Selective Inhibition of NFAT Activation by a Peptide Spanning the Calcineurin Targeting Site of NFAT

Molecular Cell, Vol. 1, 627–637, April, 1998, Copyright 1998 by Cell Press

Selective Inhibition of NFAT Activationby a Peptide Spanning the CalcineurinTargeting Site of NFAT

genes and other inducible genes (Rao, 1994; Rao et al.,1997).

The importance of calcineurin in NFAT activation hasbeen highlighted by studies on the immunosuppressivedrugs cyclosporin A (CsA) and FK506 (Liu et al., 1991;

Jose Aramburu,† Francisco Garcıa-Cozar,†Anuradha Raghavan, Heidi Okamura,Anjana Rao,* and Patrick G. Hogan*The Center for Blood Research andDepartment of PathologyHarvard Medical School Schreiber and Crabtree, 1992; Liu, 1993; Crabtree andBoston, Massachusetts 02115 Clipstone, 1994). These drugs impair NFAT activation

and NFAT-dependent gene expression by inhibiting cal-cineurin activity (Mattila et al., 1990; Bierer et al., 1993).The profound immunosuppression induced by CsA andSummaryFK506 constituted the first evidence that inhibiting cal-cineurin, and thereby inhibiting NFAT activation, couldNFAT transcription factors play a key role in the im-provide an effective therapeutic intervention. However,mune response. The activation of NFAT proteins isthe clinical use of CsA and FK506 is currently limited tocontrolled by calcineurin, the calmodulin-dependenttransplant patients, because long-term treatment withphosphatase that is inhibited by the immunosuppres-these drugs can have serious nephrotoxic and neuro-sive drugs cyclosporin A and FK506. Here, we identifytoxic effects (Sigal et al., 1991). The biochemical basisa short conserved sequence in NFAT proteins thatfor the adverse effects is not fully understood, but drug–targets calcineurin to NFAT. Mutation of a single resi-immunophilin complexes cause a broad inhibition ofdue in this sequence impairs the calcineurin-mediatedcalcineurin activity against its protein substrates, anddephosphorylation and nuclear translocation of NFAT1.more selective intervention in the calcineurin–NFAT path-Peptides spanning the region inhibit the ability of cal-way mightprovide immunosuppressive drugs for a widercineurin to bind to and dephosphorylate NFAT pro-range of clinical indications.teins, without affecting the phosphatase activity of

calcineurin against other substrates. When expressed A distinct approach to preventing activation of NFATintracellularly, a corresponding peptide inhibits NFAT would be to interfere with the protein–protein interactiondephosphorylation, nuclear translocation, and NFAT- (Loh et al., 1996b; Luo et al., 1996c; Shibasaki et al.,mediated gene expression in response to stimulation. 1996; Wesselborg et al., 1996; Masuda et al., 1997) be-Thus, disruption of the enzyme–substrate docking in- tween calcineurin and NFAT. In NFAT1, the interactionteraction that directs calcineurin to NFAT can effec- has been mapped to a regulatory domain N-terminaltively block NFAT-dependent functions. to the DNA-binding domain, which harbors the nuclear

localization signal (NLS) responsible for calcineurin-acti-vated nuclear translocation and contains multiple phos-Introductionphoserine residues that are dephosphorylated by cal-

The transcription factor NFAT has a central role in the cineurin during activation of NFAT1 in cells (Loh et al.,functioning of T cells, B cells, mast cells, and other 1996b; Luo et al., 1996c). The corresponding regions ofimmune cells. There are four known NFAT-family pro- the proteins NFAT2 and NFAT4 also bind calcineurin andteins, of which at least three (NFAT1/p, NFAT2/c, and mediate the calcineurin-sensitive localization of theseNFAT4/x) are expressed in cells of the immune system proteins (Shibasaki et al., 1996; Beals et al., 1997; Ma-(McCaffrey et al., 1993; Northrop et al., 1994; Hoey et suda et al., 1997).al., 1995; Masuda et al., 1995). Physiological activation In the current work, we have made a series of muta-of receptors such as the T cell receptor, the B cell anti- tions in the N-terminal regulatory domain of NFAT1, al-gen receptor, or the Fc receptors for IgG and IgE leads tering motifs conserved in the NFAT family, and havevia several intermediate steps toactivation of the protein defined a circumscribed region of NFAT1 that is crucialphosphatase calcineurin, which dephosphorylates NFAT for effective recognition and dephosphorylation of NFAT1(Choi et al., 1994; Venkataraman et al., 1994; Aramburu by calcineurin. Alteration of single residues in this regionet al., 1995; Hutchinson and McCloskey, 1995; Shaw et impairs the nuclear translocation of NFAT1 in stimulatedal., 1995; Loh et al., 1996a). The nuclear import, DNA- cells. A synthetic peptide spanning the region disruptsbinding activity, and transcriptional function of NFAT the interaction in vitro between NFAT proteins and cal-are regulated by calcineurin-mediated dephosphoryla- cineurin and inhibits the calcineurin-mediated dephos-tion (Shaw et al., 1995; Loh et al., 1996b; Luo et al.,

phorylation. Expression of the inhibitory peptide in cells1996b, 1996c; Shibasaki et al., 1996; Timmerman et al.,

blocks calcineurin-mediatedNFAT activation and NFAT-1996; Masuda et al., 1997). NFAT proteins, working in

driven gene expression. Our results demonstrate thatconcert with other transcription factors activated via

short peptide inhibitors may be used to disrupt a criticalreceptor or costimulatory pathways, play an essentialenzyme–substrate interaction within the NFAT activa-role in regulating the expression of certain cytokinetion pathway, and constitute a step toward developingimmunosuppressive agents that target the calcineurin–NFAT interaction without inhibiting calcineurin activity*To whom correspondence should be addressed.

†These authors contributed equally to this work. directly.

Molecular Cell628

Figure 1. Identification of an NFAT1 MutantImpaired in Calcineurin-Dependent NuclearImport

(A) Location of conserved motif 2 (CM2) inthe NFAT regulatory domain. A schematic di-agram of NFAT proteins, indicating the posi-tion of the CM2 motif and its sequence inthe four known NFAT proteins, as well as theposition of the DNA-binding domain (residuesz400–z700). Other conserved motifs in theNFAT regulatory domain are shown as closedboxes. (SR-1), a serine-rich region implicatedin controllingnuclear translocation; (NLS), nu-clear localization signal; (SP1), (SP2), and(SP3), conserved SPxx repeats; (TAD-N) and(TAD-C), N- and C-terminal transactivationdomains.(B) Mutations in the CM2 motif of NFAT1 thatwere analyzed in the course of this study.(C) Effect of the CM2 and ST21 mutationson nuclear import of full-length NFAT1. HeLacells transiently expressing wild-type HA-tagged NFAT1 or the CM2 or ST21 mutantproteins were left unstimulated or stimulatedwith ionomycin (3 mM, 10 min). NFAT1 wasdetected by immunocytochemistry with anti-HA tag.(D) Effect of the CM2 mutation on nuclearimport controlled by the NFAT1 N-terminaldomain. HeLa cells and Cl.7W2 T cells ex-pressing wild-type HA-tagged NFAT1(1–460)-GFP or the corresponding CM2 mutant wereleft unstimulated or stimulated for 10 min withionomycin (HeLa cells, 3 mM; Cl.7W2 cells, 2mM). The NFAT1 fusion protein was visualizedby GFP fluorescence.

