transcription factors of the nfat family:regulation...

P1: MBL/plb P2: MBL/rsk QC: MBL/uks T1: MBL

February 12, 1997 16:24 Annual Reviews AR026-27 AR26-27

Annu. Rev. Immunol. 1997. 15:707–47Copyright c© 1997 by Annual Reviews Inc. All rights reserved

TRANSCRIPTION FACTORS OF THENFAT FAMILY: Regulation and Function

Anjana Rao∗, Chun Luo∗, and Patrick G. HoganCenter for Blood Research and the∗Department of Pathology, Harvard MedicalSchool, 200 Longwood Avenue, Boston, Massachusetts 02115;email: [email protected]

KEY WORDS: nuclear factor of activated T cells, immunosuppression, cytokine gene regula-tion, inducible gene transcription, signal transduction, lymphocyte activation,calcineurin

ABSTRACT

As targets for the immunosuppressive drugs cyclosporin A and FK506, transcrip-tion factors of the NFAT (nuclear factor of activated T cells) family have been thefocus of much attention. NFAT proteins, which are expressed in most immune-system cells, play a pivotal role in the transcription of cytokine genes and othergenes critical for the immune response. The activity of NFAT proteins is tightlyregulated by the calcium/calmodulin-dependent phosphatase calcineurin, a pri-mary target for inhibition by cyclosporin A and FK506. Calcineurin controls thetranslocation of NFAT proteins from the cytoplasm to the nucleus of activatedcells by interacting with an N-terminal regulatory domain conserved in the NFATfamily. The DNA-binding domains of NFAT proteins resemble those of Rel-family proteins, and Rel and NFAT proteins show some overlap in their ability tobind to certain regulatory elements in cytokine genes. NFAT is also notable forits ability to bind cooperatively with transcription factors of the AP-1 (Fos/Jun)family to composite NFAT:AP-1 sites, found in the regulatory regions of manygenes that are inducibly transcribed by immune-system cells. This review dis-cusses recent data on the diversity of the NFAT family of transcription factors, theregulation of NFAT proteins within cells, and the cooperation of NFAT proteinswith other transcription factors to regulate the expression of inducible genes.

INTRODUCTION

Proteins belonging to the NFAT (nuclear factor of activated T cells) family oftranscription factors play a central role in inducible gene transcription duringthe immune response (1–4). Despite their name, NFAT proteins are expressed

7070732-0582/97/0410-0707$08.00

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708 RAO, LUO & HOGAN

Table 1 Stimuli that elicit NFAT activation

Receptor/Function Stimulus/Drug Cell type tested References

A. ImmunoreceptorsT cell receptor (TCR) Ag/MHC,αCD3 T cells (1–4, 52)B cell receptor αIg B cells (177–179)CD40 CD40L plus IL-4 B cells (178)FcεRI Ag/IgE Mast cells, basophils (173, 180, 181)FcγRIIIA (CD16) Ag/IgG,αCD16 NK cells (44)

G protein-coupled receptorsm3 Muscarinic Carbachol Stably transfected Jurkat T (66, 86, 182)

acetylcholine receptor cells and PC12pheochromocytoma cells

H1 histamine receptor Histamine Endothelial cells (human J. Carew & A. Rao,Thrombin receptor Thrombin umbilical vein) unpublished

Pharmacological agentsCalcium ionophore Ionomycin, T cells, B cells, mast cells, (1–4, 9, 22, 44, 50,

A23187 macrophages, NK cells, 52, 173, 177–181) andendothelial cells, PC12 J. Kim & P. G. Hogan,cells unpublished

Inhibitor of endoplasmic Thapsigargin T cells (71)reticulum Ca2+-ATPase

not only in T cells, but also in other classes of immune-system cells. NFATproteins are activated by stimulation of receptors coupled to calcium mobi-lization, such as the antigen receptors on T and B cells, the Fcε receptors onmast cells and basophils, the Fcγ receptors on macrophages and NK cells, andreceptors coupled to certain heterotrimeric G proteins (see Table 1). Receptorstimulation and calcium mobilization result in activation of many intracellularenzymes including the calcium- and calmodulin-dependent phosphatase cal-cineurin, a major upstream regulator of NFAT proteins (5, 6). Stimulated cellsinducibly transcribe a large array of activation-associated genes, many of whichare potential targets for NFAT; the genes encode transcription factors, signalingproteins, cytokines, cell surface receptors, and other effector proteins (7–10).

As substrates for calcineurin, NFAT proteins are major targets of the im-munosuppressive drugs cyclosporin A (CsA) and FK506 that have revolution-ized transplant surgery since the introduction of CsA in 1983 (11, 12). Thetoxicity of these drugs, which arises from their ability to inhibit calcineurinin cells outside the immune system (13, 14), has precluded their use in otherclinical situations for which immunosuppressive therapies might be warranted(such as allergy, inflammation, and autoimmune disease). Compounds thattarget NFAT directly, without affecting the phosphatase activity of calcineurin,

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NFAT FAMILY TRANSCRIPTION FACTORS 709

may lack the toxicity of CsA and FK506 while retaining their broad immuno-suppressive effects. Conceivably, more selective immunomodulatory effectsmight be achieved by ablating the functions of individual NFAT proteins inspecific cell types or on selected inducible genes.

This review describes recent advances in our understanding of the structure,regulation, and function of NFAT proteins. Because of space limitations, we donot discuss the potential involvement of NFAT proteins in many biological phe-nomena, including apoptosis, thymocyte development, and immunodeficiencydiseases. For an introduction to earlier work on NFAT and calcineurin, we referthe reader to several excellent reviews (1–7, 11, 15–19).

STRUCTURE AND DISTRIBUTION OF NFAT PROTEINS

Isolation and NomenclatureNFAT was first identified in T cells as a rapidly inducible nuclear factor bindingto the distal antigen receptor response element, ARRE-2, of the human IL-2promoter (20). The induction of NFAT in T cells required calcium-activatedsignaling pathways and was blocked by CsA and FK506 (21). The NFATcomplex that formed on this site contained a cytoplasmic component that wasexpressed in resting T cells but not in fibroblasts, and a nuclear component thatwas expressed in both T cells and fibroblasts upon stimulation with phorbolesters (22). The nuclear component was identified as the ubiquitous transcrip-tion factor AP-1, which consists of dimers of Fos- and Jun-family proteins(reviewed in 1). The cytoplasmic component (also termed preexisting, fromits presence in resting cells) was purified based on its ability to bind indepen-dently, in the absence of AP-1 proteins, to the distal NFAT site of the murineIL-2 promoter (23–25) and as an NFAT:AP-1 complex to the CLE0 elementof the human GM-CSF promoter (26). It appears in the nucleus as a result ofcalcium mobilization and calcineurin activation (22, 27) and migrates on SDSgels with an apparent molecular weight of 120–140 kDa (23, 26, 28, 29).

Over the next few years, studies from several laboratories indicated thatthe preexisting/cytoplasmic component of NFAT was a mixture of proteinsbelonging to a novel family of transcription factors [reviewed in (2–4); seeFigure 1 and Table 2]. The first member of the family (NFATp, later renamedNFAT1) was purified from cytoplasmic extracts of a murine T cell clone byaffinity chromatography using the distal NFAT site of the murine IL-2 promoter(23, 24) and cloned from murine (Ar-5) and human (Jurkat) T cell cDNAlibraries (24, 30). A distinct protein belonging to the same family, NFATc,was purified similarly from bovine thymus, and cDNA encoding it was isolatedfrom a Jurkat T cell cDNA library (25). cDNA clones encoding two otherNFAT proteins, NFAT3 and NFAT4, were isolated from Jurkat T cell and human

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Table 2 Proposed nomenclature for NFAT family proteins and chromosomal location of thegenes

Nomenclature Chromosomal locationProposed Current Human Mouse

NFAT1 20q13.1-13.31 2a (183, 184)NFAT1a NFATp (24), NFATc2 (183), NFAT1A (30) (183, 184)NFAT1b NFAT1b (30)NFAT1c NFAT1c (30)

NFAT2 18q23-qterb 18E4 (183)NFAT2a NFATc (25), NFATc1 (183), NFATc.α (34) (183)NFAT2b NFATc.β (34)

NFAT3NFAT3 NFAT3 (31)

NFAT4 16q21-22 (32) 8D (33)NFAT4x NFATx (32), NFATc3 (33)NFAT4a, b, c NFAT4a, b, c (31)

aTheNfat1 locus maps betweenAdaandGnas(expressed as genetic distance in centiMorgans): centromere-Ada-3.1-(Nfat1, Cebpb)-4.8-Gnas(184).bThe human NFAT2 gene is closely linked to STS marker D18S497 (183).

peripheral blood lymphocyte (PBL) cDNA libraries by cross-hybridization toan NFAT1 probe (31). cDNA clones encoding the NFATx isoform of NFAT4were isolated from Jurkat T cell and murine thymus cDNA libraries (32, 33).Most recently, cDNA encoding a variant of NFATc, NFATc.β, was isolatedfrom a Raji B cell library (34).

