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

Precise regulation of the NFAT ( nuclear factor of activated T cells) family of transcription factors (NFAT1 - 4) is essential for vertebrate development and function(1). 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 nucleus(1). NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)(1-5). Here we have used a genome-wide RNA interference ( RNAi) screen in Drosophila(6,7) 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 species(8,9), 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 (SRR1) 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.