exposing the nuclear localization signals on the p50-p65 heterodimer. NF-␬B is activated by a wide variety of agents, including all 18 members of the TNF superfamily, interleukin-1, interleukin-17, interleukin-18, lipopolysaccharide, H2O2, ceramide, phorbol esters, growth factors, UV, X-rays, and ␥-radiation [12] Whether all these agents activate NF-␬B through the same pathway as described above is not clear. NF-␬B activation of most agents has been shown to require the generation of reactive oxygen intermediates, in studies that used either reactive oxygen intermediate quenchers, such as

Methods

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Conclusion