Results The CM2 mutation alters the sequence 110SPRIEITPS118

of NFAT1 by replacing arginine 112, glutamate 114, andthreonine 116 with alanine (Figure 1B). The mutatedThe SPRIEIT Sequence of NFAT1 Is Required

for Nuclear Import NFAT1 was cytoplasmic in resting cells, and did nottranslocate to the nucleus upon ionomycin stimulationThe NFAT1 regulatory domain contains several motifs

that are recognizably conserved and occur in the same (Figure 1C). Another protein mutated in the same re-gion (ST21), in which the serine residues flanking thelinear order in all members of the NFAT family (Figure

1A). To examine the role of this domain in modulating SPRIEITPS motif were replaced by alanine, showed nor-mal translocation (Figure 1C). The CM2 mutation alsoNFAT1 function, we prepared a panel of NFAT1 proteins

with alanine residues substituted at 58 positions in the abrogated the nuclear translocation of a green fluores-cent protein (GFP) fusion protein containing the N-termi-conserved motifs (A. R. and P. G. H., unpublished data),

each mutated protein having 1–5 substitutions. HA- nal domain of NFAT1 (residues 1–460) in both HeLacells and Cl.7W2 T cells (Figure 1D), indicating that thetagged mutant proteins were expressed in HeLa cells

and Cl.7W2 T cells, and their subcellular distribution inhibitory effect of the CM2 mutation does not requirean intact DNA binding domain or the C-terminal domainwas assessed in resting and ionomycin-stimulated cells.

Two mutants, the KRR.AAA substitution identifying the of NFAT1.Nuclear translocation of NFAT1 was also impairedcore of the conserved NLS (Luo et al., 1996c) and a

mutant in conserved motif 2 (CM2, Figure 1A), showed by alanine replacement of the individual residues argi-nine 112, glutamate 114, and threonine 116. In Cl.7W2a striking inability to translocate to the nucleus upon

ionomycin stimulation. cells transiently expressing NFAT1(1–460)-GFP, wild-type

Targeting of Calcineurin to NFAT629

Figure 2. Mutations in the CM2 Motif Impair the Dephosphorylation of NFAT1 by Calcineurin

(A) Effect of the CM2 mutation on NFAT1 dephosphorylation in ionomycin-stimulated cells. HeLa cells and Cl.7W2 T cells expressing wild-type HA-tagged NFAT1(1–460)-GFP or the corresponding CM2 mutant were left unstimulated or stimulated with ionomycin (3 mM, 10 min).Here, and in Figures 2B–2D, cell lysates were subjected to SDS–PAGE and phosphorylation of NFAT1 was assessed by Western blotting withanti-HA tag.(B) Effect of the CM2 mutation on NFAT1 dephosphorylation in cells treated with graded concentrations of ionomycin. Cl.7W2 T cells expressingwild-type (WT) or CM2 mutant NFAT1(1–460)-GFP were left unstimulated (lane 1), stimulated with different concentrations of ionomycin for10 min (lanes 2–6), or pretreated for 30 min with 500 nM CsA before stimulation with 6 mM ionomycin (lane 7).(C) Effect of the CM2 mutation on dephosphorylation of NFAT1 by exogenous calcineurin in cell extracts. Cytoplasmic extracts from HeLacells expressing HA-tagged full-length NFAT1 (WT) or the CM2 mutant protein were prepared using iodoacetamide to inactivate endogenouscalcineurin, and then incubated in the presence of Ca21 (lane 1), CsA/CypA (lane 2), or Ca21 and calcineurin (lanes 3–5) for 30 min at 308C.(D) Effect of mutations in individual residues of the SPRIEIT motif. Cl.7W2 T cells expressing wild-type (WT) HA-tagged NFAT1(1–460)-GFPor the specified mutant protein were left unstimulated (lane 1) or stimulated with the indicated concentrations of ionomycin for 10 min(lanes 2–6).

NFAT1 translocated to the nucleus in .90% of the iono- cells stimulated with 220 nM ionomycin, and completedephosphorylation with 660 nM ionomycin. In contrast,mycin-stimulated cells. Nuclear translocation of the CM2

mutant protein was not observed, whereas nuclear dephosphorylation of the CM2 mutant was incompleteeven in cells stimulated with 6 mM ionomycin, a concen-translocation of the R112A, E114A, and T116A mutants

was detected in approximately 30%, 30%, and 15% of tration almost 10 times higher than that required to in-duce complete dephosphorylation of wild-type NFAT1.the cells (data not shown).Nevertheless, this partial dephosphorylation of the CM2mutant protein was mediated by calcineurin, since itThe SPRIEIT Sequence Is Required for Effective

Recognition and Dephosphorylation was inhibited by CsA (lane 7). The T116A mutationimpaired the ionomycin-induced dephosphorylation ofof NFAT1 by Calcineurin

The inability of the CM2 mutant to translocate to the NFAT1 in cells to the same extent as did the triple CM2mutation (Figure 2D). The R112A and E114A mutantsnucleus correlated with its very limited dephosphoryla-

tion in stimulated cells (Figure 2). Wild-type NFAT1(1– also showed impaired dephosphorylation when com-pared to wild-type NFAT1, although to a lesser extent460)-GFP, expressed either in HeLa cells or in Cl.7W2

T cells, was dephosphorylated when the cells were stim- than did the CM2 or the T116A mutant proteins (Fig-ure 2D).ulated with ionomycin, as assessed by its shift in migra-

tion on an SDS–polyacrylamide gel (Figure 2A, lanes 1, The CM2 mutant was significantly less sensitive thanwild-type NFAT1 to treatment with exogenous calci-2, 5, and 6). The more complete shift in Cl.7W2 T cells

(compare lane 2 with lane 6) may reflect a higher level neurin in vitro (Figure 2C). Lysates from HeLa cells ex-pressing wild-type NFAT1 or CM2 mutant protein wereof calcineurin activity in this cell line. In contrast, the

CM2 mutant protein was normally phosphorylated in treated with increasing concentrations of calcineurin,and NFAT dephosphorylation was analyzed by Westernresting cells, but showed no change in its migration

following ionomycin stimulation of HeLa cells (Figure blotting. As shown in Figure 2C, partial dephosphoryla-tion of wild-type NFAT1 was apparent with 200 nM cal-2A, lanes 3 and 4) and only a slight shift in stimulated

Cl.7W2 cells (lanes 7 and 8). cineurin, whereas the CM2 mutant showed equivalentlimited dephosphorylation only in thepresence of 2.5 mMThe difference in calcineurin sensitivity between wild-

type NFAT1(1–460)-GFP and the CM2 mutant was an- calcineurin.The reduced sensitivity to calcineurin can be tracedalyzed in Cl.7W2 T cells stimulated with a range of

ionomycin concentrations (Figure 2B). Significant de- to a reduction of at least 10-fold in the ability of cal-cineurin to bind mutant NFAT1 (Figure 3B). Since thephosphorylation of wild-type NFAT1 was achieved in

Molecular Cell630

Figure 3. Peptides Spanning the SPRIEIT Motif Inhibit Recognition and Dephosphorylation of NFAT by Calcineurin

(A) Sequences of the peptides. The wild-type peptides span 25 and 13 residues of murine NFAT1 including the SPRIEIT motif. The mutant25-mer peptide incorporates alanine residues at the same three positions substituted in the CM2 mutant protein. A tyrosine residue wasadded to the C terminus of the longer peptides to facilitate coupling to proteins.(B) The SPRIEIT peptides inhibit calcineurin binding to NFAT1. 125I-calcineurin binding to GST-NFAT1(1–400), either wild-type or harboring theCM2 mutation, was measured in the presence of different concentrations of the SPRIEIT-25, SPRIEIT-13, and SPAIAIA-25 peptides as indicated.The level of nonspecific binding of calcineurin was monitored by its binding to GST-LSF (LSF), a protein unrelated to NFAT1 that does notinteract with calcineurin.(C) The SPRIEIT peptides inhibit dephosphorylation of NFAT1 by calcineurin. Cytoplasmic extracts from HeLa cells stably expressing HA-tagged NFAT1(1–460)-GFP were incubated for 30 min at 308C with sodium pyrophosphate (lane 1), or with calcineurin and calmodulin (lanes2–15) in the presence of CsA/CypA (lane 2), with no added inhibitor (lane 3), or with the indicated concentrations of the peptides SPRIEIT-13(lanes 4–7), SPRIEIT-25 (lanes 8–11), or SPAIAIA-25 (lanes 12–15). NFAT1 dephosphorylation was assessed by SDS–PAGE and Western blottingwith anti-HA tag.(D) Calcineurin binds specifically to the SPRIEIT-25 peptide. Equilibrium binding of calcineurin to immobilized SPRIEIT-25 peptide wasmonitored using a BIAcore biosensor system. Where indicated, soluble SPRIEIT-25 peptide or SPAIAIA-25 peptide was included in thecalcineurin solution. The data plotted are representative of three experiments.

