The activity of transcription factors is tightly regulated by a variety of mechanisms. One class of transcription factors achieves strict regulation through control of subcellular localization, existing in an inactive or unstable state in the cytoplasm until they are activated in response to signals transduced from cell surface receptors (reviewed in reference 7
). Upon activation, the transcription factors translocate from the cytoplasm to the nucleus, where they induce gene expression. This strategy is employed by diverse families of transcription factors, including those in the Wnt, Notch, Hedgehog, STAT, SMAD, NF-κB, NFAT, and Pho4 signaling pathways (reviewed in references 7
, and 31
). In each of these cases, phosphorylation is utilized as a means of regulating nuclear translocation of the latent cytoplasmic transcription factor, although the precise mechanisms and signaling pathways involved differ widely.
NFAT is an example of a latent transcription factor whose subcellular localization, DNA binding, and transcriptional activity are all dictated by its phosphorylation state. The NFAT family is composed of four calcium-regulated members, NFAT1 (also known as NFATc2 or NFATp), NFAT2 (NFATc1, NFATc), NFAT3 (NFATc4), and NFAT4 (NFATc3, NFATx), that play an essential role in immune function as well as in development of the cardiac, vascular, and immune systems (reviewed in references 12
). NFAT proteins are located in the cytoplasm of resting cells, where they exist in a heavily phosphorylated inactive state. In response to calcium-mobilizing stimuli they are partially dephosphorylated by calcineurin, a calcium-dependent phosphatase, and translocate into the nucleus (reviewed in references 11
, and 45
). Treatment of stimulated cells with calcium chelators or the calcineurin inhibitor cyclosporin A (CsA) results in the rapid rephosphorylation of NFAT and its export out of the nucleus.
The responsiveness of NFAT proteins to calcium signaling is mediated through their regulatory domains, which contain both the calcineurin binding site and the majority of the serine residues that are phosphorylated in resting cells. Mass spectrometric analysis has demonstrated that the regulatory domain of NFAT1 is constitutively phosphorylated at 18 serine residues in resting T cells (41
). Twelve of these phosphorylated residues are contained within two distinct types of serine-rich sequence motifs, the SRR-1 and SPXX repeat motifs (referred to below as SP motifs), which are recognizably conserved throughout the NFAT family (see Fig. ). The SRR-1 region is the primary region involved in NFAT nuclear import, whereas the SP motifs have been reported to control DNA-binding affinity and nuclear export (5
). Several kinases, including glycogen synthase kinase 3 (GSK3), casein kinase 1 (CK1), p38, and c-Jun N-terminal kinase 1 (JNK1), have been suggested to regulate NFAT function (6
; reviewed in references 23
). However, no single proposed kinase displays the specificity required for full phosphorylation of both the SRR-1 and SP motifs.
FIG. 1. NFAT1 is phosphorylated by two distinct kinases. (A) N-terminal regulatory domain of NFAT1, showing the conserved serine motifs and relative positions of phosphorylated serine residues identified in NFAT1 from resting T cells. Shaded circles, conserved (more ...)
Here we demonstrate that NFAT1 regulation involves the concerted effort of distinct kinases to phosphorylate each of the two types of serine motifs. CK1 specifically phosphorylates only the NFAT1 SRR-1 motif by docking at a site that is conserved in the N-terminal regions of all four NFAT proteins, and disruption of CK1 docking is sufficient to cause aberrant nuclear translocation of NFAT1. CK1 and NFAT1 are present in a high-molecular-weight complex in resting T cells that dissociates upon activation. GSK3 is not present in this complex but can phosphorylate NFAT SP motifs if suitably primed and can synergize with CK1 to promote NFAT1 nuclear export. The NFAT pathway resembles the Wnt, Hedgehog, and circadian-rhythm pathways in being regulated by the combined activity of CK1 and GSK3, and we demonstrate that the circadian rhythm protein mPER2 also contains a functional CK1 docking motif.