1. The CBR Institute for Biomedical Research and the Departments of
2. Pathology and
3. Pediatrics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA
4. †Present addresses: Cell Signaling Technology, 166B Cummings Center, Beverly, Massachusetts 01915, USA (J.N.); AVEO Pharmaceuticals Inc., 75 Sidney Street, Cambridge, Massachusetts 02139, USA (H.O.); Department of Pediatrics, University of Washington, Seattle, Washington 98195, USA (D.B.)
5. *These authors contributed equally to this work

Correspondence to: Anjana Rao^1,2 Correspondence and requests for materials should be addressed to A.R. (Email: arao@cbr.med.harvard.edu)

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 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 (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.

1. The CBR Institute for Biomedical Research and the Departments of
2. Pathology and
3. Pediatrics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA
4. †Present addresses: Cell Signaling Technology, 166B Cummings Center, Beverly, Massachusetts 01915, USA (J.N.); AVEO Pharmaceuticals Inc., 75 Sidney Street, Cambridge, Massachusetts 02139, USA (H.O.); Department of Pediatrics, University of Washington, Seattle, Washington 98195, USA (D.B.)
5. *These authors contributed equally to this work

Correspondence to: Anjana Rao^1,2 Correspondence and requests for materials should be addressed to A.R. (Email: arao@cbr.med.harvard.edu)

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