Identification of CK1 as an NFAT1 SRR-1 kinase. (A) (Top) Silver stain gel showing kinase-active fractions from a size exclusion column. Arrow indicates the position of a band that cofractionates with SRR-1 kinase activity. (Bottom) The GST(149-183) fusion protein was incubated with an aliquot from each column fraction in the presence of [γ-³²P]ATP to assay for the presence of NFAT1 SRR-1 kinase activity. (B) Western blot analysis confirms that the distribution of CK1α coincides precisely with fractions containing NFAT1 SRR-1 kinase activity after fractionation of T-cell lysates through several columns. (C) Western blot analysis of CK1ɛ and CK1α from an early anion-exchange column shows that CK1α and CK1ɛ cofractionate with SRR-1 kinase activity, whereas both isoforms of GSK3 elute earlier and are distinct from the SRR-1 kinase activity. (D) The NFAT1 SRR-1 motif is phosphorylated by purified CK1 but not by GSK3. For priming with PKA, GST fusion proteins were prephosphorylated by PKA in the presence of cold ATP, washed thoroughly to remove PKA, and then used as a substrate for in vitro kinase assays with GSK3 and [γ-³²P]ATP. Prephosphorylation of the fusion protein by PKA enables GSK3 to target GST-NFAT1(1-415) (lanes 1 and 2). Within this domain, GSK3 is able to target the SP-2 motif when it is primed by PKA (lanes 3 and 4) but is not able to phosphorylate the SRR-1 region (lanes 5 and 6). In contrast, purified recombinant CK1 phosphorylates GST-NFAT1(1-415) (lane 7) and the SRR-1 region (lane 8) without the need for prior phosphorylation.

CK1 is complexed with NFAT1 in resting cells but dissociates upon stimulation. (A) Analysis of resting T-cell lysates fractionated over a size exclusion column shows that NFAT1 exists in a high-molecular-weight complex that coelutes with a subpopulation of CK1. GSK3 and calcineurin are not part of this complex. Aliquots from each fraction were separated by SDS-PAGE, and the elution profile of each protein was visualized by Western blotting. (B) Stimulation of T cells with the calcium ionophore ionomycin before fractionation of lysates over a size exclusion column results in dissociation of CK1 from the NFAT1 complex. CK1 no longer elutes in two peaks but elutes primarily in fractions corresponding to the expected molecular weight for a monomer of the kinase. (C) Jurkat cells that were either resting (R) or stimulated with 1 μM ionomycin (iono) in the presence of 2 mM CaCl₂ were lysed and subjected to immunoprecipitation with an antibody against the C terminus of NFAT1. Endogenous CK1α can be coimmunoprecipitated with NFAT1 from resting cells but not from cells that have been stimulated. WB, Western blot.

Docking-site mutations that disrupt NFAT1-CK1 association also result in aberrant nuclear localization of NFAT1. (A) Mutation of residues within the F-X-X-X-F motif disrupts association of NFAT1 and CK1. (Left) The mutations shown were introduced into GST proteins containing the first 100 amino acids of NFAT1. (Right) The GST proteins were incubated with cell lysates expressing CK1ɛ, and the ability of the fusion proteins to interact stably with CK1 was detected by Western blotting. Mutations that altered either phenylalanine within the docking motif of NFAT1 virtually abolished NFAT1-CK1 interactions, but substitutions in nearby amino acids not affecting these residues did not diminish the association. (B) Mutation of the CK1 docking site interferes with proper localization of NFAT1. (Top) GFP-NFAT1 proteins containing mutations in the CK1 binding motif were transfected into HeLa cells, and their localization was visualized by fluorescence. Mutations that affected the ability of CK1 and NFAT1 to associate stably, as seen in panel A, also resulted in improper localization of NFAT1. The constitutive nuclear localization of CK1 docking-site mutants is similar to that observed upon mutation of the target serine residues in the SRR-1 motif (bottom panel). Mutations that did not affect CK1-NFAT1 associations, as shown in panel A, also did not affect NFAT1 localization. (Bottom) Two representative fields from an NFAT mutant with substitutions of the target serine residues in the SRR-1 motif, showing different degrees of nuclear localization.

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 phosphoserines that become dephosphorylated upon stimulation; open circles, nonconserved or constitutively phosphorylated serines. GST(149-183) includes only the region containing the NFAT1 SRR-1 motif, while GST(1-415) encompasses the entire NFAT1 regulatory domain. (B) Fractionation of T-cell lysates over a cation-exchange column reveals two separate peaks of kinase activity when GST(1-415) is used as a substrate (shaded circles). Assay of the same fractions using GST(149-183) as a substrate shows that only one of these peaks contains an activity that is able to phosphorylate the SRR-1 motif (solid diamonds). (C) Autoradiograph of kinase assays from the fractionation shown in panel B. NFAT1 kinase activity was assessed by incubating an aliquot from each fraction with either GST(1-415) or GST(149-183) in the presence of [γ-³²P]ATP. For GST(1-415), there are two peaks of activity: fractions 33 to 37 and fractions 49 and 50 (top panel). For GST(149-183), only the activity present in fractions 49 and 50 is capable of efficiently phosphorylating the fusion protein (center panel). Mutation of the conserved serines in the SRR-1 motif to alanine completely abolishes phosphorylation of GST(149-183) by the kinase activity present in fractions 49 and 50 (bottom panel).