A unifying nomenclature for NFAT proteins is proposed in Figure 1 andTable 2, which also lists the chromosomal location of the NFAT genes. Thepresent complicated nomenclature arises largely from the isolation, in different

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1 Schematic alignment of NFAT proteins predicted from their cDNAs. For nomencla-ture, see Table 2. The region of highest homology within NFAT proteins is the DNA-bindingdomain (DBD), which shows similarity to the Rel homology region of Rel-family transcriptionfactors. A second region of homology is the NFAT homology region (NHR), which binds tocalcineurin (Cn) and is found only in NFAT family proteins. The N- and C-terminal transacti-vation domains (TADs) are indicated. The acidic/hydrophobic motifs in the N-terminal TADsare schematized as adot. The C-terminal LDQTYLDDVNEIIRKEFS motif, where present, isindicated as athick bar. For protein isoforms, identical shading patterns represent identical se-quences. The boundary of each region is labeled above the sequences with numbering referring toposition in the human protein. The lengths indicated are also for the human proteins, except forNFAT1a, which was cloned from mouse. The GenBank accession numbers for these sequences areU02079 (murine NFAT1a), U43341 (NFAT1b), U43342 (NFAT1c), U08015 (NFATc.α), U59736(NFATc.β), L41066 (NFAT3), U14510 (NFATx), L41067 (NFAT4c), U28807 (NFATc3, the murinehomolog of NFATx).

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laboratories, of multiple isoforms and species variants of the same protein; andfrom the fact that the names NFATp (preexisting) and NFATc (cytoplasmic),which were originally used to describe NFAT DNA binding activity, were car-ried over to the recombinant proteins. Because it is conceivable that somemembers of the NFAT family will be neither preexisting nor cytoplasmic incertain cell types, we suggest the simplified nomenclature used by Hoey et al(31) (NFAT1, NFAT2, NFAT3, NFAT4), which follows that employed for othertranscription factors such as those belonging to the STAT and GATA families(35, 36). For simplicity, the proposed nomenclature in Figure 1 and Table 2 isused in the remainder of this review. As we discuss in a later section, DNA-protein complexes containing NFAT proteins come in many forms, and thuswe use NFAT as a general abbreviation for NFAT-family proteins, as well asoccasionally in its original sense to refer to transcriptionally competent, NFAT-containing complexes such as NFAT:AP-1.

Sequence Homologies, Functional DomainsThe predicted primary structures of the four NFAT proteins are shown schemati-cally in Figure 1. Multiple isoforms, most likely derived by alternative splicing,have been described for NFAT1, NFAT2, and NFAT4. Two major regions ofsequence homology, comprising the DNA-binding domain (DBD) (37) and theNFAT homology region (NHR) (30), are represented in all the isoforms. TheDNA-binding domain, which lies between amino acid residues≈400 and≈700in the known isoforms, is highly conserved within the NFAT family and showsmoderate sequence similarity to the DNA-binding domains of Rel-family pro-teins (37–39). The NFAT homology region of≈300 amino acids, which islocated just N-terminal to the DNA-binding domain, shows a lesser degree ofpairwise sequence identity but strong conservation of several sequence motifscharacteristic of the NFAT family (30, 32, 33). The region of NFAT homol-ogy defines a calcineurin-activated regulatory domain that binds calcineurin, isdephosphorylated when calcineurin is activated, and controls the calcineurin-regulated nuclear translocation of NFAT1 (40). The properties of these twodomains are described in more detail in later sections.

The regions of NFAT proteins located outside the DNA-binding and regu-latory domains show relatively little sequence conservation. The≈100 aminoacid region at the very N-terminus of each protein, which behaves as a strongtransactivation domain (TAD) in NFAT1 (41), is not conserved in sequence.The most obvious instance of sequence conservation in the region C-terminalto the DBD is in a region spanning the C-terminal splice site, where NFAT3and NFAT4x contain a sequence highly homologous to the NFAT1c sequenceLDQTYLDDVNEIIRKEFS (Figure 1). A detailed discussion of the transacti-vation domains of NFAT proteins is deferred to a later section.

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Tissue Distribution, Potential Target GenesThe transcript sizes and reported sites of expression of NFAT mRNAs are listedin Table 3. Only in the case of NFAT2a and NFAT2b have distinct transcriptsbeen correlated with protein isoforms (34). The mRNA expression data shouldbe interpreted with some caution: There are significant differences in the ex-pression patterns of NFAT mRNAs reported by different laboratories (Table 3)that may reflect variable contamination with circulating lymphocytes or resi-dent mast cells. Moreover, analysis of NFAT1 indicates that protein and mRNA

Table 3 Tissue distributions of NFAT family members

Tissue distributionMember mRNA size mRNA protein

NFAT1 ∼8 kb (h, m) Thymus, spleen, heart, testis, Thymus, spleen, T cells, B cells, NKbrainr (25) cells, mast cells, monocytes,

PBL, placenta, muscle, spleen, macrophages, olfactory epithelium,thymus, pancreasn (31) olfactory bulb, and endothelial cells

spleen, leukocyte, placentan (32) w,e,i (9, 42–45, 180)B cells, mast cells, T cellsp not present in cardiac or

(173, 177) skeletal muscle, small intestine,liver, kidney, fat tissue, fibroblasts,neutrophilsw (42, 43)

NFAT2a 2.7 kb (h) PBL, testis, thymus, spleen, small T cells, mast cellse (141, 180)(NFATc.α) intestine, prostate, colonn (34), protein not detected in NK cellse (44)

NK cellsn (44) expression in other cells and tissuesspleen, thymus, skin tumorr (25) not determinedmuscle, thymus, spleen, PBL,

testisn (31)muscle, thymus, leukocyte;

low in othersn (32)

NFAT2b 4.5 kb (h) spleen, testis, ovary, small intestine,(NFATc.β) thymus, prostate, colon, PBLn (34)

Muscle, leukocyte, thymus, spleen,testis, ovary, colonn (32)

muscle; low in thymus, spleen,testis, PBLn (31)

NFAT3 3 kb (h) Placenta, lung, kidney, testis, ovary, Not determined4.5 kb (h) heart, colon;4.7 kb (h) low in brain, spleen, thymus,

PBLn (31)

NFAT4 7.0 kb (h) Thymus, leukocyte, low in othersn (32) Not determinedthymus, spleen, LNr (33)muscle, thymus, kidneyn (31)

Abbreviations: h, human; m, mouse; w, protein detected by Western blotting with specific antibodies; e, protein detected byspecific antibody supershift in EMSA; i, protein detected by immune staining; r, mRNA detected by ribonuclease protectionassays; n, mRNA detected by Northern blotting; p, mRNA detected by RT-PCR.

