Calcineurin Binds the Transcription Factor NFAT1 and Reversibly Regulates Its Activity

Abstract

NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P.G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.

Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells

“…Evidence from previous work by us and others [14,15,17], as well as the results shown here, indicate that NFAT p cellular localization depends on its phosphorylation state. Dephosphorylation of NFAT p by treatment with either St or ionomycin results in nuclear localization of NFAT p .…”

“…Constitutive cycling of NFAT p proposed in this model provides a simple mechanism consistent with the results that in the continuing presence of ionomycin, FK506 induces a complete reversal of the nuclear localization and dephosphorylation of NFAT p (Figure 1) [14]. This model suggests that in the continuing presence of ionomycin (or antigen stimulation), CaN activity is required for maintaining a net accumulation of dephosphorylated NFAT p in the nucleus by continually dephosphorylating exported NFAT p , which would then be re-imported into the nucleus.…”

“…Pretreatment of the cells with FK506 inhibits the ionomycin-induced NFAT p nuclear localization, and under these conditions NFAT p remains in the cytosol in a phosphorylated form [15]. Loh et al [14] demonstrated similar results in immunofluorescence experiments, and in addition they showed that repeated cycles of ionomycin treatment followed by washout result in the successive phosphorylation and dephosphorylation of NFAT p .…”

“…Using immunocomplex assays we have shown that NFAT p is a direct CaN substrate in itro [15]. In addition, a direct association between CaN and NFAT p has been demonstrated with immobilized CaN bound to calmodulin-Sepharose, and the site of interaction on NFAT p has been mapped to the N-terminal 415 amino acid residues [14,16]. In intact HT-2 cells we demonstrated directly, by using $#P-labelling experiments, that NFAT p is present in a phosphorylated state in the cytosol of unactivated T-cells.…”

“…The link between CaN and NFAT p (also called NFATC2 [6] and NFAT1 [14]) is provided by evidence demonstrating that NFAT p is a CaN substrate. Using immunocomplex assays we have shown that NFAT p is a direct CaN substrate in itro [15].…”