impairment of dephosphorylation is produced by mini- (Figure 3A). The peptides incorporating the wild-typeSPRIEIT sequence inhibited the binding of calcineurinmal substitutions in the SPRIEIT motif of NFAT1, such

as single substitutions for charged or polar residues, to NFAT1 with IC50 z12 mM (Figure 3B). In each case,z75% inhibition was observed with 100 mM peptide. Init is unlikely that the folding of the mutant protein is

abnormal. Moreover, a comparison of tryptic phospho- contrast, the mutant peptide incorporating the se-quence SPAIAIA did not inhibit the calcineurin–NFAT1peptide maps of the CM2 mutant protein with those of

wild-type NFAT1 (data not shown) indicates that the interaction at concentrations up to 100 mM.The SPRIEIT peptides also inhibited the dephosphory-mutant NFAT is recognized normally by intracellular ki-

nases, further evidence that it is correctly folded. The lation of NFAT1 by calcineurin in cell extracts (Figure3C). Lysates from a HeLa cell line stably expressingmost straightforward interpretation of the data is that

mutations in the CM2 motif cause a local alteration of NFAT1(1–460)-GFP were incubated with calcineurin inthe absence or presence of the peptides, and phosphor-the surface of NFAT1 recognized by calcineurin.ylation of NFAT1 was analyzed by Western blotting. Thewild-type SPRIEIT peptides at concentrations from 16–Peptides Spanning the SPRIEIT Sequence Interfere

with Calcineurin–NFAT1 Binding 400 mM inhibited dephosphorylation of NFAT1 (Figure3C, lanes 4–11), whereas the mutant SPAIAIA peptideEvidence that the SPRIEIT motif is directly involved

in NFAT–calcineurin binding was obtained by adding was inhibitory only at concentrations of 400 mM–2 mM(Figure 3C, lanes 12–15).NFAT1 peptides as competitors into an assay of radiola-

beled calcineurin binding to GST-NFAT1(1–400). The com- We examined the binding of calcineurin to immobi-lized peptide using a surface plasmon resonance (SPR)petitors used were two wild-type peptides (SPRIEIT-25

and SPRIEIT-13) spanning the SPRIEIT motif and con- technique (Figure 3D). Calcineurin bound to the immobi-lized SPRIEIT-25 peptide, giving a relatively small SPRtaining 25 residues and 13 residues of murine NFAT1,

respectively, and a mutant peptide (SPAIAIA-25) corre- signal consistent with the micromolar affinity indicatedby Figures 3B and 3C. Inclusion of soluble SPRIEIT-25sponding to the same region of the CM2 mutant protein


peptide at 25 mM or 50 mM inhibited the binding, whileinclusion of soluble SPAIAIA-25 peptide at the sameconcentrations had a minimal effect (Figure 3D). Theseresults provide evidence that the binding is specific, andthat the mode of binding is similar for the immobilizedpeptide and the NFAT protein.

Peptides Spanning the SPRIEIT SequenceDo Not Interfere with CalcineurinPhosphatase ActivityTo determine whether the SPRIEIT peptides acted asgeneral inhibitors of calcineurin phosphatase activity, weused a well-characterized calcineurin substrate, the RIIphosphopeptide. The SPRIEIT-25 peptide did not inhibitthe dephosphorylation of the RII peptide by calcineurinwhen tested in the same range of concentrations (10–400 mM) in which it inhibited NFAT1 dephosphorylation(Figure 4A). In the same experiment, calcineurin activitywas effectively inhibited by a calcineurin autoinhibitorypeptide (AI) and by FK506/FKBP12 (Figure 4A).

The SPRIEIT-25 peptide was further tested for its ef-fect on the dephosphorylation of two protein substratesof calcineurin, the PKA regulatory subunit RIIa (Blumen-thal et al., 1986) and the neuronal cytoskeletal proteintau (Drewes et al., 1993). Calcineurin dephosphorylatedboth PKA-phosphorylated RIIa (Figure 4B, lanes 1 and5) and Erk-2-phosphorylated GST-tau protein (Figure4C, lanes 1 and 4). The dephosphorylation was pre-vented by the general phosphatase inhibitor sodium py-rophosphate and by the calcineurin inhibitors CsA/cyclophilin or FK506/FKBP12. In contrast, the SPRIEIT-

Figure 4. The SPRIEIT Peptides Do Not Significantly Inhibit Cal-25 peptide did not inhibit dephosphorylation of either cineurin Phosphatase Activitysubstrate when used at concentrations (20 mM and 100 (A) Dephosphorylation of the RII peptide substrate by calcineurin.mM) sufficient to inhibit NFAT1 dephosphorylation. The 32P-RII peptide was incubated with calcineurin and calmodulin with-peptide was only minimally inhibitory at 500 mM (18% out inhibitors or in the presence of the indicated concentrations of

FK506/FKBP12, autoinhibitory peptide, or the SPRIEIT-25 peptide.inhibition of RIIa dephosphorylation, Figure 4B, lane 8;Released 32P was measured as described in Experimental Proce-and 10% inhibition of GST-tau dephosphorylation, Fig-dures.ure 4C, lane 7). Thus, the inhibition by SPRIEIT peptides(B) Dephosphorylation of RIIa protein by calcineurin. PKA-phos-

is more pharmacologically selective than the action of phorylated 32P-RIIa protein (lane 1) was incubated with calcineurinthe inhibitors CsA/cyclophilin and FK506/FKBP12, which and calmodulin in the presence of 20 mM sodium pyrophosphateinhibit calcineurin activity against all protein substrates. (lane 2), 15 mM/5 mM CsA/CypA (lane 3), 10 mM/10 mM FK506/

FKBP12 (lane 4), no added inhibitors (lane 5), or the indicated con-centrations of the SPRIEIT-25 peptide (lanes 6–9). Top panel,autora-

The CM2 Region Functions as a Calcineurin diogram of proteins separated on a 10% SDS–polyacrylamide gel.Targeting Motif in Several NFAT Proteins Bottom panel, Coomassie blue staining of the fixed gel shows that

equal amounts of the reaction were loaded in each lane. The in-The SPRIEIT sequence of NFAT1 is conserved in NFAT2,creased intensity of the stained bands in lanes 2–9 relative to thatand a similar motif is present in NFAT3 and NFAT4.in lane 1 is due to the fact that the calcineurin A chain comigratesWe therefore asked whether the SPRIEIT peptides fromwith RIIa protein under these conditions.

NFAT1 could inhibit the interaction of calcineurin with (C) Dephosphorylation of tau protein by calcineurin. ERK-2-phos-NFAT2 and NFAT4, proteins that together with NFAT1 phorylated GST-tau protein (lane 1) was incubated with calcineurinconstitute the NFAT-family members expressed in im- and calmodulin in the presence of 20 mM sodium pyrophosphate

(lane 2), 15 mM/5 mM CsA/CypA (lane 3), no added inhibitors (lanemune cells (Lyakh et al., 1997). Indeed, the wild-type4), or the indicated concentrations of the SPRIEIT-25 peptide (lanesSPRIEIT peptides from NFAT1 inhibited the ability of5–7). Top panel, autoradiogram of proteins separated on an 8%125I-calcineurin to bind to the N-terminal regions ofSDS–polyacrylamide gel. Bottom panel, Coomassie blue staining of

NFAT2 and NFAT4, while the mutant peptide SPAIAIA- the fixed gel shows that an equal amount of tau protein was loaded25 had no inhibitory effect (Figure 5A). The IC50 for the in each lane.SPRIEIT-25 peptide was z15 mM with both NFAT2 andNFAT4, very similar to the value with NFAT1.

