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Regulation of the innate immune response by threonine-phosphatase of Eyes absent

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Abstract

Innate immunity is stimulated not only by viral or bacterial components, but also by non-microbial danger signals (damage-associated molecular patterns)1. One of the damage-associated molecular patterns is chromosomal DNA that escapes degradation. In programmed cell death and erythropoiesis, DNA from dead cells or nuclei expelled from erythroblasts is digested by DNase II in the macrophages after they are engulfed. DNase II-/- (also known as Dnase2a-/- ) mice suffer from severe anaemia or chronic arthritis due to interferon-β (IFN-β) and tumour necrosis factor-α (TNF-α) produced from the macrophages carrying undigested DNA2,3 in a Toll-like receptor (TLR)-independent mechanism4. Here we show that Eyes absent 4 (EYA4), originally identified as a co-transcription factor, stimulates the expression of IFN-β and CXCL10 in response to the undigested DNA of apoptotic cells. EYA4 enhanced the innate immune response against viruses (Newcastle disease virus and vesicular stomatitis virus), and could associate with signalling molecules (IPS-1 (also known as MAVS), STING (TMEM173) and NLRX1). Three groups have previously shown that EYA has phosphatase activity5,6,7. We found that mouse EYA family members act as a phosphatase for both phosphotyrosine and phosphothreonine. The haloacid dehalogenase domain at the carboxy terminus contained the tyrosine-phosphatase, and the amino-terminal half carried the threonine-phosphatase. Mutations of the threonine-phosphatase, but not the tyrosine-phosphatase, abolished the ability of EYA4 to enhance the innate immune response, suggesting that EYA regulates the innate immune response by modulating the phosphorylation state of signal transducers for the intracellular pathogens.

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Figure 1: Identification of EYA4 as a regulator for innate immunity.
Figure 2: Activation of the signalling cascade by EYA4.
Figure 3: Two different phosphatase activities in mouse EYA.
Figure 4: The threonine-phosphatase activity of EYA4 is required for the innate immune reaction.

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Change history

  • 23 July 2009

    The figure key for Figure 2a was corrected on 23 July 2009.

References

  1. Meylan, E., Tschopp, J. & Karin, M. Intracellular pattern recognition receptors in the host response. Nature 442, 39–44 (2006)

    Article  ADS  CAS  Google Scholar 

  2. Yoshida, H., Okabe, Y., Kawane, K., Fukuyama, H. & Nagata, S. Lethal anemia caused by interferon-β produced in mouse embryos carrying undigested DNA. Nature Immunol. 6, 49–56 (2005)

    Article  CAS  Google Scholar 

  3. Kawane, K. et al. Chronic polyarthritis caused by mammalian DNA that escapes from degradation in macrophages. Nature 443, 998–1002 (2006)

    Article  ADS  CAS  Google Scholar 

  4. Okabe, Y., Kawane, K., Akira, S., Taniguchi, T. & Nagata, S. Toll-like receptor-independent gene induction program activated by mammalian DNA escaped from apoptotic DNA degradation. J. Exp. Med. 202, 1333–1339 (2005)

    Article  CAS  Google Scholar 

  5. Li, X. et al. Eya protein phosphatase activity regulates Six1–Dach–Eya transcriptional effects in mammalian organogenesis. Nature 426, 247–254 (2003)

    Article  ADS  CAS  Google Scholar 

  6. Rayapureddi, J. P. et al. Eyes absent represents a class of protein tyrosine phosphatases. Nature 426, 295–298 (2003)

    Article  ADS  CAS  Google Scholar 

  7. Tootle, T. L. et al. The transcription factor Eyes absent is a protein tyrosine phosphatase. Nature 426, 299–302 (2003)

    Article  ADS  CAS  Google Scholar 

  8. Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. Cell 124, 783–801 (2006)

    Article  CAS  Google Scholar 

  9. Medzhitov, R. Recognition of microorganisms and activation of the immune response. Nature 449, 819–826 (2007)

    Article  ADS  CAS  Google Scholar 

  10. Jemc, J. & Rebay, I. The eyes absent family of phosphotyrosine phosphatases: properties and roles in developmental regulation of transcription. Annu. Rev. Biochem. 76, 513–538 (2007)

