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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.

Author information

Author notes

    • Subhra Biswas
    • , Sathyavageeswaran Shreeram
    • , Mahathir Humaidi
    •  & Ee Tsin Wong

    These authors contributed equally to the work.

Affiliations

  1. Laboratory of NF- κB signalling, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.

    • Joanne Chew
    • , Mahathir Humaidi
    • , Ee Tsin Wong
    • , Hsiangling Teo
    • , Amit Hazra
    •  & Vinay Tergaonkar
  2. Cell Cycle Control and Tumorigenesis Group, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.

    • Sathyavageeswaran Shreeram
    •  & Dmitry V. Bulavin
  3. Laboratory of Cell Cycle Control, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.

    • Cheok Chit Fang
  4. Singapore Immunology Network, Biomedical Sciences Institute (A*STAR), 8A Biomedical Grove, #04 Immunos, Singapore 138648.

    • Subhra Biswas
    •  & Manprit Kaur Dhillion
  5. Unidad de Investigación, Hospital Universitario La Paz, Pseo de La Castellana 261, Madrid 28046, Spain.

    • Eduardo López-Collazo
  6. Department of Biochemistry, National University of Singapore, Singapore 117597.

    • Vinay Tergaonkar

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Vinay Tergaonkar.

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DOI

https://doi.org/10.1038/ncb1873

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