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Nemo-like kinase suppresses Notch signalling by interfering with formation of the Notch active transcriptional complex

Nature Cell Biology volume 12, pages 278–285 (2010)Cite this article

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Abstract

The Notch signalling pathway has a crucial function in determining cell fates in multiple tissues within metazoan organisms1. On binding to ligands, the Notch receptor is cleaved proteolytically and releases its intracellular domain (NotchICD). The NotchICD enters the nucleus and acts cooperatively with other factors to stimulate the transcription of target genes. High levels of Notch-mediated transcriptional activation require the formation of a ternary complex consisting of NotchICD, CSL (CBF-1, suppressor of hairless, LAG-1) and a Mastermind family member2,3,4,5. However, it is still not clear how the formation of the ternary complex is regulated. Here we show that Nemo-like kinase (NLK) negatively regulates Notch-dependent transcriptional activation by decreasing the formation of this ternary complex. Using a biochemical screen, we identified Notch as a new substrate of NLK. NLK-phosphorylated Notch1ICD is impaired in its ability to form a transcriptionally active ternary complex. Furthermore, knockdown of NLK leads to hyperactivation of Notch signalling and consequently decreases neurogenesis in zebrafish. Our results both define a new function for NLK and reveal a previously unidentified mode of regulation in the Notch signalling pathway.

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Figure 1: Identification of Notch as a new NLK substrate.
Figure 2: NLK binds to and phosphorylates Notch1ICD.
Figure 3: NLK suppresses Notch signalling by means of Notch phosphorylation.
Figure 4: NLK reduces the Notch1ICD–CSL–Mastermind ternary complex formation.
Figure 5: NLK promotes primary neurogenesis by inhibiting Notch signalling in zebrafish.

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References

  1. Artavanis-Tsakonas, S., Rand, M. D. & Lake, R. J. Notch signaling: cell fate control and signal integration in development. Science 284, 770–776 (1999).

    Article  CAS  Google Scholar 

  2. Bray, S. J. Notch signalling: a simple pathway becomes complex. Nature Rev. Mol. Cell Biol. 7, 678–689 (2006).

    CAS  Google Scholar 

  3. Wu, L. et al. MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nature Genet. 26, 484–489 (2000).

    Article  CAS  Google Scholar 

  4. Kitagawa, M. et al. A human protein with sequence similarity to Drosophila mastermind coordinates the nuclear form of notch and a CSL protein to build a transcriptional activator complex on target promoters. Mol. Cell. Biol. 21, 4337–4346 (2001).

    Article  CAS  Google Scholar 

  5. Oyama, T. et al. Mastermind-1 is required for Notch signal-dependent steps in lymphocyte development in vivo. Proc. Natl Acad. Sci. USA 104, 9764–9769 (2007).

    Article  CAS  Google Scholar 

  6. Choi, K. W. & Benzer, S. Rotation of photoreceptor clusters in the developing Drosophila eye requires the nemo gene. Cell 78, 125–136 (1994).

    Article  CAS  Google Scholar 

  7. Meneghini, M. D. et al. MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans. Nature 399, 793–797 (1999).

    Article  CAS  Google Scholar 

  8. Thorpe, C. J. & Moon, R. T. nemo-like kinase is an essential co-activator of Wnt signaling during early zebrafish development. Development 131, 2899–2909 (2004).

    Article  CAS  Google Scholar 

  9. Kortenjann, M. et al. Abnormal bone marrow stroma in mice deficient for nemo-like kinase, Nlk. Eur. J. Immunol. 31, 3580–3587 (2001).

    Article  CAS  Google Scholar 

  10. Ishitani, T. et al. The TAK1–NLK–MAPK-related pathway antagonizes signalling between β-catenin and transcription factor TCF. Nature 399, 798–802 (1999).

    Article  CAS  Google Scholar 

  11. Ishitani, T., Ninomiya-Tsuji, J. & Matsumoto, K. Regulation of lymphoid enhancer factor 1/T-cell factor by mitogen-activated protein kinase-related Nemo-like kinase-dependent phosphorylation in Wnt/β-catenin signaling. Mol. Cell. Biol. 23, 1379–1389 (2003).