    Article  CAS  Google Scholar 

  11. Kim, T. K. & Maniatis, T. The mechanism of transcriptional synergy of an in vitro assembled interferon-β enhanceosome. Mol. Cell 1, 119–129 (1997)

    Article  CAS  Google Scholar 

  12. Ohmori, Y. & Hamilton, T. A. Cooperative interaction between interferon (IFN) stimulus response element and κB sequence motifs controls IFNγ- and lipopolysaccharide-stimulated transcription from the murine IP-10 promoter. J. Biol. Chem. 268, 6677–6688 (1993)

    CAS  PubMed  Google Scholar 

  13. Sharma, S. et al. Triggering the interferon antiviral response through an IKK-related pathway. Science 300, 1148–1151 (2003)

    Article  ADS  CAS  Google Scholar 

  14. Iwamura, T. et al. Induction of IRF-3/-7 kinase and NF-κB in response to double-stranded RNA and virus infection: common and unique pathways. Genes Cells 6, 375–388 (2001)

    Article  CAS  Google Scholar 

  15. Kawai, T. et al. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nature Immunol. 6, 981–988 (2005)

    Article  CAS  Google Scholar 

  16. Meylan, E. et al. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167–1172 (2005)

    Article  ADS  CAS  Google Scholar 

  17. Ohto, H. et al. Cooperation of six and eya in activation of their target genes through nuclear translocation of Eya. Mol. Cell. Biol. 19, 6815–6824 (1999)

    Article  CAS  Google Scholar 

  18. Ishikawa, H. & Barber, G. N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678 (2008)

    Article  ADS  CAS  Google Scholar 

  19. Moore, C. B. et al. NLRX1 is a regulator of mitochondrial antiviral immunity. Nature 451, 573–577 (2008)

    Article  ADS  CAS  Google Scholar 

  20. Tattoli, I. et al. NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-κB and JNK pathways by inducing reactive oxygen species production. EMBO Rep. 9, 293–300 (2008)

    Article  CAS  Google Scholar 

  21. Jin, L. et al. MPYS, a novel membrane tetraspanner, is associated with major histocompatibility complex class II and mediates transduction of apoptotic signals. Mol. Cell. Biol. 28, 5014–5026 (2008)

    Article  CAS  Google Scholar 

  22. Cook, P. J. et al. Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions. Nature 458, 591–596 (2009)

    Article  ADS  CAS  Google Scholar 

  23. Das, A. K., Helps, N. R., Cohen, P. T. & Barford, D. Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 Å resolution. EMBO J. 15, 6798–6809 (1996)

    Article  CAS  Google Scholar 

  24. Lee, M. S. & Kim, Y. J. Signaling pathways downstream of pattern-recognition receptors and their cross talk. Annu. Rev. Biochem. 76, 447–480 (2007)

    Article  CAS  Google Scholar 

  25. Michallet, M. C. et al. TRADD protein is an essential component of the RIG-like helicase antiviral pathway. Immunity 28, 651–661 (2008)

    Article  CAS  Google Scholar 

  26. Abdelhak, S. et al. A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. Nature Genet. 15, 157–164 (1997)

    Article  CAS  Google Scholar 

  27. Orten, D. J. et al. Branchio-oto-renal syndrome (BOR): novel mutations in the EYA1 gene, and a review of the mutational genetics of BOR. Hum. Mutat. 29, 537–544 (2008)

    Article  CAS  Google Scholar 

  28. Mutsuddi, M. et al. Using Drosophila to decipher how mutations associated with human branchio-oto-renal syndrome and optical defects compromise the protein tyrosine phosphatase and transcriptional functions of eyes absent. Genetics 170, 687–695 (2005)

    Article  CAS  Google Scholar 

  29. Borsani, G. et al. EYA4, a novel vertebrate gene related to Drosophila eyes absent. Hum. Mol. Genet. 8, 11–23 (1999)

    Article  CAS  Google Scholar 

  30. Takeshita, S., Kaji, K. & Kudo, A. Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts. J. Bone Miner. Res. 15, 1477–1488 (2000)

