The cytokine-induced conformational switch of nuclear NF-{kappa}B p65 is mediated by p65 phosphorylations

The transcription factor NF-κB serves to upregulate gene expression in response to precarious signals such as the proinflammatory cytokines TNF and IL-1. Here we show that stimulation of cells with TNF or IL-1 results in a profound conformational switch of the NF-κB subunit p65, as revealed by limited proteolysis assays. We also describe the identification of a conformation-specific monoclonal antibody that preferentially immunoprecipitates the inducibly refolded p65 protein. The cytokine-triggered reconfiguration of p65 mainly occurs for p65 contained in the nuclear fraction. Phosphorylations serve as the central driving force for the inducible reconfiguration of p65. Accordingly, mutation of single phosphorylation sites in the C-terminal transactivation domain lead to large conformational changes which result in strongly decreased ubiquitination and also in differential protein/protein interactions. Induced conformational changes of p65 thus increase the intramolecular flexibility and therefore expand and specify the repertoire of possible protein/protein interactions. Constitutively bound chaperones of the Hsp/Hsc70 family are not important for the cytokine-induced conformational switch per se, but rather control the fidelity of protein rearrangement. Accordingly, pharmacologic inhibition of Hsp/Hsc70 interferes with p65-triggered gene expression.

Source: BJ Cell - October 31, 2013 Category: Biochemistry Authors: M Milanovic, M Kracht, M Lienhard Schmitz Tags: BJ Gene Source Type: research