The heat shock response inhibits phosphorylation of I-KBA

INTRODUCTION: I-κBα is a major intracellular protein that inhibits activation of the proinflammatory transcription factor, NF-κB. In response to proinflammatory signals I-κBα undergoes sequential phosphorylation, ubiquitination, and rapid degradation by the 26S proteasome, thus allowing for activation of NF-κB. We previously demonstrated that the heat shock response, a fundamental cellular defense mechanism, inhibits degradation of I-κBα and subsequent activation of NF-κB. ¹ The purpose of the current study was to determine the proximal mechanism by which the heat shock response inhibits degradation I-κBα. METHODS: RAW 264.7 murine macrophages were used in all experiments. Degradation of I-κBα was induced by treating cells with 100 ng/ml of endotoxin for 5 to 60 minutes. The heat shock response was induced by incubating cells at 43 degrees Celsius for 45 minutes. Degradation of I-κBα was detected by Western blot analyses. Ubiquitination of I-κBα was detected by immunoprecipitation with an anti-I-κBα IgG, followed by Western blot analyses using an anti-ubiquitin IgG. Phosphorylation of I-κBα was detected by Western blot analyses using an anti-phospho-I-κBα IgG. RESULTS: Treatment with endotoxin caused complete degradation of I-κBα within 10 minutes. Induction of the heat shock response, 1 hour before treatment with endotoxin, completely inhibited degradation of I-κBα at all time points tested. Five minutes after endotoxin treatment phosphorylated and ubiquitinated I-κBα was readily detected. Induction of the heat shock response, 1 hour before treatment with endotoxin, blocked both phosphorylation and ubiquitination of I-κBα. CONCLUSIONS: These data demonstrate a novel proximal mechanism by which the heat shock response inhibits degradation of I-κBα and subsequent activation of NF-κB. The heat shock response inhibits phosphorylation and subsequent ubiquitination of I-κBα. Ongoing experients are aimed at determining if the heat shock response inhibits activation of I-κB kinase, and/or increases intracellular phosphatase activity.