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Table 4 Involvement of calcineurin and NFAT proteins in expression of selected inducible genes

Effects of CsA or NFATCell type testeda FK506b implicatedc References

A. CytokinesIL-1β macrophages (bacteria) partial inhibition n.t. (185)

mast cells (FcεRI) partial inhibition n.t. (186)IL-2 T cells inhibition yes (2–4, 112, 187)IL-3 T cells inhibition yesd (187, 188)

eosinophils, neutrophils inhibition n.t. (189)IL-4 T cells inhibition yes (152, 180, 190)

mast cells inhibition yes and referencestherein

IL-5 T cells inhibition n.t. (191)T cells (Ag/APC) partial inhibition n.t. (188)T cells (ConA/αTCR) no inhibition n.t. (192)T cells (PMA/dbcAMP) inhibition yes (193, 194)mast cells (Ag/IgE) inhibition yes (173, 195)

IL-6 mast cells (FcεRI) inhibition n.t. (186, 196)IL-8 T cells inhibition yese (155)IL-10 T cell (Ag/APC) partial inhibition n.t. (188)

spleen cells partial inhibitionf n.t. (197)IL-13 T cells inhibition yes (198)

T cells(αCD28/PMA± αCD3) stimulation n.t. (199)

GM-CSF T cells inhibition yesg (58, 59, 141, 187,200–202)

endothelial cells partial inhibition yes (9)NK cells (Ag/IgG) inhibition yes (44)eosinophils, neutrophils inhibition n.t. (189)

IFN-γ T cells inhibition yes (203–206)TGF-β T cells stimulation n.t. (207, 208)TNF-α T cells inhibition yes (57, 136, 153, 187)

B cells inhibition yes (57, 137)macrophages (bacteria) partial inhibition n.t. (185)mast cells (Ag/IgE) inhibition n.t. (186, 196)NK cells (Ag/IgG) inhibition yes (44)

B. Surface ReceptorsCD40L T cells inhibition yes (150, 151, 209)IL-2Rα T cells inhibition n.t.h (210)

(CD25)CD69 T cells partial inhibition n.t. (211)CTLA-4 spleen cellsi n.t. altered in (73, 176)

NFAT1−/− miceFasL spleen cellsi n.t. decreased in (176)

NFAT1−/− miceT cell hybridoma inhibition n.t. (212)

(Continued)

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Table 4 Continued

Effects of CsA or NFATCell type testeda FK506b implicatedc References

C. Transcription FactorsNF-κB1 T cells inhibition n.t. (8, 210)c-Rel T cells inhibitionj n.t. (213)Oct-2 T cells inhibition n.t. (214)NFATc T cells inhibition n.t. (25)Nur77 T cells inhibitionk n.t.l (215, 216)

aUnless otherwise specified (in parentheses), T cells were stimulated withαCD3, ConA, or ionomycin, alone or in combi-nation with phorbol ester PMA, and other cells were stimulated with PMA plus ionomycin.bThe effect of CsA or FK506 on the induction of protein, mRNA, or promoter/enhancer activity was assessed.cn.t., not tested. yes, at least one of the criteria is met experimentally: (i) identification of NFAT-binding sites in the pro-moter/enhancer, (ii) mutation of the sites diminishes reporter expression driven by the promoter/enhancer, (iii) multimers ofthe sites confer CsA/FK506-sensitive induction on a linked reporter gene, (iv) overexpression of NFAT proteins enhancesreporter activity.dA T cell-specific enhancer has recently been identified 14 kb upstream of the IL-3 gene. Like the GM-CSF enhancer, theIL-3 enhancer contains three NFAT binding sites that function as enhancer elements (P Cockerill, personal communication).eS. Okamoto & K. Matsushima, personal communication.fCsA inhibited in vivo induction of mRNA in spleen cells and serum IL-10 levels by anti-CD3.gThe NFAT binding site (CLE0) in the GM-CSF promoter also binds the Ets protein, Elf-1 (217).hCsA inhibited the induction of NF-κB1 mRNA.imRNA induction was assessed in spleen cells from mice injected with anti-CD3 antibody.jCsA inhibits c-Rel mRNA induction as well as the nuclear translocation of c-Rel protein.kCsA inhibits the DNA binding activity of Nur77 as well as Nur77 promoter-driven reporter gene expression.lA distinct nuclear factor, belonging to the RSRF/MEF2 family, has been implicated.

expression do not always correlate: Although NFAT1 mRNA has been variablydetected in brain, heart, and skeletal muscle, NFAT1 protein expression has notbeen detected in bulk extracts of these tissues (42, 43). Consistent findings arethat NFAT1 and NFAT2 mRNAs are expressed in peripheral lymphoid tissue(spleen, PBL), and that NFAT4 mRNA is expressed at high levels in the thy-mus, suggesting a role in T cell development. NFAT3 mRNA is expressed atlow levels in lymphoid tissues (31), and thus this protein may be preferentiallyexpressed outside the immune system. NFAT2 mRNA is upregulated in acti-vated T cells and NK cells via a CsA-sensitive mechanism that may involve anNFAT-regulated positive feedback loop (25, 32, 44).

At the protein level, NFAT2 and the major NFAT1 isoforms NFAT1b andNFAT1c are expressed in peripheral T cells and T cell lines (Table 3), but theirrelative expression may vary in different cell lines or T cell subsets. NFAT1 isalso expressed in other classes of immune-system cells (B cells, mast cells, NKcells, some monocytes and macrophages), consistent with NFAT activation andCsA-sensitive gene expression in these cells (Table 1). Additionally, NFAT1 isexpressed in olfactory epithelium (45) and in some endothelial cell lines (9, 42).

Some potential target genes for NFAT proteins are listed in Table 4. NFATinvolvement is reasonably well established for the IL-2, IL-4, GM-CSF, and

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TNF-α cytokine genes in T cells. There is good but less extensive evidencefor NFAT regulation of the IL-3, IL-5, IL-8, interferon-γ (IFN-γ ) and CD40Lgenes in T cells, the TNF-α gene in B cells, and the IL-4 and IL-5 genes inmast cells. In many of the other examples listed in Table 4, inducible gene ex-pression is inhibited by CsA and FK506, suggesting but by no means provingthe participation of calcineurin and NFAT. High concentrations of these im-munosuppressants are known to affect cell metabolism by interfering with thechaperone (peptidyl prolyl isomerase) functions of their immunophilin receptor(46). Further, calcineurin is known to modulate the functions of transcriptionfactors other than NFAT (reviewed in 3). A recent example relates to the CsA-sensitive induction of Nur77 mRNA in activated T cells, which is thought to bemediated by RSRF (MEF2), a nuclear factor distinct from NFAT (47).

REGULATION OF NFAT ACTIVATION

Early Stages of NFAT Activation: Role of CalcineurinThe early activation of NFAT proteins has been investigated for NFAT1 (43,48–52), recombinant NFAT2 (25), and recombinant NFAT4 (33, 53). Threedifferent steps of activation have been defined: dephosphorylation, nucleartranslocation, and increase in affinity for DNA (Figure 2). The consensusfrom these studies is that, in resting cells, NFAT proteins are phosphorylated,reside in the cytoplasm, and show low affinity for DNA. Stimuli that elicitcalcium mobilization (see Table 1) result in the rapid dephosphorylation ofNFAT proteins and their translocation to the nucleus; the dephosphorylatedproteins show increased affinity for DNA. Each step of activation is blocked

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 2 A model for activation of NFAT proteins. (1) NFAT interacts with calcineurin (CnAand CnB) and is retained in the cytoplasm by a complex of other proteins (shaded) in restingT cells. Without calcium and calmodulin (CaM), the autoinhibitory domain of CnA blocks thecatalytic active site of CnA and calcineurin is inactive. The identified functional regions of NFAT aredenoted TADn for N-terminal transactivation domain, NLS for nuclear localization sequence, DBDfor DNA-binding domain, and TADc for C-terminal transactivation domain. In the resting state,phosphorylation of NFAT proteins masks the NLS and the residues in the DBD that will contactDNA. (2) The binding of calmodulin triggered by increase of intracellular calcium ([Ca2+]i ) leadsto a conformational change in calcineurin that moves the autoinhibitory domain of calcineurin awayfrom the catalytic active site. Activated calcineurin dephosphorylates NFAT. (3) Dephosphorylationcauses a conformational change in NFAT proteins, which exposes the NLS and the residues that willcontact DNA, resulting in nuclear import and increase in DNA binding activity. (4) Withdrawal ofcalcium or inhibition of calcineurin leads to rephosphorylation of NFAT proteins by unidentifiedkinase(s). The rephosphorylated NFAT proteins return to the original state in the cytoplasm. Seetext for details.

