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A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT

Nature volume 441, pages 646–650 (2006)Cite this article

Abstract

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1–4) is essential for vertebrate development and function1. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus1. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)1,2,3,4,5. Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila6,7 to identify additional regulators of the signalling pathway leading from Ca2+–calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+–calmodulin–calcineurin signalling and NFAT kinases) are conserved across species8,9, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.

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Figure 1: The NFAT regulatory domain and the genome-wide RNAi screen in Drosophila.
Figure 2: Screening for NFAT phosphorylation: DYRK kinase negatively regulates NFAT activation.
Figure 3: Depletion of endogenous DYRK1A potentiates NFAT activation.
Figure 4: Pre-phosphorylation by DYRK primes the NFAT regulatory domain for subsequent phosphorylation by GSK3 and CK1.
Figure 5: DYRK2 inhibits IL-2 transcription.

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References

  1. Hogan, P. G., Chen, L., Nardone, J. & Rao, A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 17, 2205–2232 (2003)

    Article  CAS  Google Scholar 

  2. Beals, C. R., Sheridan, C. M., Turck, C. W., Gardner, P. & Crabtree, G. R. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science 275, 1930–1934 (1997)

    Article  CAS  Google Scholar 

  3. Zhu, J. et al. Intramolecular masking of nuclear import signal on NF-AT4 by casein kinase I and MEKK1. Cell 93, 851–861 (1998)

    Article  CAS  Google Scholar 

  4. Okamura, H. et al. Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity. Mol. Cell 6, 539–550 (2000)

    Article  CAS  Google Scholar 

  5. Okamura, H. et al. A conserved docking motif for CK1 binding controls the nuclear localization of NFAT1. Mol. Cell. Biol. 24, 4184–4195 (2004)

    Article  CAS  Google Scholar 

  6. Boutros, M. et al. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832–835 (2004)

    Article  ADS  CAS  Google Scholar 

  7. Armknecht, S. et al. High-throughput RNA interference screens in Drosophila tissue culture cells. Methods Enzymol. 392, 55–73 (2005)

    Article  CAS  Google Scholar 

  8. Myers, E. W. et al. A whole-genome assembly of Drosophila. Science 287, 2196–2204 (2000)

    Article  ADS  CAS  Google Scholar 

  9. Yeromin, A. V., Roos, J., Stauderman, K. A. & Cahalan, M. D. A store-operated calcium channel in Drosophila S2 cells. J. Gen. Physiol. 123, 167–182 (2004)

    Article  CAS  Google Scholar 

  10. Aramburu, J. et al. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science 285, 2129–2133 (1999)

    Article  CAS  Google Scholar 

  11. Neal, J. W. & Clipstone, N. A. Glycogen synthase kinase-3 inhibits the DNA binding activity of NFATc. J. Biol. Chem. 276, 3666–3673 (2001)

    Article  CAS  Google Scholar 

  12. Carafoli, E. The calcium signalling saga: tap water and protein crystals. Nature Rev. Mol. Cell Biol. 4, 326–332 (2003)

    Article  CAS  Google Scholar 

  13. Roderick, H. L. & Bootman, M. D. Calcium influx: is Homer the missing link? Curr. Biol. 13, R976–R978 (2003)

    Article  CAS  Google Scholar 

  14. Roos, J. et al. STIM1, an essential and conserved component of store-operated Ca2+ channel function. J. Cell Biol. 169, 435–445 (2005)

    Article  CAS  Google Scholar 

  15. Liou, J. et al. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr. Biol. 15, 1235–1241 (2005)

    Article  CAS  Google Scholar 

  16. Zhang, S. L. et al. STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 437, 902–905 (2005)

    Article  ADS  CAS  Google Scholar 

  17. Suzuki, N. et al. Severe impairment of IL-1 and TLR signalling in mice lacking IRAK-4. Nature 416, 750–756 (2002)

    Article  ADS  CAS  Google Scholar 

  18. Kannan, N. & Neuwald, A. F. Evolutionary constraints associated with functional specificity of the CMGC protein kinases MAPK, CDK, GSK, SRPK, DYRK, and CK2α. Protein Sci. 13, 2059–2077 (2004)

    Article  CAS  Google Scholar 

  19. Lochhead, P. A., Sibbet, G., Morrice, N. & Cleghon, V. Activation-loop autophosphorylation is mediated by a novel transitional intermediate form of DYRKs. Cell 121, 925–936 (2005)

    Article  CAS  Google Scholar 

  20. Becker, W. & Joost, H. G. Structural and functional characteristics of Dyrk, a novel subfamily of protein kinases with dual specificity. Prog. Nucleic Acid Res. Mol. Biol. 62, 1–17 (1999)

    CAS  PubMed  Google Scholar 

  21. Kentrup, H. et al. Dyrk, a dual specificity protein kinase with unique structural features whose activity is dependent on tyrosine residues between subdomains VII and VIII. J. Biol. Chem. 271, 3488–3495 (1996)