Interaction of NFAT family members with CK1. (A) GST fusion proteins containing the regulatory domains of NFAT proteins bind CK1 isoforms. GST or GST fusion proteins encompassing the regulatory domains of NFAT1, NFAT2, or NFAT4 were incubated with HEK 293 cell lysates that were either mock transfected or transfected with CK1 isoform α or ɛ. Bound proteins were visualized by Western blotting and sequential probing with antibodies against CK1α and CK1ɛ. (B) NFAT1 interacts with the kinase domain of CK1. Full-length HA-tagged NFAT1 was expressed in HEK 293 cells either alone, in combination with HA-tagged full-length CK1ɛ, or in combination with C-terminally truncated CK1ɛ [CK1ɛ(319)]. NFAT1 was immunoprecipitated with an anti-NFAT1 antibody, and precipitated proteins were visualized by Western blotting with an antibody against the HA tag. (C) Endogenous NFAT1 and CK1 stably associate. NFAT1 was immunoprecipitated from Jurkat T cells with either an anti-NFAT1 antibody or preimmune serum. The presence of associated CK1 in the immunoprecipitates was detected by Western blotting (WB) with antibodies against CK1α or CK1ɛ. (D) The region of NFAT1 that mediates CK1 binding maps to the N-terminal transactivation domain. GST fusion proteins encompassing various regions of the NFAT1 regulatory domain were incubated with cell lysates transfected with CK1ɛ. Fusion proteins able to associate with CK1 were detected by Western blot analysis using anti-CK1ɛ.

CK1 and GSK3 coordinate to effect rephosphorylation and nuclear export of NFAT1. (A) D5 T cells were preincubated for 1 h with CKI-7, LiCl, or a combination of both inhibitors. Control cells were incubated with a corresponding amount of DMSO and/or NaCl. Cells were stimulated with ionomycin to induce dephosphorylation and nuclear translocation of NFAT1. Calcineurin activity was subsequently inhibited by addition of CsA to the cells, which were attached to coverslips and fixed in paraformaldehyde at the indicated time points. The subcellular localization of endogenous NFAT1 was visualized by indirect immunofluorescence. (B) D5 T cells were preincubated with inhibitors and thenstimulated with ionomycin (iono) as for panel A. CsA was added to cells, and the phosphorylation state of NFAT1 at various time points after CsA addition was determined by quickly lysing cells in boiling SDS sample buffer. Western blotting was performed with an anti-NFAT1 antibody. (C) CKI-7 does not inhibit GSK3 kinase activity. GST(1-415) was prephosphorylated by use of PKA with cold ATP before being washed extensively and incubated with increasing concentrations of CKI-7 in the presence of [γ-³²P]ATP and either GSK3 or CK1. LiCl and NaCl (each at 10 mM) were used as positive and negative controls, respectively, for inhibition of GSK3 activity. (D) Quantitation of the ability of CKI-7 to inhibit GSK3 and CK1. GST(1-415) was incubated with either GSK3 or CK1 in the presence of CKI-7 as described for panel C. Radiolabeled GST(1-415) was then separated by SDS-PAGE, and incorporation of ³²P into the fusion protein was quantified by phosphorimager analysis. (E) Inhibition of CK1 or GSK3 mimics the effect of mutating the SRR-1 or SP motifs. Resting HeLa cells stably expressing the NFAT1(1-460)-GFP fusion protein were incubated for 2 h in the presence of LMB in addition to the inhibitor DMSO, LiCl, or CKI-7 before being fixed and visualized by GFP fluorescence. Inhibition of CK1 results in partial NFAT1 nuclear localization in resting cells, reminiscent of NFAT1 localization after SRR-1 mutation. LiCl has no effect on endogenous NFAT1 localization in resting cells, in agreement with a role for GSK3 in phosphorylation of the SP motifs but not the NFAT1 SRR-1 motif.

The F-X-X-X-F motif also serves as a CK1 docking site in the PER circadian-rhythm proteins. (A) Sequences of F-X-X-X-F motifs in NFAT and PER proteins thought to interact with CK1. (B) Mutation of the F-X-X-X-F motif in mPER2 abolishes binding to CK1ɛ. Mutations targeting the phenylalanines shown in panel A were introduced into GST fusion proteins containing mPER2 residues 583 to 765, which encompass most of the CK1 binding domain. Fusion proteins were incubated with HEK cell lysates and analyzed for the ability to interact with endogenous CK1 by Western blotting (WB) using an antibody against CK1ɛ. GST fusion proteins containing the wild-type CK1 binding domain of mPER2 (amino acids 583 to 765) or NFAT1 (amino acids 1 to 100) were able to associate with CK1ɛ, while GST alone or fusion proteins carrying mutations in the F-X-X-X-F motif were unable to interact with the kinase. (C) Processive phosphorylation by CK1. See Discussion for details.