Further, the NFAT1 SPRIEIT-13 peptide inhibited de- shift in mobility of theprotein onan SDS–polyacrylamidegel (Figure 5B, lanes 1–4). The wild-type SPRIEIT-13phosphorylation of the N-terminal domain of NFAT4 by

calcineurin (Figure 5B). Addition of calcineurin to lysates peptide inhibited dephosphorylation in a dose-depen-dent manner, with 400 mM peptide preventing dephos-from HEK-293T cells expressing NFAT4(3–407)-GFP

caused dephosphorylation of NFAT4, detectable by a phorylation more effectively than 100 mM peptide at all

Molecular Cell632

that the GFP-derived proteins were expressed at com-parable levels. Inhibition by GFP-SPRIEIT was essen-tially complete at low concentrations of stimulus, butwas incomplete under stronger stimulation (comparelanes 6 and 7 with lane 8). This result is consistent withthe in vitro assays shown in Figure 5B.

The ability of the GFP-SPRIEIT protein to inhibit nu-clear translocationof endogenous NFAT1was examinedin Cl.7W2 T cells by immunocytochemistry (Figure 6B).Expression of GFP-SPRIEIT impaired the ionomycin-induced nuclear translocation of NFAT1 (middle panels),whereas expression of GFP and GFP-SPAIAIA did not(top and bottom panels). Consistent with data in Figures5B and 6A, translocation was completely inhibited onlyin those cells expressing high levels of the GFP-SPRIEITprotein, indicating that effective inhibition required ahigh intracellular concentration of the peptide. In sepa-rate experiments (see Experimental Procedures), we es-timated the average intracellular concentration of theGFP-SPRIEIT and GFP-SPAIAIA fusion proteins as 50–200 mM in Cl.7W2 T cells, comparable to the 16–400mM required to inhibit the dephosphorylation of NFATproteins in vitro (Figures 3C and 5B).

Finally, we tested the effect of the GFP-SPRIEIT fusionFigure 5. The CM2 Region Functions as a Common Calcineurin- proteins on NFAT-activated gene expression in Jurkat TTargeting Motif in NFAT-Family Proteins

cells. Plasmids encoding GFP, GFP-SPRIEIT, and GFP-(A) The SPRIEIT peptides of NFAT1 inhibit calcineurin binding to

SPAIAIA proteins were introduced into Jurkat cells to-NFAT2 and NFAT4. Binding of 125I-calcineurin to GST-NFAT2(1–418)gether with an NFAT-driven luciferase reporter plasmid,and GST-NFAT4(11–419) was assessed in the presence of SPRIEIT-and a day later the cells were stimulated with PMA and13, SPRIEIT-25, or SPAIAIA-25 peptide at the concentrations indi-

cated. Binding to GST-LSF was measured to monitor the level of ionomycin. GFP-SPRIEIT caused a dose-dependent re-nonspecific binding. duction in reporter activity, whereas GFP-SPAIAIA had(B) The SPRIEIT peptide of NFAT1 inhibits dephosphorylation of little effect (Figure 6C). Western blotting with anti-GFPNFAT4. Cytoplasmic extractsof HEK-293T cells transiently express-

confirmed that the amounts of GFP fusion proteins ex-ing HA-tagged NFAT4(3–407)-GFP were incubated for 30 min atpressed were equivalent, and were proportional to the308C with sodium pyrophosphate (lane 1), or with increasing concen-amount of plasmid DNA used (data not shown). Thetrations of calcineurin/calmodulin in the absence of inhibitors (lanes

2–4) or in the presence of the SPRIEIT-13 peptide (100 mM, lanes estimated intracellular concentration of GFP-peptide in5–7, and 400 mM, lanes 8–10) or the SPAIAIA-25 peptide (400 mM, Jurkat cells was 15–80 mM. The GFP-SPRIEIT fusionlanes 11–13). Phosphorylation of NFAT4 was assessed by SDS– protein was not a general inhibitor of transcription sincePAGE and Western blotting with anti-HA tag.

it did not inhibit its own expression, the expression ofcotransfected NFAT1(1–460)-GFP (Figure 6A), or the in-ducible expression of a luciferase reporter gene con-

calcineurin concentrations tested (Figure 5B, compare trolled by AP-1 sites (data not shown). The incompletelanes 8–10 with lanes 5–7). In contrast, the mutant pep- inhibition of NFAT activity by GFP-SPRIEIT, z65% intide SPAIAIA-25 displayed little or no inhibition even at the experiment shown, is probably related to the varying400 mM (lanes 11–13). The results are indicative of a levels of expression of the GFP fusion protein in individ-common calcineurin targeting mechanism in the differ- ual cells (Figure 6B).ent NFAT proteins.

Discussion

Expression of a SPRIEIT Peptide in CellsWe have identified a conserved sequence motif of NFATInhibits the Activation of NFAT1 In Vivothat is critical for effective recognition and dephosphor-To assess the ability of the SPRIEIT peptide to inhibitylation of NFAT proteins by calcineurin. Effective recog-NFAT1 activation in vivo, GFP fusion proteins contain-nition reflects a docking interaction that directs cal-ing one C-terminal copy of the SPRIEIT (wild-type) orcineurin to NFAT1. Selective disruption of this dockingSPAIAIA (CM2 mutant) sequences were expressed ininteraction in cells interrupts the calcineurin–NFAT sig-Cl.7W2 T cells together with HA-tagged NFAT1(1–460)-naling pathway and blocks gene expression induced byGFP. Cells were stimulated with a range of ionomycinNFAT.concentrations, and cell lysates were analyzed for de-

phosphorylation of NFAT1 by Western blotting with anti-HA tag. As shown in Figure 6A, the GFP-SPRIEIT fusion The Calcineurin–NFAT Interaction Is Criticalprotein inhibited ionomycin-induced dephosphorylation for Activation of NFATof NFAT1, whereas the GFP-SPAIAIA protein had no Experiments in cells and biochemical studies in vitro

demonstrate that the SPRIEIT motif is essential for thesignificant effect. Western blotting with anti-GFP showed


Figure 6. Intracellular Delivery of a SPRIEITPeptide of NFAT1 Inhibits NFAT1 ActivationIn Vivo

(A) A GFP-SPRIEIT fusion protein inhibits ion-omycin-induced dephosphorylation of NFAT1in T lymphocytes. HA-tagged NFAT1 (1–460)-GFP was expressed in Cl.7W2 T cells to-gether with GFP (lanes 1–4), GFP-SPRIEIT(lanes 5–8), or GFP-SPAIAIA (lanes 9–12).Twenty-four hours later, cells were left un-treated or were stimulated for 10 min withthe indicated concentrations of ionomycin.Lysates were analyzed by SDS–PAGE andWestern blotting for dephosphorylation ofNFAT1 (top panel) and for expression of GFPand GFP-peptide fusion proteins (bottompanel).(B) The GFP-SPRIEIT fusion protein blocksthe nuclear translocation of NFAT1 in iono-mycin-stimulated T cells. Cl.7W2 T cells tran-siently expressing GFP, GFP-SPRIEIT, orGFP-SPAIAIA were stimulated with iono-mycin (2 mM, 10 min) and processed for im-munocytochemistry. For each experiment,the same microscope field is shown viewedwith a rhodamine filter set to visualize NFAT1(left panels) and with a fluorescein filter set toidentify cells expressing GFP or GFP-peptidefusion proteins (right panels). Arrows indicatecells expressing high levels of GFP. NeitherGFP nor the GFP-peptide fusion proteins af-fected the cytoplasmic localization of NFAT1in unstimulated cells (not shown).(C) The GFP-SPRIEIT fusion protein inhibitsNFAT-mediated gene expression in T cells.Jurkat T cells cotransfected with an NFAT-driven luciferase reporter plasmid and withGFP-SPRIEIT or GFP-SPAIAIA expressionplasmids were stimulated with 20 nM PMAand 1 mM ionomycin, and luciferase activity(Luo et al., 1996a) in cell extracts was mea-sured after 6 hr. The level of reporter activityis expressed as percent inhibition relative toa sample cotransfected with the NFAT-drivenluciferase reporter plasmid and 9 mg of GFPexpression plasmid.

interaction of NFAT proteins with calcineurin. In vitro, interaction with calcineurin (Masuda et al., 1997), theyemphasize the primacyof theSPRIEIT motif, since NFATNFAT1 mutated in the SPRIEIT motif is deficient in bind-

ing to calcineurin and is inefficiently dephosphorylated mutated in the SPRIEIT motif is not dephosphorylatedand is not activated in cells.by calcineurin. In cells, the mutated NFAT1 is defective

in the earliest manifestations of NFAT activation, its de- Specific protein–protein contacts are a general or-ganizing feature of intracellular signaling pathways. Inphosphorylation and its recruitment to the cell nucleus.

