IKKβ phosphorylation regulates RPS3 nuclear translocation and NF-κB function during infection with Escherichia coli strain O157:H7

Abstract

NF-κB is a major gene regulator in immune responses, and ribosomal protein S3 (RPS3) is an NF-κB subunit that directs specific gene transcription. However, it is unknown how nuclear translocation of RPS3 is regulated. Here we report that phosphorylation of RPS3 Ser209 by the kinase IKKβ was crucial for nuclear localization of RPS3 in response to activating stimuli. Moreover, virulence protein NleH1 of the foodborne pathogen Escherichia coli strain O157:H7 specifically inhibited phosphorylation of RPS3 Ser209 and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but resulting in less mortality in a gnotobiotic piglet-infection model. Thus, the IKKβ-dependent modification of a specific amino acid in RPS3 promoted specific NF-κB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.

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Figure 1: RPS3 is phosphorylated and associates with IKKβ in response to NF-κB activation.
Figure 2: IKKβ kinase activity is required for the nuclear translocation of RPS3.
Figure 3: Importin-α-mediated nuclear translocation of RPS3 is dependent on degradation of IκBα.
Figure 4: IKKβ phosphorylates RPS3 at Ser209.
Figure 5: Phosphorylation of RPS3 at Ser209 is critical for its nuclear translocation and NF-κB-specifier function.
Figure 6: NleH1 blocks the phosphorylation of RPS3 Ser209.
Figure 7: NleH1 alters the substrate specificity of IKKβ to block IKKβ-mediated phosphorylation of RPS3.

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Acknowledgements

We thank T. Huxford (San Diego State University) and C. Wu (National Cancer Institute) for Flag-tagged SSEE and SSAA IKKβ mutants; U. Siebenlist (National Institute of Allergy and Infectious Diseases) for the hemagglutinin-tagged SSAA IκBα mutant; M. Biancalana (National Institute of Allergy and Infectious Diseases) for recombinant RPS3 protein with the tag cleaved; S. Porcella for DNA sequencing; O. Schwartz, L. Koo and S. Becker for assistance with fluorescence microscopy; and D. Levens, A. Snow and U. Siebenlist for critical reading of the manuscript. Supported by the US National Institutes of Health (R00CA137171 to F.W., a subaward of P20 RR016443, R03AI076227 and R56AI087686 to P.R.H.) and the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health.

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F.W. and M.J.L. designed the experiments; F.W., A.W., X.G. and M.B. did the experiments; and F.W., P.R.H. and M.J.L. analyzed data and wrote the manuscript.

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Correspondence to Michael J Lenardo.

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The authors declare no competing financial interests.

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Wan, F., Weaver, A., Gao, X. et al. IKKβ phosphorylation regulates RPS3 nuclear translocation and NF-κB function during infection with Escherichia coli strain O157:H7. Nat Immunol 12, 335–343 (2011). https://doi.org/10.1038/ni.2007

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