Abstract
Post-translational modifications of NF-κB through phosphorylations enhance its transactivation potential. Much is known about the kinases that phosphorylate NF-κB, but little is known about the phosphatases that dephosphorylate it. By using a genome-scale siRNA screen, we identified the WIP1 phosphatase as a negative regulator of NF-κB signalling. WIP1-mediated regulation of NF-κB occurs in both a p38-dependent and independent manner. Overexpression of WIP1 resulted in decreased NF-κB activation in a dose-dependent manner, whereas WIP1 knockdown resulted in increased NF-κB function. We show that WIP1 is a direct phosphatase of Ser 536 of the p65 subunit of NF-κB. Phosphorylation of Ser 536 is known to be essential for the transactivation function of p65, as it is required for recruitment of the transcriptional co-activator p300. WIP1-mediated regulation of p65 regulated binding of NF-κB to p300 and hence chromatin remodelling. Consistent with our results, mice lacking WIP1 showed enhanced inflammation. These results provide the first genetic proof that a phosphatase directly regulates NF-κB signalling in vivo.
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Acknowledgements
We wish to thank the Agency for Science Technology and Research (A*star) for funding and support. We are grateful to David Lane for the use of his laboratory resources during the course of this work. We acknowledge the help of Walter Blackstock and Jayantha Gunaratne for analysing the phosho peptides by mass spectrometry. We thank G. Natoli for the CHIP protocol and Minami Y for the WIP1 antibody.
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J.C., V.T., D.B. and S.B. conceived the project and planned experiments and analysis; J.C., S.S., M.H., E.T.W., V.T. and H.T. performed all the biochemical and gene expression analyses; C.C.F. helped with the genome scale screening; E.L.C. provided patient samples; S.B., M.K.D. analysed the sepsis samples; S.B., M.K.D. and A.H. performed the endotoxin experiments on the mice and all related gene expression experiments; V.T. oversaw the project and in consultation with D.B. wrote the manuscript.
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Chew, J., Biswas, S., Shreeram, S. et al. WIP1 phosphatase is a negative regulator of NF-κB signalling. Nat Cell Biol 11, 659–666 (2009). https://doi.org/10.1038/ncb1873
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DOI: https://doi.org/10.1038/ncb1873
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