The findings provide strong support for the idea that particular, protein serine/threonine phosphatases, in-cluding calcineurin, are targeted to selected substratesbinding of calcineurin to NFAT precedes dephosphory-

lation, which precedes all other activation events. through protein–protein interactions (Hubbard and Co-hen, 1993; Somlyo and Somlyo, 1994). Docking of pro-We have shown that calcineurin binds directly to the

SPRIEIT peptide, indicating that the SPRIEIT motif of tein phosphatase 1 (PP1) through a glycogen-targetingsubunit directs it to its substrates on glycogen particlesNFAT forms a part of the calcineurin–NFAT contact.

SPRIEIT peptides do not occupy the catalytic site of (Tang et al., 1991; Doherty et al., 1995), while dockingthrough a myosin-targeting subunit directs PP1 to smoothcalcineurin (Figure 4A), but rather this contact serves as

a docking site for calcineurin that ensures the efficient muscle myosin light chain (Alessi et al., 1992). In eachcase, the targeting subunit increases the efficiency ofdephosphorylation of substrate sites elsewhere in the

N-terminal domain of NFAT. Although our results do not dephosphorylation of the substrate. The contact surfaceon PP1 for these targeting subunits is a surface grooveexclude the possibility that NFAT has additional sites of

Molecular Cell634

at some distance from the PP1 catalytic site (Egloff et The Intracellular Calcineurin–NFAT SignalingPathway Is Inhibited by Blockingal., 1997). This contact region is also a docking surfacethe Protein–Protein Interactionfor the enzyme inhibitors DARPP-32 and inhibitor-1,A central finding of this work is that calcineurin-medi-each of which regulates the activity of the phosphataseated activation of NFAT in cells, and NFAT-driven geneby positioning a second inhibitor loop in the PP1 cata-expression, can be blocked by disrupting the protein–lytic site (Endo et al., 1996; Kwon et al., 1997). In theprotein interaction of calcineurin with NFAT. The SPRIEITcase of calcineurin, it has been proposed that FKBP12peptide was expressed in these experiments as a fusionplays the role of a targeting subunit directing calcineurinprotein with the carrier protein GFP, but since shortto some substrate proteins including the IP3 receptorSPRIEIT peptides are effective in disrupting the protein–(Cameron et al., 1997), the ryanodine receptor (Cameronprotein interaction in vitro, the implication is that a pep-et al., 1995; Timerman et al., 1995), and the TGFb typetide, peptide mimetic, or other small analog that can beI receptor (Wang et al., 1996). The calcineurin–NFATdelivered into cells at similar concentrations will likewiseinteraction represents a different mode of calcineurin–block NFAT function. Such compounds would constitutesubstrate interaction, in that a targeting subunit is nota new class of immunosuppressants, whose mechanismessential. Yet another mode of targeting is illustratedwould contrast with that of the current immunosuppres-by AKAP-79, which binds calcineurin and other signalingsive drugs CsA and FK506.enzymes in an inactive form, localizing them to specified

The presence of a conserved threonine residue, andcellular sites (Coghlan et al., 1995; Klauck et al., 1996;of less-conserved serine residues, in the motif recog-Faux and Scott, 1997).nized by calcineurin raises the question whether thecalcineurin–NFAT interaction in cells is itself regulatedThe Specialized Calcineurin–NFAT Dockingby phosphorylation. In this regard, there is convincingInteraction Is Not Characteristic of Allevidence that phosphorylation of theSPRIEIT motifdoesCalcineurin Substratesnot limit the interaction of calcineurin with NFAT1 inThe specialized docking interaction that allows efficientresting cells. The SPRIEIT motif, including the flanking

dephosphorylation of NFAT proteins is not required forserine residues, is not constitutively phosphorylated in

dephosphorylation of calcineurin substrates in general.resting cells (H. O., J. Viola, J. Qin, P. H., and A. R.,

Dephosphorylation of two protein substrates unrelatedunpublished data). Moreover, mutation of the serine res-

to NFAT, the RIIa protein and the tau protein, was not idues flanking the SPRIEIT motif, serine 110 and serinesignificantly inhibited by the SPRIEIT peptide at concen- 118, did not affect the localization of NFAT1 in restingtrations that efficiently inhibited dephosphorylation of cells (Figure 1C). Previous work has shown that cal-NFAT1. Nor does the NFAT docking interaction have cineurin binds to fully phosphorylated NFAT1 extractedevident similarities with other known protein–protein in- from unstimulated cells (Loh et al., 1996b; Wesselborgteractions of calcineurin. The regions of FKBP12 that et al., 1996), further supporting the idea that the inactivitycontact calcineurin (Griffith et al., 1995; Kissinger et al., of calcineurin in resting cells, rather than its inability to1995) show no resemblance to the SPRIEIT motif of recognize NFAT, maintains NFAT in an inactive form.NFAT1. The region of AKAP-79 that is thought to bind On the other hand, numerous precedents, including theto calcineurin is similar in sequence to one of the contact physiological regulation of PP1 in striated muscle byregions of FKBP12 (Coghlan et al., 1995), but not to the reversible phosphorylation of its docking site on theNFAT motif. Neither a SPRIEIT sequence nor the strictly glycogen-targeting subunit (Hubbard and Cohen, 1993),conserved IxIT sequence appears generally in calci- indicate that inducible phosphorylation within or adja-neurin substrates, but given that the permissible degen- cent to the SPRIEIT motif could be a mechanism foreracy of the recognized sequence has yet to be deter- modulating the calcineurin–NFAT interaction.mined, further tests will be needed to determine whetherinhibitors based on the SPRIEIT peptides will interfere Conclusionwith dephosphorylation of a subset of the protein sub- We have identified a conserved sequence motif of NFATstrates of calcineurin. proteins, the SPRIEIT motif, that is a docking site for

It is, however, likely that the SPRIEITP motif serves a calcineurin. This conserved motif is located near the Nsimilar docking function in all NFAT proteins. First, there terminus of the NFAT regulatory domain in the fouris recognizable conservation of the motif as SPRIEITS known NFAT proteins, and its integrity is necessary

for the effective recognition and dephosphorylation ofin NFAT2, CPSIRITS in NFAT3, and CPSIQITS in NFAT4.NFAT1 by calcineurin. Synthetic peptides spanning thisIn particular, the functionally critical residue threoninemotif inhibit calcineurin binding to NFAT and prevent116 inNFAT1 is conserved in all the other NFAT proteins.NFAT dephosphorylation. Expression of the peptides inSecond, the SPRIEIT peptides based on NFAT1 inhibitcells inhibits activation of NFAT assessed in assays ofcalcineurin binding to NFAT2 and NFAT4, as well asdephosphorylation, nuclear import, and NFAT transcrip-calcineurin-mediated dephosphorylation of these NFAT-tional function. Thus, disruption of the enzyme–sub-family proteins (Figure 5; and J. A., unpublished data).strate docking interaction that directs calcineurin toThird, peptides incorporating the PSIRIT and PSIQITNFAT is an effective way of interrupting inducible genesequences of NFAT3 and NFAT4 inhibit the binding ofexpression dependent on NFAT.calcineurin to NFAT1 (F. G.-C., unpublished data). It is

plausible that the sequence IxIT, positioned by adjacentExperimental Procedureshydrophilic residues, forms the core contact, although

residues flanking the conserved motif or residues lo- Reagentscated in other loops in the NFAT proteins may contribute NFAT1 peptides, RII peptide (Blumenthal et al., 1986), and AI peptide

(Hashimoto et al., 1990) were synthesized at the Tufts–New Englandto stabilizing the contact.