    Article  CAS  Google Scholar 

  31. Lam, K. M. & Hao, Q. Induction of lymphocyte agglutination and lysis by Newcastle disease virus. Vet. Microbiol. 15, 49–56 (1987)

    Article  CAS  Google Scholar 

  32. Shiraishi, T. et al. Increased cytotoxicity of soluble Fas ligand by fusing isoleucine zipper motif. Biochem. Biophys. Res. Commun. 322, 197–202 (2004)

    Article  CAS  Google Scholar 

  33. Hanayama, R. et al. Identification of a factor that links apoptotic cells to phagocytes. Nature 417, 182–187 (2002)

    Article  ADS  CAS  Google Scholar 

  34. Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000)

    Article  ADS  CAS  Google Scholar 

  35. Ray, S. & Diamond, B. Generation of a fusion partner to sample the repertoire of splenic B cells destined for apoptosis. Proc. Natl Acad. Sci. USA 91, 5548–5551 (1994)

    Article  ADS  CAS  Google Scholar 

  36. Kitamura, T. et al. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Exp. Hematol. 31, 1007–1014 (2003)

    Article  CAS  Google Scholar 

  37. Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063–1066 (2000)

    Article  CAS  Google Scholar 

  38. Higuchi, R. in PCR Protocols: a Guide to Methods and Applications 177–188 (Academic, 1990)

    Google Scholar 

  39. Mizushima, S. & Nagata, S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 18, 5322 (1990)

    Article  CAS  Google Scholar 

  40. Sawasaki, T., Ogasawara, T., Morishita, R. & Endo, Y. A cell-free protein synthesis system for high-throughput proteomics. Proc. Natl Acad. Sci. USA 99, 14652–14657 (2002)

    Article  ADS  CAS  Google Scholar 

  41. Donella Deana, A. et al. An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A. Biochim. Biophys. Acta 1051, 199–202 (1990)

    Article  CAS  Google Scholar 

  42. Umeda, I. O., Nakata, H. & Nishigori, H. Identification of protein phosphatase 2C and confirmation of other protein phosphatases in the ocular lenses. Exp. Eye Res. 79, 385–392 (2004)

    Article  CAS  Google Scholar 

  43. Van Veldhoven, P. P. & Mannaerts, G. P. Inorganic and organic phosphate measurements in the nanomolar range. Anal. Biochem. 161, 45–48 (1987)

    Article  CAS  Google Scholar 

  44. Yoneyama, M. et al. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. EMBO J. 17, 1087–1095 (1998)

    Article  CAS  Google Scholar 

  45. Fujisawa-Sehara, A., Hanaoka, K., Hayasaka, M., Hiromasa-Yagami, T. & Nabeshima, Y. Upstream region of the myogenin gene confers transcriptional activation in muscle cell lineages during mouse embryogenesis. Biochem. Biophys. Res. Commun. 191, 351–356 (1993)

    Article  CAS  Google Scholar 

  46. Kawane, K. et al. Structure and promoter analysis of murine CAD and ICAD genes. Cell Death Differ. 6, 745–752 (1999)

    Article  CAS  Google Scholar 

  47. Shapiro, D. J., Sharp, P. A., Wahli, W. W. & Keller, M. J. A high-efficiency HeLa cell nuclear transcription extract. DNA 7, 47–55 (1988)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank T. Fujita and M. Yoneyama for the NDV, A. Sehara-Fujisawa for the myogenin promoter, and M. Fujii and M. Harayama for secretarial assistance. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture in Japan. T.S. is a Research Assistant for Kyoto University Global COE program (Center for Frontier Medicine).

Author Contributions Y.O. screened the cDNA library, and identified EYA4 as a regulator of the innate immune reaction. T.S. produced recombinant EYA in 293T cells and wheat-germ extracts, biochemically characterized, and found its threonine-phosphatase activity. S.N. designed the research and wrote the paper.

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Correspondence to Shigekazu Nagata.

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Okabe, Y., Sano, T. & Nagata, S. Regulation of the innate immune response by threonine-phosphatase of Eyes absent. Nature 460, 520–524 (2009). https://doi.org/10.1038/nature08138

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