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Control of NF-kappa B activity by the I kappa B beta inhibitor
R Weil1, S T Whiteside, A Israël
1Unité de Biologie Moléculaire de l'Expression Génique, UMR 321 Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France.
Insights
Inhibitors of nuclear factor kappa B (NF-kappa B), I kappa B alpha and I kappa B beta, are equally associated with p65. I kappa B beta is degraded by TNF alpha, contrary to previous findings.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- The transcription factor NF-kappa B regulates immune responses and is kept inactive by I kappa B inhibitors.
- I kappa B alpha and I kappa B beta are known inhibitors of NF-kappa B in mammalian cells, binding to p65 or c-Rel subunits.
- A third inhibitor, I kappa B epsilon, has also been identified.
Purpose of the Study:
- To investigate the association and degradation of I kappa B alpha and I kappa B beta with p65 in E29.1 T cell hybridoma.
- To clarify the role of I kappa B beta degradation in response to TNF alpha.
- To examine the phosphorylation status of I kappa B beta and its relation to proteolysis.
Main Methods:
- Co-immunoprecipitation assays to assess protein-protein interactions.
- Western blotting to detect protein degradation and phosphorylation.
- Analysis of protein forms in response to TNF alpha stimulation.
Main Results:
- I kappa B alpha and I kappa B beta are equally associated with the p65 subunit.
- I kappa B beta undergoes degradation in response to TNF alpha, contradicting prior reports.
- I kappa B beta is constitutively phosphorylated and resynthesized as a hypophosphorylated form, suggesting phosphorylation is not the primary signal for degradation.
Conclusions:
- The findings challenge previous understandings of I kappa B beta regulation.
- I kappa B beta's degradation by TNF alpha is a significant event in NF-kappa B pathway activation.
- Constitutive phosphorylation of I kappa B beta does not appear to be the trigger for its proteolysis.
Abstract:
The transcription factor NF-kappa B is maintained in an inactive cytoplasmic state by I kappa B inhibitors. In mammalian cells, I kappa B alpha and I kappa B beta proteins have been purified and shown to be the inhibitors of NF-kappa B through their association with the p65 or c-Rel subunits. In addition, we have isolated a third NF-kappa B inhibitor, I kappa B epsilon (1). Upon treatment with a large variety of inducers, I kappa B alpha, I kappa B beta are proteolytically degraded, resulting in NF-kappa B translocation into the nucleus. Here we show that in E29.1 T cell hybridoma I kappa B alpha and I kappa B beta are equally associated with p65 and that I kappa B beta is degraded in response to TNF alpha in contrast to what has been originally published. Our data also suggest that, unlike I kappa B alpha, I kappa B beta is constitutively phosphorylated and resynthesized as a hypophosphorylated form. The absence of slow migrating forms of I kappa B beta following stimulation suggests that the phosphorylation does not necessarily constitute the signal-induced event which targets the molecule for proteolysis.