Medical Center Peptide Synthesis Facility. Ionomycin and the phor- blotting (2 3 104–5 3 104 cell equivalents/lane). Intracellular levelsof GFP, GFP-SPRIEIT, and GFP-SPAIAIA fusion proteins (150–600bol ester PMA were from Calbiochem, calmodulin and calcineurin

from Sigma, and protease and phosphatase inhibitors from Sigma ng/106 cells [Cl.7W2] and 50–200 ng/106 cells [Jurkat]) were esti-mated from Western blots by comparison with recombinant GFPand Calbiochem. CsA was from Sandoz and FK506 from Fujisawa.

Cyclophilin A (CypA) was provided by Dr. Christopher Walsh (Har- standards (rEGFP, Clontech). These amounts were converted toconcentrations assuming a mass of 27 kDa for GFP and 30 kDa forvard Medical School) and FKBP12 by Dr. Stuart Schreiber (Harvard

University). GFP-SPRIEIT and GFP-SPAIAIA and a cellular volume of 0.4 ml/106

cells, and correcting for the percentage of cells expressing GFP(20%–30%).Expression Plasmids

The expression vector pEFTAGmNFAT1-C, encoding murine NFAT1NFAT Dephosphorylation in Cell Extractsisoform C with three tandem copies of the influenza virus hae-Cells (5 3 106/ml) were resuspended in 100 mM HEPES (pH 7.4), 10magglutinin (HA) epitope at its N terminus, has been described (LuomM K acetate, 2 mM Mg acetate, 2 mM EGTA, 50 mg/ml aprotinin,et al., 1996a). The expression vectors HA-NFAT1(1–460)-GFP and25 mM leupeptin, 2 mMPMSF, 0.05% Nonidet P-40,10 mM iodoacet-HA-NFAT4(3–407)-GFP were generated by subcloning fragments ofamide (IAM) for 10 min on ice. IAM prevents protein degradation andmurine NFAT1 (Luo et al., 1996a) and human NFAT4 (Hoey et al.,inhibits endogenous calcineurin in the lysates. Nuclei-free extracts1995) into pEGFP-N1 (Clontech). The fusion proteins have threewere supplemented with 100 mM NaCl and 10 mM dithiothreitolcopies of the HA tag at the N terminus and GFP at the C terminus.(DTT) to neutralize unreacted IAM. Typically, a 50 ml reaction con-A HeLa cell line stably expressing HA-NFAT1(1–460)-GFP wastained 25 ml of cell extract, 200 nM calcineurin, 600 nM calmodulin,cloned by limiting dilution after G418 selection and maintained in2 mM DTT, 2 mM CaCl2. Incubation was for 30 min at 308C. Inhibitors,the presence of 1 mg/ml G418 (Sigma). The GFP-SPRIEIT and GFP-preincubated with calcineurin/calmodulin for 30 min on ice, were atSPAIAIA plasmids were made by introducing oligonucleotides en-final concentrations 20 mM sodium pyrophosphate, 15 mM/5 mMcoding KPAGASGPSPRIEITPSHEAYD and KPAGASGPSPAIAIAPSCsA/CypA, and peptides as indicated.HEAYD into pEGFP-N1. Details of subcloning and mutagenesis are

available from the authors.RII Peptide Dephosphorylation32P-RII phosphopeptide, labeled with PKA catalytic subunit (Sigma)Transfectionsto z800 cpm/pmol, was incubated with 100 nM calcineurin and 600Plasmid DNA (0.7–1 mg/106 cells) was introduced into Jurkat T cellsnM calmodulin for 30 min at 308C in 60 ml of 12 mM Tris-Cl (pH 7.5),and Cl.7W2 T cells (Valge-Archer et al., 1990) by electroporation,3.5 mM MgCl2, 8 mM 2-mercaptoethanol, 58 mg/ml BSA, 117 mMand into HeLa cells and HEK-293T cells by precipitation with calciumCaCl2. Where indicated, calcineurin/calmodulin was preincubatedphosphate. Jurkat cells (15 3 106) were transfected with 2 mg ofwith FK506/FKBP12 (10 mM/10 mM), AI peptide, or SPRIEIT-25 pep-NFAT-luciferase reporter plasmid pNF-AT3-luc (Hedin et al., 1997)tide. Released 32P was quantified in the supernatant after additionand 9 mg of GFP expression plasmids as indicated.of 500 ml of 0.1% TCA and incubation with 100 ml of a 50% slurryof AG 50W resin (BioRad).Immunocytochemistry

Cl.7W2 cells and HeLa cells on coverslips were stimulated in me-Dephosphorylation of RIIa and Tau Proteinsdium supplemented to 5 mM Ca21, which did not alter the cyto-Hexahistidine-tagged RIIa protein (Hemmer et al., 1997) immobilizedplasmic localization of NFAT1 in resting cells. Endogenous NFAT1on Ni-NTA resin (Qiagen) was phosphorylated (120 cpm/pmol) byin Cl.7W2 cells was detected with rabbit anti-NFAT1 isoform CPKA catalytic subunit (Sigma). GST-tau (Lee et al., 1989) immobilized(Wang et al., 1995) and HA-tagged proteins with monoclonal anti-on glutathione-Sepharose was phosphorylated (140 cpm/pmol) bybody 12CA5 (Boehringer-Mannheim), followed in each case by anERK-2 (MAPK, New England Biolabs). A 40–50 ml reaction containedappropriate Cy3-conjugated anti-IgG (Jackson ImmunoResearch).32P-labeled RIIa (90 nM, in solution) or 32P-GST-tau (22 pmol, boundto beads), calcineurin/calmodulin (200 nM/600 nM) that had beenCalcineurin–NFAT Bindingpreincubated 20–30 min on ice without or with calcineurin inhibitors1 mg of GST-NFAT1(1–400), GST-NFAT2(1–418) (Luo et al., 1996c),or peptides, 2 mM CaCl2 (RIIa) or 2 mM CaCl2 and 2 mM MnCl2 (tau),GST-NFAT4(11–419), or GST-LSF (Shirra et al., 1994) fusion protein100 mM HEPES (pH 7.4), 100 mM NaCl, 20 mM K acetate, 2 mMwas immobilized on gluthathione-Sepharose by incubation for 30Mg acetate, 2 mM DTT. After 45 min (RIIa) or 90 min (tau) at 308C,min at 48C in 50 mM Tris-phosphate (pH 8.0), 150 mM NaCl, 5 mMthe extent of dephosphorylation was quantified by SDS–PAGE andMgCl2, 20 mM 2-mercaptoethanol, 1% Triton X-100, 1 mM sodiumPhosphorImager (Molecular Dynamics) analysis.o-vanadate, 20 mM leupeptin, 10 mg/ml aprotinin, 2 mM PMSF. The

resin was washed, resuspended in 250 ml of buffer supplementedAcknowledgmentswith 200 mM CaCl2 and 14 nM 125I-calcineurin (150–300 cpm/fmol),

and incubated for 30 min at 48C. SPRIEIT or SPAIAIA peptides wereWe thank Dr. Gloria Lee, Dr. Susan Taylor, Dr. David McKean, Dr.included as indicated. After incubation, binding reactions were fil-Christopher Walsh, and Dr. Stuart Schreiber for generous gifts oftered through a 5 mm hydrophilic PVDF filter (Millipore), filters werereagents. We are grateful to Dr. Cristina Lopez-Rodriguez for advicewashed rapidly, and bound 125I-calcineurin was determined.on mutagenesis and to Dr. Joao Viola for advice on phosphopeptidemapping and for unpublished data. J. A. is a recipient of an ArthritisCalcineurin–Peptide BindingFoundation Postdoctoral Fellowship. F. G.-C. was supported by aBinding of calcineurin to the SPRIEIT-25 peptide was analyzed inPostdoctoral Fellowship from the Ministry of Science and Educationa BIAcore instrument (Pharmacia Biosensor). SPRIEIT-25 peptideof Spain. H. O. was supported by a Cancer Research Institute Fel-(300–4000 resonance units) was immobilized on a CM5 sensor chiplowship. A. R. is a Scholar of the Leukemia Society of America. Thisusing the amine coupling kit (Pharmacia Biosensor). Calcineurinwork was supported in part by NIH grant AI 40127 to A. R.(4.75 mM) was injected at 5 ml/min for 4 min at 258C in 20 mM TES

