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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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.
Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
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