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by CsA or FK506, implying that calcineurin is involved, and suggesting thatdephosphorylation is the initial step of activation. It should be noted thatcytokine and LPS receptors, which are not linked to Ca2+ mobilization, are notexpected to activate calcineurin or NFAT.

The activation of NFAT proteins follows precisely the activation of cal-cineurin. In T cells, if intracellular free calcium levels ([Ca2+]i ) remain el-evated, calcineurin activity remains high, and NFAT1 remains activated andnuclear for many hours (43, 51, 52). Conversely if calcineurin activity isinhibited by removal of the calcium-mobilizing stimulus, or by addition ofthe calcineurin inhibitors CsA and FK506 to stimulated T cells, NFAT1 re-verts to its original phosphorylated state within 5–15 min, returns to the cyto-plasm, and again shows a low affinity for DNA (43, 51, 52). Similar resultshave been obtained for recombinant NFAT4 expressed in fibroblasts (53). Thedephosphorylation/rephosphorylation and nuclear import/export cycle can berepeated many times by removal and readdition of ionomycin (51, 53), in-dicating that the activation of NFAT1 and NFAT4 is regulated by a dynamicinterplay between calcineurin and constitutively active kinases. In an analo-gous physiological situation, nuclear NFAT1 returns slowly to the cytoplasmin antigen-stimulated cells as a result of feedback mechanisms that cause a de-cline in capacitative calcium entry and the eventual repletion of calcium stores(52).

Overexpression of mutant calcineurins has pronounced effects on NFATfunction. Constitutively active calcineurin substituted partially for the cal-cium requirement for NFAT-dependent gene expression (54–60). Conversely,an inactive calcineurin, mutated in an essential active site histidine, inhibitedthe movement of recombinant NFAT4 from the cytoplasm to the nucleus ofstimulated fibroblasts (53). Further, a calcineurin A chain fragment containingonly the B subunit-binding region suppressed NFAT-dependent reporter geneexpression in Jurkat T cells, apparently by competing with the endogenous Achain for the regulatory B chain and thereby reducing the calcineurin activityin stimulated cells (61). However, mice deficient in the expression of the majorcalcineurin Aα isoform showed only a moderate impairment of the secondaryimmune response, suggesting that NFAT activation could be mediated by othercalcineurin isoforms (62).

Requirement for Capacitative Calcium EntryA prolonged elevation of [Ca2+]i levels, beyond the initial transient resultingfrom the emptying of calcium stores, is required in T cells to maintain cal-cineurin and NFAT proteins in an activated state. This plateau phase of calciummobilization is the outcome of capacitative calcium entry, a process that cou-ples the depletion of intracellular calcium stores to the influx of extracellular

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calcium through specialized calcium channels (reviewed in 63–65). Capacita-tive calcium entry can be elicited by stimulation of each of the receptors listedin Table 1, as a result of their ability to activate phospholipase C, increase theintracellular concentration of inositol trisphosphate, and deplete intracellularcalcium stores (5, 6, 66). It can also be elicited pharmacologically by treatingcells with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPaseneeded for repletion of calcium stores, or with calcium ionophores such as ion-omycin, which deplete the stores directly (67, 68). Addition of SKF96365,an inhibitor of capacitative calcium entry, to stimulated T cells resulted in animmediate deactivation of NFAT1 (52) that resembled the deactivation causedby the calcineurin inhibitors CsA and FK506 (51).

The importance of capacitative calcium entry for NFAT activation is furtheremphasized by the finding that among mutant T cells selected for their inabil-ity to promote NFAT-dependent expression of a toxin gene, a high proportionwere defective in their ability to increase [Ca2+]i in response to ionomycin orthapsigargin (69, 70). Moreover, parallel measurements of [Ca2+]i and NFAT-dependent reporter gene expression in individual T cells indicated that effectiveactivation of the transcriptional functions of NFAT proteins required capacita-tive calcium entry (71). In cells stimulated with thapsigargin or immobilizedanti-CD3, NFAT-dependent reporter gene expression was observed only in thecells that also showed a sustained increase in [Ca2+]i . In T cells that were lesseffectively stimulated, as with soluble rather than immobilized anti-CD3, orunder conditions that would decrease the driving force for calcium influx, onlythe transient peak of calcium mobilization was apparent, and NFAT-dependenttransactivation was less effective or did not occur (71).

Dephosphorylation of NFAT Proteins by CalcineurinIt is likely that NFAT proteins are directly dephosphorylated by calcineurin inactivated cells. When tested in vitro, calcineurin dephosphorylated purifiedNFAT1 (23), NFAT1 in immunoprecipitates (40, 43), recombinant NFAT1 inCOS cell extracts (40), and endogenous NFAT1 in T cell nuclear or cytoplas-mic extracts (49). The phosphatase inhibitor okadaic acid, at a concentration(300 nM) known to inhibit protein phosphatase-1 (PP1) and PP2A completely,did not inhibit the dephosphorylation of recombinant NFAT1 by added cal-cineurin in COS cell extracts (40). This result argues against the involvementof a phosphatase cascade initiated by calcineurin and resulting in PP1 or PP2Aactivation, such as that previously described in hippocampus (72).

The evidence relating dephosphorylation to changes in NFAT function isstrong but still circumstantial. Dephosphorylation is a major biochemicalchange detected in NFAT1, NFAT2, and NFAT4 after cell stimulation (25, 33,43, 50–53) and clearly precedes the nuclear import of endogenous NFAT1 (50),

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suggesting that dephosphorylation is required for nuclear import. Dephospho-rylation may also be required for optimal DNA binding. Stimulation of T cellswith ionomycin or through the T cell receptor (TCR) results in a substantialincrease in the intrinsic DNA-binding affinity of NFAT1 (50–52) and NFAT2(73) for the distal NFAT site of the murine IL-2 promoter. Treatment of cellextracts with calcineurin or alkaline phosphatase also results in increased DNAbinding of NFAT1 (49), suggesting that phosphorylated residues directly orindirectly mask a region of DNA contact in NFAT1. A model incorporatingthese findings is discussed below (Figure 2).

Targeting of Calcineurin to the NFAT Regulatory DomainThere is evidence that NFAT proteins interact directly with calcineurin withinthe cell. NFAT1 in T cell extracts binds to calcineurin immobilized on calmodu-lin-sepharose beads (48, 51). Binding can be demonstrated using both thephosphorylated form of NFAT1 from resting cells and the physiologically de-phosphorylated form from stimulated cells (48, 51). The ability of bacteriallyexpressed recombinant NFAT1 to bind to calcineurin in vitro (40) shows thatthe interaction is not mediated by other proteins in cell lysates.

Analysis of recombinant NFAT fragments shows that the NFAT-calcineurininteraction involves the N-terminal≈400 residues of NFAT1, NFAT2, andNFAT4 (40, 53), and thus the interaction maps to the conserved NHR (30). Theconserved residues in this region are clustered in several short sequence motifs(30), which include the three SP motifs (consensus SPxxSPxxSPxxxxx[D/E][D/E]) noted earlier (32, 33). In NFAT1 and NFAT4, the N-terminal region con-taining the conserved residues functions as a calcineurin-activated regulatorydomain: It contains phosphorylated serine residues that are dephosphorylatedby calcineurin, and it regulates the subcellular localization of covalently linkedprotein domains in a calcium- and calcineurin-dependent way (40, 53). The reg-ulatory domain also contains a candidate nuclear localization sequence (NLS)whose mutation prevents the nuclear import of full-length NFAT1 (40). Whenoverexpressed, a fragment of NFAT2 containing this regulatory domain has a“dominant negative” effect, in that it downregulates NFAT-dependent transac-tivation mediated by endogenous or recombinant NFAT proteins (25, 74).

The NFAT-calcineurin interaction appears to be another example of target-ing interactions involving protein phosphatases (40). For instance, the catalyticsubunit of PP1, which shows little substrate specificity in vitro, is targeted tosubstrates in glycogen, in myofibrils, and in sarcoplasmic reticulum by distincttargeting subunits (75). Calcineurin itself is directed to the inositol trispho-sphate and ryanodine receptors by an interaction with FKBP12 (76), and tothe RII subunit of protein kinase A and relevant subsynaptic substrates by itsinteraction with the A-kinase anchor protein AKAP-79 (77, 78).

