Concerted Dephosphorylation of the Transcription Factor NFAT1 Induces a Conformational Switch that Regulates Transcriptional Activity

Abstract

NFAT transcription factors are highly phosphorylated proteins that are regulated by the calcium-dependent phosphatase calcineurin. We show by mass spectrometry that NFAT1 is phosphorylated on fourteen conserved phosphoserine residues in its regulatory domain, thirteen of which are dephosphorylated upon stimulation. Dephosphorylation of all thirteen residues is required to mask a nuclear export signal (NES), cause full exposure of a nuclear localization signal (NLS), and promote transcriptional activity. An inducible phosphorylation site in the transactivation domain contributes to transcriptional activity. Our data suggest that dephosphorylation promotes NFAT1 activation by increasing the probability of an active conformation, in a manner analogous to that by which depolarization increases the open probability of voltage-gated ion channels. This conformational switch paradigm may explain modification-induced functional changes in other heavily phosphorylated proteins.

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].…”

Transcriptional complexes formed by NFAT dimers regulate the induction of T cell tolerance

“…We have previously shown that these active NFAT proteins, which contain several serine-toalanine substitutions in key residues located in the regulatory region of the NHR domain, are constitutively nuclear and insensitive to the inhibitory effect of cyclosporine A (CsA) (19). Therefore, to assess the effect of the plasmid-encoded NFAT proteins, transfected cells were treated with CsA to block endogenous NFAT activation.…”

DNA-PK phosphorylation sites on Oct-1 promote cell survival following DNA damage

“…Multi-site phosphorylation has been documented for several factors, including Sic1 (Nash et al, 2001), NFAT (Okamura et al, 2000), Stat6 (Maiti et al, 2005) and Ets-1 (Pufall et al, 2005). It has been suggested to overcome a threshold needed to establish a biological response (Nash et al, 2001;Gunawardena, 2005;Pufall et al, 2005).…”