(pH 7.5), 120 mM NaCl, 10 mM 2-mercaptoethanol, 5 mM MgCl2,Received December 4, 1997; revised February 17, 1998.300 mM CaCl2, 100mM EGTA, 0.1% NaN3, 0.005% BIAcore surfactant

P20. For competition measurements, soluble SPRIEIT-25 or SPAIAIA-References25 peptide was added to the calcineurin solution. Calcineurin bind-

ing at 165 s was monitored as the steady-state increase in SPRAlessi, D.R., McDougall, L.K., Sola, M.M., Ikebe, M., and Cohen, P.signal relative to the signal from buffer alone, or from buffer con-(1992). The control of protein phosphatase-1 by targeting subunits.taining peptide, as appropriate.Eur. J. Biochem. 210, 1023–1035.

Aramburu, J., Azzoni, L., Rao, A., and Perussia, B. (1995). ActivationWestern BlottingCells collected by centrifugation were lysed (1 3 106/ml) in sample and expression of the nuclear factors of activated T cells, NFATp

and NFATc, in human natural killer cells: regulation upon CD16buffer supplemented with 20 mM sodium pyrophosphate and 10mM EDTA, the lysates boiled, and samples analyzed by Western ligand binding. J. Exp. Med. 182, 801–810.

Molecular Cell636

Beals, C.R., Clipstone, N.A., Ho, S.N., and Crabtree, G.R. (1997). Nu- Hutchinson, L.E., and McCloskey, M.A. (1995). FceRI-mediated in-duction of nuclear factor of activated T-cells. J. Biol. Chem. 270,clear localization of NF-ATc by a calcineurin-dependent, cyclosporin-16333–16338.sensitive intramolecular interaction. Genes Dev. 11, 824–834.

Kissinger, C.R., Parge, H.E., Knighton, D.R., Lewis, C.T., Pelletier,Bierer, B.E., Hollander, G., Fruman, D., and Burakoff, S.J. (1993).L.A., Tempczyk, A., Kalish, V.J., Tucker, K.D., Showalter, R.E., Moo-Cyclosporin A and FK506: molecular mechanisms of immunosup-maw, E.W., et al. (1995). Crystal structures of human calcineurinpression and probes for transplantation biology. Curr. Opin. Immu-and the human FKBP12–FK506–calcineurin complex. Nature 378,nol. 5, 763–773.641–644.Blumenthal, D.K., Takio, K., Hansen, R.S., and Krebs, E.G. (1986).Klauck, T.M., Faux, M.C., Labudda, K., Langeberg, L.K., Jaken, S.,Dephosphorylation of cAMP-dependent protein kinase regulatoryand Scott, J.D. (1996). Coordination of three signaling enzymes bysubunit (type II) by calmodulin-dependent protein phosphatase. De-AKAP79, a mammalian scaffold protein. Science 271, 1589–1592.terminants of substrate specificity. J. Biol. Chem. 261, 8140–8145.Kwon, Y.-G., Huang, H.-B., Frederic, D., Girault, J.-A., Greengard, P.,Cameron, A.M.,Nucifora, F.C.J., Fung, E.T., Livingston,D.J., Aldape,and Nairn, A.C. (1997). Characterization of the interaction betweenR., Ross, C.A., and Snyder, S.H. (1997). FKBP12 binds the inositolDARPP-32 and protein phosphatase 1 (PP-1): DARPP-32 peptides1,4,5-triphosphate receptor at leucine-proline (1400–1401) and an-antagonize the interaction of PP-1 with binding proteins. Proc. Natl.chors calcineurin to this FK506-like domain. J. Biol. Chem. 272,Acad. Sci. USA 94, 3536–3541.27582–27588.Lee, G., Neve, R.L., and Kosik, K.S. (1989). The microtubule bindingCameron, A.M., Steiner, J.P., Roskams, A.J., Ali, S.M., Ronnett, G.V.,domain of tau protein. Neuron 2, 1615–1624.and Snyder, S.H. (1995). Calcineurin associated with the inositol

1,4,5-triphosphate receptor–FKBP12 complex modulates Ca21 flux. Liu, J. (1993). FK506 and cyclosporin, molecular probes for studyingCell 83, 463–472. intracellular signal transduction. Immunol. Today 14, 290–295.Choi, M.S., Brines, R.D., Holman, M.J., and Klaus, G.G. (1994). Induc- Liu, J., Farmer, J.D., Jr., Lane, W.S., Friedman, J., Weissman, I., andtion of NF-AT in normal B lymphocytes by anti-immunoglobulin or Schreiber, S.L. (1991). Calcineurin is a common target of cyclophilin–CD40 ligand in conjunction with IL-4. Immunity 1, 179–187. cyclosporin A and FKBP–FK506 complexes. Cell 66, 807–815.Coghlan, V.M., Perrino, B.A., Howard, M., Langeberg, L.K., Hicks, Loh, C., Carew, J.A., Kim, J., Hogan, P.G., and Rao, A. (1996a). T-cellJ.B., Gallatin, W.M., and Scott, J.D. (1995). Association of protein receptor stimulation elicits an early phase of activation and a laterkinase A and protein phosphatase 2B with a common anchoring phase of deactivation of the transcription factor NFAT1. Mol. Cell.protein. Science 267, 108–111. Biol. 16, 3945–3954.Crabtree, G.R., and Clipstone, N.A. (1994). Signal transmission be- Loh, C., Shaw, K.T., Carew, J., Viola, J.P., Luo, C., Perrino, B.A.,tween the plasma membrane and nucleus of T-lymphocytes. Annu. and Rao, A. (1996b). Calcineurin binds the transcription factorRev. Biochem. 63, 1045–1083. NFAT1 and reversibly regulates its activity. J. Biol. Chem. 271,

10884–10891.Doherty, M.J., Moorhead, G., Morrice, N., Cohen, P., and Cohen,P.T.W. (1995). Amino acid sequence and expression of the hepatic Luo, C., Burgeon, E., Carew, J.A., McCaffrey, P.G., Badalian, T.M.,glycogen-binding GL-subunit of PP1. FEBS Lett. 375, 294–298. Lane, W.S., Hogan, P.G., and Rao, A. (1996a). Recombinant NFAT1

(NFATp) is regulated by calcineurin in T cells andmediates transcrip-Drewes, G., Mandelkov, E.-M., Baumann, K., Goris, J., Merlevede,tion of several cytokine genes. Mol. Cell. Biol. 16, 3955–3966.W., and Mandelkov, E. (1993). Dephosphorylation of tau protein and

Alzheimer paired helical filaments by calcineurin and phosphatase- Luo, C., Burgeon, E., and Rao, A. (1996b). Mechanisms of transacti-2A. FEBS Lett. 336, 425–432. vation by nuclear factor of activated T cells-1. J. Exp. Med. 184,