    Article  CAS  Google Scholar 

  12. Kanei-Ishii, C. et al. Wnt-1 signal induces phosphorylation and degradation of c-Myb protein via TAK1, HIPK2, and NLK. Genes Dev. 18, 816–829 (2004).

    Article  CAS  Google Scholar 

  13. Kojima, H. et al. STAT3 regulates Nemo-like kinase by mediating its interaction with IL-6-stimulated TGFβ-activated kinase 1 for STAT3 Ser-727 phosphorylation. Proc. Natl Acad. Sci. USA 102, 4524–4529 (2005).

    Article  CAS  Google Scholar 

  14. Ohkawara, B. et al. Role of the TAK1–NLK–STAT3 pathway in TGF-β-mediated mesoderm induction. Genes Dev. 18, 381–6 (2004).

    Article  CAS  Google Scholar 

  15. Verheyen, E. M., Purcell, K. J., Fortini, M. E. & Artavanis-Tsakonas, S. Analysis of dominant enhancers and suppressors of activated Notch in Drosophila. Genetics 144, 1127–1141 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Kankel, M. W. et al. Investigating the genetic circuitry of mastermind in Drosophila, a notch signal effector. Genetics 177, 2493–2505 (2007).

    Article  CAS  Google Scholar 

  17. Zhou, S. et al. SKIP, a CBF1-associated protein, interacts with the ankyrin repeat domain of NotchIC to facilitate NotchIC function. Mol. Cell. Biol. 20, 2400–2410 (2000).

    Article  CAS  Google Scholar 

  18. Takke, C., Dornseifer, P., v. Weizsacker, E. & Campos-Ortega, J. A. her4, a zebrafish homologue of the Drosophila neurogenic gene E(spl), is a target of NOTCH signalling. Development 126, 1811–1821 (1999).

    CAS  PubMed  Google Scholar 

  19. Yeo, S., Kim, M. J., Kim, H., Huh, T. & Chitnis, A. B. Fluorescent protein expression driven by her4 regulatory elements reveals the spatiotemporal pattern of Notch signaling in the nervous system of zebrafish embryos. Dev. Biol. 301, 555–567 (2007).

    Article  CAS  Google Scholar 

  20. Hyodo-Miura, J. et al. Involvement of NLK and Sox11 in neural induction in Xenopus development. Genes Cells 7, 487–496 (2002).

    Article  CAS  Google Scholar 

  21. Bigas, A., Martin, D. I. & Milner, L. A. Notch1 and Notch2 inhibit myeloid differentiation in response to different cytokines. Mol. Cell Biol. 18, 2324–2333 (1998).

    Article  CAS  Google Scholar 

  22. Inglés-Esteve, J., Espinosa, L., Milner, L. A., Caelles, C. & Bigas, A. Phosphorylation of Ser2078 modulates the Notch2 function in 32D cell differentiation. J. Biol. Chem. 276, 44873–44880 (2001).

    Article  Google Scholar 

  23. Kurooka, H., Kuroda, K. & Honjo, T. Roles of the ankyrin repeats and C-terminal region of the mouse notch1 intracellular region. Nucleic Acids Res. 26, 5448–5455 (1998).

    Article  CAS  Google Scholar 

  24. Kurooka, H. & Honjo, T. Functional interaction between the mouse notch1 intracellular region and histone acetyltransferases PCAF and GCN5. J. Biol. Chem. 275, 17211–17220 (2000).

    Article  CAS  Google Scholar 

  25. Pufall, M. A. et al. Variable control of Ets-1 DNA binding by multiple phosphates in an unstructured region. Science 309, 142–145 (2005).

    Article  CAS  Google Scholar 

  26. Zhao, Y., Tong, C. & Jiang, J. Hedgehog regulates smoothened activity by inducing a conformational switch. Nature 450, 252–258 (2007).

