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

Abstract

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

Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex

“…In resting cells, NFAT proteins are located in the cytoplasm, where they are heavily phosphorylated through synergistic actions of three different families of kinases, casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), and dual specificity tyrosine phosphorylation regulated kinase (DYRK) (2)(3)(4)(5). Phosphorylation results in masking of a nuclear localization sequence (NLS), exposure of a nuclear export sequence (NES), and cytoplasmic localization of NFAT (1,2).…”

Increased dosage of DYRK1A and DSCR1 delays neuronal differentiation in neocortical progenitor cells

“…Furthermore, we linked increased dosage of DYRK1A and DSCR1 to reduced nuclear levels of transcription factor NFATc. It is known that nuclear export of the NFATc family of transcription factors is accelerated upon its phosphorylation by DYRK1A (Arron et al 2006;Gwack et al 2006) and that their nuclear import is suppressed by DSCR1 through deactivation of calcineurin (Ryeom et al 2008), an important regulator for nuclear entry of NFATc. Consistently, we found that overexpression of DYRK1A and DSCR1 diminished nuclear occupancy of NFATc in progenitors.…”

“…In this activation process, DSCR1 inhibits calcineurin and thereby suppresses dephosphorylation and nuclear entry of NFATc (Rothermel et al 2000). On the other hand, nuclear export of NFATc is accelerated upon its priming phosphorylation of the serine/proline repeats by DYRK1A (Arron et al 2006;Gwack et al 2006) and subsequent phosphorylation by GSK3 (Graef et al 1999). Of note, in HEK cells, overexpression of DYRK1A and DSCR1 synergistically prevents NFATc-dependent transcription (Arron et al 2006).…”

Calcium signaling in lymphocytes

“…Dephosphorylation of cytoplasmic NFAT proteins by calcineurin unmasks the nuclear localization sequence and then NFAT proteins translocate into nucleus [4]. NFAT-driven gene expression is highly dependent on sustained Ca 2+ influx and calcineurin activity, because a decrease in intracellular Ca 2+ levels or treatment with the calcineurin inhibitor cyclosporin A results in the immediate export of NFAT from nucleus by NFAT kinases, which include GSK3 (glycogen synthase kinase 3), CK1 (casein kinase 1) and DYRK1A (dual-specificity tyrosine-phosphorylation regulated kinase 1A) [28][29][30].…”