141–147.Egloff, M.-P., Johnson, D.F., Moorhead, G., Cohen, P.T.W., Cohen,P., and Barford, D. (1997). Structural basis for the recognition of Luo, C., Shaw, K.T., Raghavan, A., Aramburu, J., Garcıa-Cozar, F.,regulatory subunits by the catalytic subunit of protein phosphatase Perrino, B.A., Hogan, P.G., and Rao, A. (1996c). Interaction of cal-1. EMBO J. 16, 1876–1887. cineurin with a domain of the transcription factor NFAT1 that con-

trols nuclear import. Proc. Natl. Acad. Sci. USA 93, 8907–8912.Endo, S., Zhou, X., Connor, J., Wang, B., and Shenolikar, S. (1996).Multiple structural elements define the specificity of recombinant Lyakh, L., Ghosh, P., and Rice, N.R. (1997). Expression of NFAT-human inhibitor-1 as a protein phosphatase-1 inhibitor. Biochemis- family proteins in normal human T cells. Mol. Cell. Biol. 17, 2475–try 35, 5220–5228. 2484.Faux, M.C., and Scott, J.D. (1997). Regulation of the AKAP79-protein Masuda, E., Naito, Y., Tokumitsu, H., Campbell, D., Saito, F., Han-kinase C interaction by Ca21/calmodulin. J. Biol. Chem. 272, 17038– num, C., Arai, K.-I., and Arai, N. (1995). NFATx, a novel member of17044. the NFAT family that is expressed predominantly in the thymus. Mol.

Cell. Biol. 15, 2697–2706.Griffith, J.P., Kim, J.L., Kim, E.E., Sintchak, M.D., Thomson, J.A.,Fitzgibbon, M.J., Fleming, M.A., Caron, P.R., Hsiao, K., and Navia, Masuda, E.S., Liu, J., Imamura, R., Imai, S.I., Arai, K.I., and Arai, N.M.A. (1995). X-ray structure of calcineurin inhibited by the immu- (1997). Control of NFATx1 nuclear translocation by a calcineurin-nophilin–immunosuppressant FKBP12–FK506 complex. Cell 82, regulated inhibitory domain. Mol. Cell. Biol. 17, 2066–2075.507–522. Mattila, P.S., Ullman, K.S., Fiering, S., Emmel, E.A., McCutcheon,Hashimoto, Y., Perrino, B.A., and Soderling, T.R. (1990). Identifica- M., Crabtree, G.R., and Herzenberg, L.A. (1990). The actions oftion of an autoinhibitory domain in calcineurin. J. Biol. Chem. 265, cyclosporin A and FK506 suggest a novel step in the activation of1924–1927. T lymphocytes. EMBO J. 9, 4425–4433.Hedin, K.E., Bell, M.P., Kalli, K.R., Huntoon, C.J., Sharp, B.M., and McCaffrey, P.G., Luo, C., Kerppola, T.K., Jain, J., Badalian, T.M.,McKean, D.J. (1997). d-opioid receptors expressed by Jurkat T cells Ho, A.M., Burgeon, E., Lane, W.S., Lambert, J.N., Curran, T., etenhance IL-2 secretion by increasing AP-1 complexes and activity al. (1993). Isolation of the cyclosporin-sensitive T cell transcriptionof the NFAT/AP-1-binding promoter element. J. Immunol. 159, 5431– factor NFATp. Science 262, 750–754.5440. Northrop, J.P., Ho, S.N., Chen, L., Thomas, D.J., Timmerman, L.A.,Hemmer, W., McGlone, M., and Taylor, S.S. (1997). Recombinant Nolan, G.P., Admon, A., and Crabtree, G.R. (1994). NF-AT compo-strategies for rapid purification of catalytic subunits of cAMP- nents define a family of transcription factors targeted in T-cell activa-dependent protein kinase. Anal. Biochem. 245, 115–122. tion. Nature 369, 497–502.Hoey, T., Sun, Y.L., Williamson, K., and Xu, X. (1995). Isolation of Rao, A. (1994). NF-ATp: a transcription factor required for the co-two new members of the NF-AT gene family and functional charac- ordinate induction of several cytokine genes. Immunol. Today 15,terization of the NF-AT proteins. Immunity 2, 461–472. 274–281.

Hubbard, M.J., and Cohen, P. (1993). On target with a new mecha- Rao, A., Luo, C., and Hogan, P.G. (1997). Transcription factors ofnism for the regulation of protein phosphorylation. Trends Biochem. the NFAT family: regulation and function. Annu. Rev. Immunol. 15,

707–747.Sci. 18, 172–177.


Schreiber, S.L., and Crabtree, G.R. (1992). The mechanism of actionof cyclosporin A and FK506. Immunol. Today 13, 136–142.

Shaw, K.T., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J.,Sharma, S., Rao, A., and Hogan, P.G. (1995). Immunosuppressivedrugs prevent a rapid dephosphorylation of transcription factorNFAT1 in stimulated immune cells. Proc. Natl. Acad. Sci. USA 92,11205–11209.

Shibasaki, F., Price, E.R., Milan, D., and McKeon, F. (1996). Role ofkinases and the phosphatase calcineurin in the nuclear shuttling oftranscription factor NF-AT4. Nature 382, 370–373.

Shirra, M.K., Zhu, Q., Huang, H.-C., Pallas, D., and Hansen, U. (1994).One exon of the human LSF gene includes conserved regions in-volved in novel DNA-binding and dimerization. Mol. Cell. Biol. 14,5076–5087.

Sigal, N.H., Dumont, F., Durette, P., Siekierka, J.J., Peterson, L.,Rich, D.H., Dunlap, B.E., Staruch, M.J., Melino, M.R., Koprak, S.L.,et al. (1991). Is cyclophilin involved in the immunosuppressive andnephrotoxic mechanism of action of cyclosporin A? J. Exp. Med.173, 619–628.

Somlyo, A.P.,and Somlyo, A.V. (1994). Signal transduction andregu-lation in smooth muscle. Nature 372, 231–236.

Tang, P.M., Bondor, J.A., Swiderek, K.M., and dePaoli-Roach, A.A.(1991). Molecular cloning and expression of the regulatory (RGL) sub-unit of glycogen-associated protein phosphatase. J. Biol. Chem.266, 15782–15789.

Timerman, A.P., Wiederrecht, G., Marcy, A., and Fleischer, S. (1995).Characterization of an exchange reaction between soluble FKBP-12 and the FKBP·ryanodine receptor complex. J. Biol. Chem. 270,2451–2459.

Timmerman, L.A., Clipstone, N.A., Ho, S.N., Northrop, J.P., andCrabtree, G.R. (1996). Rapid shuttling of NF-AT in discrimination ofCa21 signals and immunosuppression. Nature 383, 837–840.

Valge-Archer, V.E., de Villiers, J., Sinskey, A.J., and Rao, A. (1990).Transformation of T lymphocytes by thev-fos oncogene. J. Immunol.145, 4355–4364.

Venkataraman, L., Francis, D.A., Wang, Z., Liu, J., Rothstein, T.L.,and Sen, R. (1994). Cyclosporin-A sensitive induction of NF-AT inmurine B cells. Immunity 1, 189–196.

Wang, D.Z., McCaffrey, P.G., and Rao, A. (1995). The cyclosporin-sensitive transcription factor NFATp is expressed in several classesof cells in the immune system. Ann. NY Acad. Sci. 766, 182–194.

Wang, T., Li, B.-Y., Danileson, P.D., Shah, P.C., Rockwell, S., Lech-leider, R.J., Martin, J., Mangarano, T., and Donahoe, P.K. (1996).The immunophilin FKBP12 functions as a common inhibitor of theTGFb family type I receptors. Cell 86, 435–444.

Wesselborg, S., Fruman, D.A., Sagoo, J.K., Bierer, B.E., and Bura-koff, S.J. (1996). Identification of a physical interaction betweencalcineurin and nuclear factor of activated T cells (NFATp). J. Biol.Chem. 271, 1274–1277.

Documents

Selective Inhibition of NFAT Activation by a Peptide Spanning the Calcineurin Targeting Site of NFAT