    Article  CAS  Google Scholar 

  27. Minoguchi, S. et al. RPB-L, a transcription factor related to RBP-Jκ. Mol. Cell. Biol. 17, 2679–2687 (1997).

    Article  CAS  Google Scholar 

  28. Raya, A. et al. Notch activity induces Nodal expression and mediates the establishment of left-right asymmetry in vertebrate embryos. Genes Dev. 17, 1213–1218 (2003).

    Article  CAS  Google Scholar 

  29. Kim, C. H. et al. Zebrafish elav/HuC homologue as a very early neuronal marker. Neurosci. Lett. 216, 109–112 (1996).

    Article  CAS  Google Scholar 

  30. Mizutani, T. et al. Conservation of the biochemical mechanisms of signal transduction among mammalian Notch family members. Proc. Natl Acad. Sci. USA 98, 9026–9031 (2001).

    Article  CAS  Google Scholar 

  31. Shimizu, K. et al. Functional diversity among Notch1, Notch2, and Notch3 receptors. Biochem. Biophys. Res. Commun. 291, 775–779 (2002).

    Article  CAS  Google Scholar 

  32. Lin, S. E. et al. Identification of new human mastermind proteins defines a family that consists of positive regulators for notch signaling. J. Biol. Chem. 277, 50612–50620 (2002).

    Article  CAS  Google Scholar 

  33. Wettstein, D. A., Turner, D. L. & Kintner, C. The Xenopus homolog of Drosphila Suppressor of Hairless mediates Notch signaling during primary neurogenesis. Development 124, 693–702 (1997).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank T. Honjo, T. C. SĂĽdhof, U. Lendahl, K. Yasutomo, C. Kintner, D. Hayward, J. D. Griffin, L. Wu, M. Takeichi, S. Chiba, A. Kikuchi, H. Fujisawa and K. Hozumi for providing plasmid vectors, antibody and cultured cells; H. Matsuo for technical assistance; members of the H. Aiba laboratory (especially T. Sunohara) for technical advice; and J. Ninomiya-Tsuji for helpful discussions. This research was supported by the Yamada Science Foundation (T.I.), the Astellas Foundation for Research on Metabolic Disorders (T.I. and M.I.), the Program for Improvement of Research Environment for Young Researchers from SCF commissioned by MEXT of Japan (T.I. and M.I.), and the Grants-in-Aid for Scientific Research programs in Japan (T.I., K.M. and M.I.).

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Authors and Affiliations

  1. Unit on Nervous Development Systems, 464-8602, Nagoya, Japan

    Tohru Ishitani, Tomoko Hirao, Miho Isoda & Motoyuki Itoh

  2. Division of Biological Science, Group of Signal Transduction, Laboratory of Cell Regulation, Graduate School of Science, 464-8602, Nagoya, Japan

    Tohru Ishitani, Maho Suzuki & Kunihiro Matsumoto

  3. Institute for Advanced Research, Nagoya University, 464-8602, Nagoya, Japan

    Motoyuki Itoh

  4. Division of Cell Regulation Systems, Department of Post-Genome Science Center, Medical Institute of Bioregulation, Kyushu University, 812-8582, Fukuoka, Japan

    Tohru Ishitani & Shizuka Ishitani

  5. Department of Molecular and Tumor Pathology, Chiba University Graduate School of Medicine, 260-8670, Chiba, Japan

    Kenichi Harigaya & Motoo Kitagawa

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  1. Tohru Ishitani
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Contributions

T.I. designed the research, performed most of the experiments, analysed data and wrote the paper. T.H., M.S., M. Isoda and S.I. performed experiments and analysed data. K.H. participated in discussions and helped write the paper. M.K. designed the research and performed the experiments, analysed data and wrote the paper. K.M. designed the initial experiments to identify NLK substrates, participated discussions, and helped write the paper. M. Itoh designed the project and experiments, supervised the research, coordinated experiments and wrote the paper.

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Correspondence to Tohru Ishitani or Motoyuki Itoh.

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Ishitani, T., Hirao, T., Suzuki, M. et al. Nemo-like kinase suppresses Notch signalling by interfering with formation of the Notch active transcriptional complex. Nat Cell Biol 12, 278–285 (2010). https://doi.org/10.1038/ncb2028

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