    Article  CAS  Google Scholar 

  22. Wiechmann, S. et al. Unusual function of the activation loop in the protein kinase DYRK1A. Biochem. Biophys. Res. Commun. 302, 403–408 (2003)

    Article  CAS  Google Scholar 

  23. Himpel, S. et al. Specificity determinants of substrate recognition by the protein kinase DYRK1A. J. Biol. Chem. 275, 2431–2438 (2000)

    Article  CAS  Google Scholar 

  24. Campbell, L. E. & Proud, C. G. Differing substrate specificities of members of the DYRK family of arginine-directed protein kinases. FEBS Lett. 510, 31–36 (2002)

    Article  CAS  Google Scholar 

  25. Woods, Y. L. et al. The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bɛ at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase. Biochem. J. 355, 609–615 (2001)

    Article  CAS  Google Scholar 

  26. Nishi, Y. & Lin, R. DYRK2 and GSK-3 phosphorylate and promote the timely degradation of OMA-1, a key regulator of the oocyte-to-embryo transition in C. elegans. Dev. Biol. 288, 139–149 (2005)

    Article  CAS  Google Scholar 

  27. Shirayama, M. et al. The conserved kinases CDK-1, GSK-3, KIN-19, and MBK-2 promote OMA-1 destruction to regulate the oocyte-to-embryo transition in C. elegans. Curr. Biol. 16, 47–55 (2006)

    Article  CAS  Google Scholar 

  28. Harwood, A. J. Regulation of GSK-3: a cellular multiprocessor. Cell 105, 821–824 (2001)

    Article  CAS  Google Scholar 

  29. Flotow, H. et al. Phosphate groups as substrate determinants for casein kinase I action. J. Biol. Chem. 265, 14264–14269 (1990)

    CAS  PubMed  Google Scholar 

  30. Bhattacharyya, A. & Svendsen, C. N. Human neural stem cells: a new tool for studying cortical development in Down's syndrome. Genes Brain Behav. 2, 179–186 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Mathey-Prevot, N. Perrimon, S. Armknecht, N. Ramadan, J. Murphy, K. Richardson, M. Booker and staff at the Drosophila RNAi Screening Centre at Harvard Medical School for valuable assistance with the screens. We also thank M. Dziadek for the gift of antibody against STIM1, and W. C. Yeh and T. Mak for sending spleen and lymph nodes isolated from Irak4+/- and Irak4-/-mice. This work was supported by NIH grants to A.R. and S.F. S. Sharma is supported by a postdoctoral fellowship from the Canadian Institutes of Health Research. Author Contributions The Drosophila RNAi screen was designed, validated and performed by Y.G., and positive candidates were identified through visual screening performed by Y.G., J.N. and H.O. S. Sharma identified DYRK as a functional NFAT kinase through biochemical and functional analyses of the human homologues of candidate Drosophila kinases, drawing in part on unpublished data and reagents provided by H.O. Selected other candidates were investigated in detail by Y.G., S. Sharma, D.B. and S. Srikanth. A.I. and S.F. performed measurements of intracellular calcium concentration in mammalian cells. J.N. and B.T. were responsible for bioinformatic analysis. P.G.H. and A.R. provided overall direction and supervised project planning and execution.

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Author notes

  1. Julie Nardone

    Present address: Cell Signaling Technology, 166B Cummings Center, Beverly, Massachusetts, 01915, USA

  2. Heidi Okamura

    Present address: AVEO Pharmaceuticals Inc., 75 Sidney Street, Cambridge, Massachusetts, 02139, USA

  3. Diana Bolton

    Present address: Department of Pediatrics, University of Washington, Seattle, Washington, 98195, USA

  4. Yousang Gwack and Sonia Sharma: *These authors contributed equally to this work

Authors and Affiliations

  1. The CBR Institute for Biomedical Research,

    Yousang Gwack, Sonia Sharma, Julie Nardone, Bogdan Tanasa, Alina Iuga, Sonal Srikanth, Heidi Okamura, Diana Bolton, Stefan Feske, Patrick G. Hogan & Anjana Rao

  2. Departments of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts, 02115, USA

    Yousang Gwack, Sonia Sharma, Alina Iuga, Sonal Srikanth, Heidi Okamura & Anjana Rao

  3. Departments of Pediatrics, Harvard Medical School, 200 Longwood Avenue, Massachusetts, 02115, Boston, USA

    Stefan Feske

  4. Cell Signaling Technology, 166B Cummings Center, Beverly, Massachusetts, 01915, USA

    Heidi Okamura & Diana Bolton

  5. AVEO Pharmaceuticals Inc., 75 Sidney Street, Cambridge, Massachusetts, 02139, USA

    Julie Nardone & Diana Bolton

  6. Department of Pediatrics, University of Washington, Seattle, Washington, 98195, USA

    Julie Nardone & Heidi Okamura

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Correspondence to Anjana Rao.

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Gwack, Y., Sharma, S., Nardone, J. et al. A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT. Nature 441, 646–650 (2006). https://doi.org/10.1038/nature04631

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