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Models and SpeculationsA model for NFAT activation is depicted in Figure 2. NFAT proteins are tar-geted via their conserved regulatory domains to inactive calcineurin in restingcells; depending on the affinity, these interactions may be either transient orstable. Increases in [Ca2+]i lead to the activation of calmodulin (79); binding ofcalmodulin to a region near the C-terminus of calcineurin displaces the autoin-hibitory domain of calcineurin and exposes the calcineurin active site (80). Ac-tivated calcineurin dephosphorylates associated NFAT proteins at multiple sitesin their regulatory domains. The NLS in the regulatory domain, and potentiallya DNA recognition region in the DNA-binding domain, become accessible inthe dephosphorylated proteins. One possibility (not shown) is that the residuesimmediately adjacent to the NLS or to the region of DNA contact are directlymodified with phosphates and thus masked. Alternatively, dephosphorylationmay expose these regions by producing an intrinsic conformational change inNFAT proteins (Figure 2). The model also depicts a calcineurin-dependentinteraction of the regulatory domain with a larger protein complex (possiblycalcineurin itself) in the cytoplasm. This is inferred from the fact that althoughthey are small enough to diffuse through nuclear pores, N-terminal fragmentsof NFAT1 and NFAT4 are retained in the cytoplasm of resting cells and moveto the nucleus only upon activation (40, 53). Dephosphorylated NFAT proteinsare still capable of binding calcineurin and may continue to bind it in activatedcells.

The rapid reversal of NFAT activation by CsA and FK506 implies a con-tinuing interplay between NFAT kinases and calcineurin (51, 53). Both setsof enzymes can act on NFAT proteins in the nucleus: Activated calcineurinmaintains nuclear NFAT proteins in their activated state, while the kinasesrephosphorylate the proteins when calcineurin is inhibited with CsA or FK506.An interesting possibility is that the enzymes move to the nucleus upon activa-tion, as demonstrated for calcineurin overexpressed with recombinant NFAT4in fibroblasts (53).

OTHER SIGNALING PATHWAYS THAT INFLUENCENFAT ACTIVATION

Many signaling pathways and proteins, other than calcineurin, modulate NFATfunction in T cells (see Figure 3). Since most of the experiments involvemeasuring reporter gene expression driven by a composite NFAT:AP-1 site,their interpretation is necessarily complex, and it is not always apparent whethercalcineurin, NFAT proteins, or AP-1 proteins are affected. For the sake ofbrevity, we have limited our discussion to the effects of pathways normally

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Figure 3 A simplified view of some signaling pathways reported to influence NFAT-dependenttranscription (see text). Molecules discussed in the text are boxed. Unidentified signaling moleculesand pathways are indicated by question marks. Key: CaMK, multifunctional Ca2+/calmodulin-dependent protein kinases; CAML, calcium-signal modulating cyclophilin ligand; Cn, calcineurin;1Cn, constitutively active calcineurin lacking the autoinhibitory domain; JNK, c-Jun N-terminalkinase; DN, dominant negative; SRF, serum response factor.

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activated in antigen-stimulated cells, concentrating on cases in which NFATfunction has been specifically addressed, and where there is an indication of themechanisms involved. Experiments involving transgenic or “knockout” miceare not described except where a mechanistic interpretation is possible.

TCR-Proximal Steps: Lck, ZAP-70, VavThe earliest consequence of TCR cross-linking is the activation of tyrosine ki-nases including Lck and ZAP-70, and the tyrosine phosphorylation of multipleintracellular substrates including Vav (81, 82). Stimulation of T cells with acombination of a calcium ionophore and PMA bypasses these early steps butmimics the effects of TCR cross-linking on gene transcription (6). Both Lckand ZAP-70 are required for calcium mobilization in T cells (82), and overex-pression of wild-type or constitutively active Lck upregulated NFAT-dependentreporter gene expression in Lck JCam.1 cells (83). Likewise, overexpressionof kinase-deficient ZAP-70 inhibited NFAT activity in Jurkat T cells stimulatedwith anti-TCR antibodies but did not affect NFAT activity in PMA/ionomycin-stimulated cells, suggesting that an early TCR-proximal step was involved (84).

ZAP-70 interacts with Vav, an SH2 domain–containing protein that is rapidlyphosphorylated on tyrosine residues upon T cell activation (85). In turn, Vav in-teracts through its SH2 domain with SLP-76, a tyrosine-phosphorylated proteinthat also binds the adapter protein Grb2 (85a). Overexpression of Vav and/orSLP-76 augmented basal and TCR-stimulated NFAT-dependent reporter geneexpression in Jurkat cells (85a, 86). The effect of Vav was specific for T cellreceptor stimulation, since NFAT activation through the muscarinic receptorwas not augmented by Vav. Likewise, there was no augmentation in cells lack-ing Lck, CD45, or the TCRβ chain, and Vav overexpression did not result inincreases in [Ca2+]i or in the levels of tyrosine-phosphorylated proteins. Vav-induced augmentation was blocked by the calcineurin inhibitors CsA and FK506and by overexpression of dominant negative forms of Ras and Raf, suggestingthat Vav functioned upstream of these molecules or in a parallel pathway.

PMA-Activated Signaling PathwaysSingle-cell measurements indicate that PMA enhances the calcium sensitivityof NFAT-dependent reporter gene expression, as assessed by the number of cellsexpressing the reporter gene at different [Ca2+]i levels (71). The magnitude ofthis effect is striking: In the presence of PMA, an average threefold increasein the sustained plateau level of [Ca2+]i results in reporter gene expression inhalf of the responsive cells (71). The most likely effect of PMA is to inducethe de novo synthesis and posttranslational modification of AP-1 and othertranscription factors (6), thereby increasing the transcriptional effectiveness ofnuclear NFAT proteins. PMA-activated kinases may also directly phosphorylate

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NFAT2 (25), although there are no data on whether this influences NFAT2function. In contrast, PMA stimulation does not, under the conditions so fartested, alter the mobility of NFAT1 on SDS gels, its subcellular distribution, orthe function of its major transactivation domain (41, 52).

Mechanistically, the major known effect of PMA in T cells is to activateprotein kinase C, Ras, and Raf; the effects of activated and dominant nega-tive versions of these proteins on NFAT-dependent transactivation in T cellshave been previously reviewed (6). A major conclusion is that Ras is indis-pensable for NFAT function: Activated Ras substitutes effectively for PMA,and together with constitutively active calcineurin it provides a full stimulusfor NFAT-dependent reporter gene expression, whereas dominant negative Rasinhibits NFAT activation in response to TCR stimulation (6, 87, 88). MultipleRas effectors including Raf and the Ras-related GTPases Rac and Rho havebeen described (88–94). The Raf pathway is clearly important for AP-1 induc-tion, via activation of the Raf substrate MEK1, a dual specificity kinase, andits substrates the MAP kinases ERK-1 and ERK-2 (reviewed in 95). In T cells,however, constitutively active Raf or MEK1 was not as effective as activated Rasin substituting for PMA and synergizing with ionomycin to stimulate NFAT-driven transcription. Consistent with these findings, dominant negative versionsof these proteins only partially inhibited Ras-mediated activation (88, 96). Aconstitutively active form of another Ras effector, Rac-1, activated AP-1-drivenreporter gene expression maximally by a pathway not involving ERK but didnot activate an NFAT-driven reporter gene efficiently, even in combination withionomycin and constitutively active MEK1 (88). Based on these results, it hasbeen proposed that yet another Ras effector pathway exists in T cells and isneeded for maximal expression of a reporter gene driven by NFAT:AP-1 (88;see Figure 3). It will be interesting to determine whether this effector pathwaytargets NFAT proteins, AP-1 proteins, or both.

