title: The cytokine-induced conformational switch of nuclear factor κB p65 is mediated by p65 phosphorylation.
abstract: The transcription factor NF-κB (nuclear factor κB) serves to up-regulate gene expression in response to precarious signals such as the pro-inflammatory cytokines TNF (tumour necrosis factor) and IL-1 (interleukin 1). In the present study 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 led 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 (heat-shock protein)/Hsc70 (heat-shock cognate protein, 73 kDa) family are not important for the cytokine-induced conformational switch, but rather control the fidelity of protein rearrangement. Accordingly, pharmacological inhibition of Hsp/Hsc70 interferes with p65-triggered gene expression.
authors.affiliation: *Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Justus-Liebig-University, 35392 Giessen, Germany.
authors.forename: Maja
authors.initials: M
authors.lastname: Milanovic
authors.affiliation: †Rudolf-Buchheim-Institute for Pharmacology, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Kracht
authors.affiliation: *Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Justus-Liebig-University, 35392 Giessen, Germany.
authors.forename: M Lienhard
authors.initials: ML
authors.lastname: Schmitz
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology; D050883:HSC70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051996:Transcription Factor RelA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D015854:Up-Regulation
chemical_list: D016207:Cytokines; D050883:HSC70 Heat-Shock Proteins; D050505:Mutant Proteins; C498143:RELA protein, human; D011994:Recombinant Proteins; D051996:Transcription Factor RelA