Two’s Company, Three’s a Complex

Abstract

Activation of gene transcription by members of the nuclear factor κB (NF-κB) family of transcription factors is crucial to such processes as proliferation and inflammation. Perhaps the best-characterized NF-κB is a heterodimer composed of p50 and p65; however, some aspects of its regulation remain poorly understood. Differences between the properties of NF-κB from nuclear extracts and those of recombinant NF-κB led Wan et al . to search for other proteins that might form part of endogenous NF-κB complexes. They affinity-purified and analyzed by mass spectrometry those proteins from 293T cells that bound to a tagged form of p65 and identified ribosomal protein S3 (RPS3) as a major binding partner. Reporter assays showed that knockdown of RPS3 by small inhibitory (si) RNAs impaired T cell receptor (TCR)-stimulated NF-κB activation in Jurkat T cells. Electrophoretic mobility shift assays (EMSAs) demonstrated the TCR-stimulated formation of complexes containing p50, p65, and RPS3. Chromatin immunoprecipitation (ChIP) assays showed that recruitment of p65 to target genes was greatly diminished in RPS3-knockdown cells compared with that in control cells. TCR-stimulated activation of these genes was also lower in RPS3-knockdown cells than in control cells, as assessed by real-time reverse transcription polymerase chain reaction assays. Comparison of RPS3-knockdown with p65-knockdown T cells by microarray assays showed that RPS3 was necessary for the expression of a subset of p65 target genes, one of which encodes the cytokine interleukin-2 (IL-2). Consistent with this, TCR-stimulated proliferation, which is dependent on IL-2, was blocked in RPS3-knockdown T cells. These results challenge the current dimer-based model of NF-κB activity and suggest that RPS3 is a required subunit of NF-κB that provides target specificity. F. Wan, D. E. Anderson, R. A. Barnitz, A. Snow, N. Bidere, L. Zheng, V. Hegde, L. T. Lam, L. M. Staudt, D. Levens, W. A. Deutsch, M. J. Lenardo, Ribosomal protein S3: A KH domain subunit in NF-κB complexes that mediates selective gene regulation. Cell 131 , 927-939 (2007). [PubMed]