Pathways Related to Calcium MobilizationIn addition to activating calcineurin, increases in [Ca2+]i lead to the activationof other calmodulin-dependent enzymes (79). Among these, two multifunc-tional Ca2+/calmodulin-dependent protein kinases (CaMK) may regulate NFATactivity. A constitutively active form of a T cell CaMKII, CaMKIIγB, partiallyinhibited both NFAT- and AP-1-driven reporter gene expression in Jurkat T cellsand blocked the ability of constitutively active calcineurin to augment IL-2 pro-moter function (97, 98). The opposite effect was observed for constitutivelyactive CaMKIV/Gr, which enhanced NFAT activity in large part by activatingAP-1 (99). The enhancement by CaMKIV/Gr, which was not blocked by domi-nant negative Ras, may be mediated through upregulation of the transcriptionalfunction of serum response factor (SRF) and the consequent induction of c-Fos

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(99, 100). Curiously, both CaMKs are activated by CaM and Ca2+ during T cellactivation, exhibit broad substrate specificity, and phosphorylate the same min-imal consensus substrate sequence (101), suggesting that the opposite effects ofthe two CaMKs may involve their targeting to different intracellular substrates.

Overexpression of a cyclophilin-binding protein, CAML, significantly in-creased the basal level of intracellular calcium in transfected T cells and par-tially substituted for the calcium requirement for NFAT-dependent transcription(102). This effect required extracellular calcium and was inhibited by CsA orFK506, suggesting that CAML functioned by facilitating calcium entry intocells (102).

Costimulatory PathwaysThe T cell surface molecule CD28 delivers a costimulatory signal necessaryfor maximal activation through the TCR (103). CD28 stimulation results in astriking augmentation of IL-2, TNF-α, GM-CSF, and IFN-γ production (104),which has its basis in a large increase in steady state mRNA levels (105). Asignificant component of the effect is posttranscriptional (106), possibly arisingfrom mRNA stabilization (105). However, CD28 stimulation also results inthree- to eightfold increases in reporter gene expression driven by the IL-2,IL-3, GM-CSF, and IFN-γ promoters (107, 108). The CD28 response elements(CD28REs) in these genes have been mapped (107, 108) and shown to bindinducible factors variously identified as Rel proteins (109–111), NFAT proteins(112, 113), and≈35-kDa protein(s) visualized by UV cross-linking to a labelledoligonucleotide probe but not yet characterized molecularly (108, 114). It isinteresting that in both the IL-2 and GM-CSF promoters, the sequences thatconfer responsiveness to HTLV-1 Tax map to the CD28REs (113, 115).

The nature of the signaling pathways involved in CD28 signaling has beenstudied in many laboratories (116). Stimulation of T cells with anti-CD28antibodies elicits a spectrum of responses, in part resembling those elicited byactivation of the TCR, but differing from those resulting from stimulation withthe physiogical ligand of CD28, B7.1 (117). The consensus is that the CD28pathway is not CsA sensitive (104, 118).

In considering whether NFAT proteins play a role in CD28-mediated costim-ulatory pathways, two observations are relevant. First, at least some NFAT pro-teins may be activated via CD28-dependent, calcineurin-independent pathways,as judged by the CsA-insensitive induction of low levels of NFAT-dependentreporter gene expression and nuclear NFAT DNA-binding activity in T cellsstimulated with anti-CD28 and PMA (74). Second, NFAT proteins can bind tothe CD28REs of both the IL-2 and GM-CSF promoters, which bind Rel-familyproteins (108–111, 115). Thus a plausible mechanism linking CD28 stimulationto cytokine gene transcription would be activation of NFAT proteins through

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a distinct pathway that may synergize with the calcium/calcineurin pathway,followed by binding of the activated proteins to CD28REs and conventionalNFAT sites in the promoter and enhancer regions of NFAT-inducible genes.The potential interplay between NFAT and Rel proteins at the CD28REs andotherκB-like sites is discussed in a later section.

Other Signaling PathwaysA variety of biological and pharmacological agents including protein kinase A(PKA) activators (119–123), glucocorticoids (124–126), transforming growthfactor-β (TGF-β) (127), retinoic acid (128), and vitamin D3 (129) are knownto inhibit IL-2 production by activated T cells. Of these, PKA activators andvitamin D3 have been reported to affect NFAT function.

PKA In a mouse T cell hybridoma stimulated with thapsigargin, dibutyrylcAMP inhibited NFAT:AP-1-dependent reporter gene expression, and the inhi-bition was substantially reversed by PMA, suggesting either that AP-1 proteinswere limiting in the treated cells or that a PKA-mediated inhibition of NFATfunction could be compensated for by an increase in AP-1 (71). Whether PKAactivators inhibit AP-1 function, NFAT function, or both remains unclear. Inseveral studies in which EL-4 mouse thymoma cells were used, PKA activa-tors had no effect on NFAT DNA-binding activity (119, 121, 122); this findingcould reflect the fact that NFAT is constitutively nuclear in EL-4 cells (130),in which the calcineurin A chain bears a spontaneous activating mutation in itsautoinhibitory domain (60). In primary human T cells, however, prostaglandinE2 (PGE2) significantly inhibited the induction of both NFAT:AP-1 and AP-1DNA-binding activity (123); whereas in primary CD4+ murine T cells, choleratoxin, one of whose several effects is to elevate cAMP (131), inhibited theinduction of NFAT but not AP-1 DNA-binding activity (120). The inhibitoryeffects of PKA may not involve AP-1, however. An established target for PKAis the transcription factor CREB, which becomes phosphorylated on serine-133;based on the findings that CREB is phosphorylated at the same site, presumablyby other kinases, upon stimulation through the TCR (132, 133), and that thymo-cytes from transgenic mice expressing a dominant negative version of CREBshowed significantly decreased induction of several Fos- and Jun-family m-RNAs (132), it has been suggested that CREB is involved in upregulating AP-1activity in stimulated T cells. Clearly, further experiments are necessary to elu-cidate the molecular effects (if any) of PKA on the phosphorylation status andfunction of NFAT proteins in cells. If the evidence favors a consistent effectof PKA on NFAT activity, a further point that will need to be considered isthat PKA activators upregulate IL-4 and IL-5 production while inhibiting IL-2production (120, 121).

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VITAMIN D3 The natural immunosuppressant vitamin D3 downregulates IL-2production by activated T cells. The inhibitory effects of vitamin D3 are me-diated through the vitamin D3 receptor, which binds as a heterodimer with itspartner the retinoid X receptor to sequences overlapping the distal NFAT siteof the human IL-2 promoter, thereby preventing the binding of the NFAT:AP-1complex to the same site (129).

GLUCOCORTICOIDS NFAT and NFAT:AP-1 complexes do not appear to bedirect targets for inhibition by glucocorticoids, because neither the inductionof NFAT DNA-binding activity nor the level of NFAT:AP-1-dependent reportergene expression was affected by dexamethasone treatment of Jurkat T cells(124–126).

RETINOIC ACID, TGF-β The inhibitory effects of retinoic acid and TGF-β havebeen mapped to the octamer element of the IL-2 promoter (127, 128), whichis now known to be a composite NFAT:AP-1:Oct site (112). However, neithercompound influenced NFAT:AP-1-dependent reporter gene expression, sug-gesting that neither directly affected the function of NFAT.

DNA BINDING AND TRANSACTIVATION

DNA BindingThe DNA-binding domains of NFAT proteins are likely to be similar in three-dimensional structure to the DNA-binding domains of Rel-family proteins, andto utilize corresponding loops to make some of their contacts with DNA (37–39).The domains (≈300 amino acids) are highly conserved within each family (60–70% pairwise identity for NFAT proteins; 41–61% for mammalian Rel proteins)and show a low but significant level of sequence identity (≈17%) in pairwisecomparisons between individual members of the NFAT family and the Relfamily (31). The minimal DNA-binding domain (≈190 amino acids) of NFAT1(37) corresponds almost exactly to the N-terminal specificity domain of NF-κBp50, which makes the majority of base-specific contacts with DNA (134, 135).This domain contains the highly conserved recognition loop (RFRYxCEG)of Rel-family proteins, represented in the NFAT family as the equally highlyconserved RAHYETEG sequence in which each of the underlined residues islikely to contact DNA (37, 39).

