NFAT regulation: DYRK kinases prime inhibition

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A genome-wide RNAi screen in Drosophila identifies the kinases DYRK1A and DYRK2 as negative regulators of NFAT in vertebrates.

Nuclear factor of activated T cells (NFAT) proteins are Ca²⁺-regulated pro-inflammatory transcription factors that are also important in musculoskeletal development, vascular patterning and osteoclast differentiation. NFAT proteins are phosphorylated and reside in the cytoplasm in resting cells. An increase in intracellular Ca²⁺ leads to their dephosphorylation by the Ca²⁺/calmodulin-dependent serine phosphatase calcineurin and translocation to the nucleus. When in the nucleus they induce target gene transcription, thus providing a direct link between intracellular Ca²⁺ signaling and gene expression. NFAT proteins are only found in vertebrates, but the pathways that regulate NFAT phosphorylation and localization are conserved across species. Now, Yousang Gwack et al. identify two members of the dual-specificity tyrosine-phosphorylation regulated kinases (DYRKs)-family as novel regulators of NFAT using a genome-wide RNA interference (RNAi) screen in Drosophila.

The authors first had to ensure that an NFAT-GFP fusion protein was appropriately regulated in response to Ca²⁺ when expressed in Drosophila S2R⁺ cells. Indeed, Ca²⁺ store depletion by thapsigargin led to NFAT dephosphorylation and its translocation to the nucleus, whereas inhibition of calcineurin with cyclosporin A or RNAi-mediated depletion prevented NFAT dephosphorylation and nuclear import.

The RNAi screen identified 738 potential regulators of NFAT that impaired the thapsigargin-dependent NFAT dephosphorylation and nuclear import. Among them were Na⁺/Ca²⁺ exchangers, the scaffold protein Homer, stromal interaction protein (STIM) and several kinases including casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3), which are well established inhibitors of NFAT. To determine whether any of the novel NFAT-regulating kinases could directly phosphorylate NFAT in vertebrates, the authors performed immunoprecipitation experiments and in vitro kinase assays in HEK293 cells, and found that DYRK1A and DYRK2, were able to counter calcineurin mediated dephosphorylation of NFAT by directly phosphorylating NFAT’s regulatory domain. Conversely, DYRK1A depletion by RNAi in HeLa cells led to NFAT activation further indicating that DYRKs are negative regulators of NFAT in vivo.

Interestingly, DYRKs were found to prime NFAT for further phosphorylation by GSK3 and CK1. The authors showed that pre-phosphorylation by DYRK2 accelerated CK1-mediated phosphorylation by two-fold. However, unlike other ‘priming kinases’ DYRKs phosphorylate a distinct motif (SP-3) from those targeted by CK1 (the SRR-1 motif) or GSK3 (the SP-2 motif), thus, the authors suggest that DYRKs are involved in a ‘discontiguous priming’ mechanism.

In addition to showing that DYRK1A and DYRK2 are novel negative regulators of NFAT, this study demonstrates that provided the key members of a signal transduction pathway are represented in Drosophila, the relative ease of carrying out genome-wide screens in S2R⁺ cells can be harnessed to accelerate the identification of vertebrate signaling pathway regulators.

Monica Hoyos-Flight
Cell Migration Gateway

References

Yousang Gwack, Sonia Sharma, Julie Nardone, Bogdan Tanasa, Alina Iuga, Sonal Srikanth, Heidi Okamura, Diana Bolton, Stefan Feske, Patrick G Hogan & Anjana Rao. A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT. Nature 441, 646 - 650 (01 Jun 2006); | Article | PubMed |