A striking difference between NFAT and Rel proteins is that whereas Rel pro-teins are dimeric in solution and bind DNA as obligate dimers, NFAT proteinsare monomeric in solution and when bound to DNA (31). NFAT proteins canalso bind to certainκB-like sites (see below): in this case, an NFAT monomerbinds to the 5′ half-site, and a second NFAT monomer binds to the symmetrical

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Table 5 Site selectivities of NFAT proteins

IL-2 (distal NFAT)NFAT −AP-1 +AP-1 IL-4 (P1)a TNF-α (κ3) References

NFAT1 +++ ++++ +++ ++ (31, 33, 34)NFAT2 ++ ++++ +++ − (31–34)NFAT3 + ++++ +++ n.t. (31)NFAT4 ± ++++ ± − (31–33)

aResults from Hoey et al (31) using bacterially-expressed recombinant DNA-bindingdomains. Using full-length recombinant proteins expressed in COS cells, Ho et al (33)found that NFAT2 bound least well to the IL-4 P1 site (binding decreases in the orderNFAT1> NFAT4> NFAT2).

3′ half-site, without stabilization of the binding by protein-protein interactionsbetween the monomers (136). This property has led to the classification ofNFAT proteins as “monomeric Rel proteins” (39). The monomeric nature ofNFAT proteins explains why the minimal DNA-binding fragment of NFAT1,which lacks the region corresponding to the C-terminal dimerization domainof Rel proteins, can bind independently to DNA (37). Moreover, it providesa rationale for why the N-terminal two thirds of the Rel similarity region ofNFAT proteins is more highly conserved in sequence than the C-terminal onethird (31).

Although all four NFAT proteins are capable of binding cooperatively withAP-1 proteins to the distal IL-2 promoter NFAT site (31, 32, 34), they showcharacteristic site preferences when tested for DNA binding in the absence ofAP-1 (Table 5). Of the four NFAT proteins, NFAT1 displays the highest affinityfor each of the three sites shown in Table 5, binding in the order IL-4 P1>

distal IL-2> TNF-α κ3. In contrast, NFAT4 binds poorly or not at all to thedistal IL-2 site (31–33), and neither NFAT2 nor NFAT4 bind under standardconditions to the TNF-α promoterκ3 site (33, 34). As a consequence, NFAT2bfails to activate the TNF-α promoter when transiently expressed in T or B celllines, while NFAT1 is highly active in this respect (34). Curiously, overall TNF-α production is not impaired in NFAT1-deficient mice (73), possibly becausethe TNF-α promoter contains additional NFAT sites capable of binding theother NFAT proteins (137), or because the other NFAT proteins do function attheκ3 site in the genomic context.

Transactivation DomainsAnalysis of NFAT1 suggests that both the N- and C-terminal regions of NFATproteins contain transcriptional activation domains (30). The N-terminal TADof NFAT1 has been localized to the N-terminal≈100 residues of NFAT1, whichlike the corresponding regions of NFAT2b, NFAT3, and NFAT4 is rich in acidic

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residues and proline (30–32, 34). In this region, NFAT1 contains at least oneacidic/hydrophobic patch,25QDELDFSILFDYEYL39, that resembles those im-plicated in transactivation by acidic activation domains (138, 139). Similaracidic/hydrophobic patches are present in NFAT2b (4LEDQEFDFEFLFEF17),NFAT3 (8DEELEFKLVFGEE20), and NFAT4 (9HDELDFKLVFGED21) (31,32, 34). Despite the very limited sequence conservation, therefore, theN-terminal regions of all these proteins could conceivably function as acidictransactivation domains. The corresponding region of NFAT2a, which lacksthe acidic/hydrophobic patches, is also capable of transactivation (C Luo, un-published), perhaps through interaction with a distinct set of nuclear proteins.

When expressed as a GAL4 fusion protein, the region of NFAT1c locatedC-terminal to the DNA-binding domain is also capable of transactivation (41).Again, this region shows only limited sequence similarity to the correspondingregions of NFAT2b, NFAT3, or NFAT4, except for the LDQTYLDDVNEI-IRKEFS sequence mentioned previously, which spans a C-terminal splice siteand is represented in NFAT3 and NFAT4x (see Figure 1). Although more ex-periments are needed to establish the significance of this conserved segment,it is interesting that the NFAT1c isoform, which possesses this sequence, isreproducibly more active in transactivation assays than the NFAT1b isoform,which differs in this portion of the C-terminal sequence (41).

Cooperation with AP-1NFAT proteins show a characteristic ability to cooperate with AP-1 in DNAbinding and transactivation (reviewed in 1). The minimal DNA-binding domainof NFAT1 suffices for cooperative binding with AP-1 dimers to the distal NFATsite of the murine IL-2 promoter (37). The interaction between NFAT proteinsand AP-1 involves binding of these unrelated transcription factors to adjacentsites on DNA and results in an approximately 20-fold increase in the stabilityof the NFAT1:AP-1:DNA complex compared with the DNA-protein complexcontaining NFAT1 alone (23, 27, 140). In DNA-protein complexes containingNFAT1, Fos, and Jun, the Fos-Jun dimer shows a remarkable orientationalpreference that is not apparent in the absence of NFAT1, in that the AP-1 half-siteproximal to the bound NFAT1 monomer is occupied exclusively by Jun (140).The paradigm of NFAT-AP-1 cooperation has been recognized in a number ofdifferent cytokine promoter/enhancer regions (reviewed in 1) (Table 6). Notethat cooperative binding implies not merely that two transcription factors canoccupy adjacent sites on DNA, but that the DNA-protein complex containingboth transcription factors is significantly more stable or of higher affinity thanthose containing the individual proteins (27, 140, 141).

When expressed without its transactivation and regulatory domains in T cells,the DNA-binding domain of NFAT1 is found in the nucleus and can mediate

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Table 6 Selected NFAT binding sites in gene regulatory regions

Promoter Species Site Sequences References

Group I NFAT AP-1

IL-2 m −280 AGGAAA ATT TGTTTCA (20, 27,h −280 AGGAAA AAC TGTTTCA 162, 163)m −135 AGGAAA AAC AA AGGTA (21, 162,h −135 AGGAAA AAT GAAGGTA 163)

m, h −90 TTGAAA ATA TGTGTAA (112, 218)m −45 TGGAAA AAT AATA TGGr (112)h −45 TGGAAA AAT ATTA TGGr

IL-4 m P1 TGGAAA ATT TTATTACAr (219)h P TGGAAA TTT TCGTTACAr (161)

IL-5 m P TGGAAA CCC TGAGT TTr (173, 195)h P TGGAAA CAT TTAGTTTr

GM-CSF h GM330 CGGAGCCCC TGAGTCA (141)enhancer h GM420 TGGAAA GA− TGACATCA r

h GM550 AGGAAA GCA AGAGTCA r

CD40L m, h NM-1 TGGAAA ATG TGCTTCGr (150, 151)

IFN-γ h TGGAAA TTT TTTTGTAr (205, 206)

IL-13 h TGGAAA ATC CAGTGTCGCAr (198)

CTLA-4 m −207 TGGAAA AT− GTACTCAAr (220)h −195 TGGAAA AT− GTATTCAAr

T AConsensus GGAAA ATN TGAGTCA TGACGTCA

A NC (AP-1) (CREB/ATF)

Group II κB like sites adjacent sites

hTNF-α κ3/CRE GGAGAAACCC ATGAGCTCA r (ATF-2/Jun) (136, 154)hIL-8 −81/−65 TGGAATTTCC TCTGACATAA (155). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E-selectin PDI GGGGATTTCC TCTTTACTGGATGT (9, 157). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

HIV-1 LTR κB/SP-1 GGGACTTTCC AGGGAGGCGTGGCC (Sp1) (201)hGM-CSF CK-1 (CD28RE) AGGAGATTCC ACAGTTCA (115, 201)mIL-2 CD28RE/AP-1 AAGAAATTCC AGAGAGTCA (AP-1) (112, 113)

For each element, a continuous sequence is divided by inspection into adjacent NFAT and AP-1 sites in Group I, and aκB-like site and the adjacent sequence in Group II. Introduced gaps are indicated by dashes. Nucleotides known or presumedto interact with NFAT proteins are shown in boldface type. Known and probable binding sites for AP-1/CREB/ATF proteinsare underlined, with nucleotides fitting the consensus shown in boldface type. The Group I NFAT consensus is based on thesequences shown. The human IL-4 P element and the IL-13 element could equally well be classified in Group II.m, mouse; h, human; r, sequence shown is for the noncoding strand.

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strong NFAT-dependent transactivation in cells stimulated with ionomycin andPMA (41). Under these circumstances, mutation of either the NFAT site orthe adjacent AP-1 site abrogated reporter gene expression, indicating that tran-scription required recruitment of AP-1 proteins by the NFAT1 DNA-bindingdomain. Recruitment of unrelated transcription factors by strong cooperativeinteractions involving DNA-binding domains could be a general theme in tran-scriptional regulation: The isolated DNA-binding domains of GATA proteinsand MyoD1 induce cell differentiation (142, 143), and protein-protein interac-tions have been detected between the DNA-binding domains of AP-1 and Etsproteins (144), AP-1 and NF-κB (145), C/EBP and NF-κB (146), and c-Junand the glucocorticoid receptor (147).

NFAT BINDING SITES IN GENE REGULATORY REGIONS

Table 6 presents a compilation of known and potential NFAT binding sitesin the regulatory regions of selected inducible genes. The largest and best-documented class consists of sites on which NFAT proteins form a cooperativecomplex with AP-1 or other bZIP proteins (Group I). NFAT proteins also bindto sites that are, or resemble, binding sites for conventional Rel-family proteins(Group II). Sites that bind NFAT proteins with relatively high affinity in theapparent absence of an adjacent AP-1 site have also been described (112).

NFAT:AP-1 SitesThis subject has been extensively reviewed (1, 141), and only salient points areemphasized here. Composite elements that support the cooperative binding ofNFAT and AP-1 proteins have been noted in the promoter regions of the IL-2,IL-4, IL-5, and CD40L genes, and in the GM-CSF enhancer (Table 6, Group I).Based on a comparison of these sequences, the NFAT binding site is presentedas a 9-bp element positioned next to a 7–8-bp element capable of binding dimersof AP-1, CREB/ATF, or other bZIP-family proteins. The spacing of the NFATand AP-1 elements appears to be narrowly specified, with no more than a singlebase pair insertion or deletion permitted between the two sites. Sites in whichthe GGAAA core sequence is preceded by a T rather than an A appear to bindNFAT proteins more strongly. The identified AP-1/CRE sites range from strongelements, as in the GM330 AP-1 and GM420 CRE sites, to barely recognizablevariants of the consensus sequence, as in the IL-4 and CD40L sites. The relativestrengths of the NFAT and AP-1 elements can vary reciprocally over a widerange: The−90 IL-2 site does not bind NFAT proteins independently andbinds AP-1 proteins only moderately well, but supports a strong cooperativeinteraction between these two proteins. Conversely the CD40L element pairsa strong NFAT site with a relatively weak AP-1 site. Note that the CTLA-4

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sites in Table 6 were identified by inspection, within the region shown to befunctional by promoter deletions. Also note that NFAT:AP-1 cooperation at theIFN-γ , IL-13, and CTLA-4 sites has not yet been established.

The possibility that NFAT binding to Group I-like sites in vivo always in-volves AP-1 or bZIP proteins deserves serious consideration. In the absenceof NFAT, AP-1 proteins do not bind detectably to the distal NFAT site of themurine IL-2 promoter (23, 140), a moderately high-affinity NFAT-binding sitethat is the prototype for a cooperative NFAT:AP-1 site (23). It is possible, there-fore, that even strong NFAT sites with no obvious adjacent AP-1 sites, such asthe−45 site of the IL-2 promoter (Table 6), do in fact support the formationof cooperative NFAT:AP-1 complexes in the context of larger transcriptionalcomplexes assembled on genomic promoters and enhancers. Conversely, re-porter gene expression driven by multiple AP-1 sites has been reported to beselectively sensitive to CsA and FK506 in T cells (148, 149); this may reflect,in part, the ability of AP-1 proteins to recruit NFAT proteins to adjacent sites.An important technical point when assessing protein binding to these sites byelectrophoretic mobility shift assay is that the use of insufficiently long oligonu-cleotides may lead to the classification of an NFAT site as independent of AP-1(see 150 and 151 for the CD40L site), or an AP-1 site as independent of NFAT.Carrying this reasoning further, if either half of a composite NFAT:AP-1 siteis of low affinity, the weak binding of the relevant protein to that site may beentirely missed if the adjacent site or the partner protein that binds to it is notrepresented in the assay.

The relative order of the NFAT and AP-1 sites is fixed as shown in Table 6:reversal of the order, such that the NFAT site lies 3′ to the AP-1 site on the DNAstrand represented, permits independent but not cooperative binding (J Jain,A Rao, unpublished). By this analysis, the interaction of the bZIP protein c-Mafwith NFAT1 on the IL-4 promoter (152) is unlikely to involve the same type ofprotein-protein interactions as in prototype NFAT:AP-1 complexes (140), sinceit occurs on the sequence AGTTGCTGAAACCAAGGGAAAAT GAGTT (non-coding strand), with the order of NFAT (bold and underlined) and c-Maf (un-derlined) sites reversed with respect to the canonical NFAT:AP-1 site.

κB-Like SitesNFAT proteins also bind several sequences that resemble binding sites for Relfamily proteins (Group II). Theκ3 element in the TNF-α promoter, whichbehaves as a strong CsA-sensitive element in stimulated T cells (153), is thebest-documented example of a functional site in this group (136). As mentionedabove, NFAT1 appears to bind with much higher affinity than NFAT2 andNFAT4 to this site (33, 34). Detailed analysis indicates that the GGA sequence(bold in Table 6) is a nucleating site that is preferentially occupied at low NFAT1

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concentration; mutation of this sequence eliminates binding to the site (136). Athigher concentrations, a second molecule of NFAT1 binds to the other half-site,contacting the GGG sequence on the opposite strand, but the monomers boundto the two half-sites make few if any cooperative interactions with each other.An adjacent site that binds ATF-2/Jun dimers is required for maximal functionof theκ3 element in reporter assays, although there are no stabilizing protein-protein interactions between NFAT1 and ATF-2/Jun bound to the adjacent sites(154).

The IL-8 site, which was originally identified by inspection as aκB-likesite (155), may also fit the criteria for classification in Group II. The distancebetween the presumed CRE and the GGAA sequence identified by mutation asfunctional (155) matches the spacing in the TNF-α κ3 element more closelythan the spacing in any of the Group I sites. The IL-8 site does not bind any ofthe known Rel proteins in electrophoretic mobility shift assays using nuclearextracts from stimulated Jurkat T cells, but the complex formed reacts withanti-NFAT1 antibodies (S Okamoto, K Matsushima, personal communication).Moreover, production of IL-8 and induction of the IL-8 promoter are bothstrongly inhibited by FK506 in Jurkat T cells (155), in which NF-κB inductionis not sensitive to CsA or FK506 (21), implying that this site behaves in vivoas a functional NFAT site.

The κB sites in the E-selectin promoter and the HIV-1 LTR are other ex-amples ofκB-like sites that bind NFAT1 in vitro (Table 6). Where known,the nucleotides (TTCC) that are complementary to those contacted by boundNFAT1 are shown in bold, and comprise the nucleating site. Both sites are wellestablished as Rel binding sites (156, 157), but it is not known whether NFATbinding at these sites has a functional effect.

CD28 Response ElementsThe CD28REs of the IL-2 and GM-CSF promoters bind Rel-family proteins aswell as NFAT proteins (109–113, 115), and we have therefore classified themasκB-like elements in Group II. The CD28RE of the GM-CSF promoter isthe same as its CK-1 site (115). Like the IL-8 site, however, these elementshave characteristics of both Group I and Group II sites, although the Rel-likecharacteristics may predominate.

The binding of NFAT proteins to the IL-2 promoter CD28RE has been sug-gested to involve their cooperation with AP-1 proteins bound to a strong adja-cent AP-1 site (112). Based on our earlier discussion of composite NFAT:AP-1sites, this would only be possible if the NFAT protein bound to the weak NFATsite, AAGAAA, at the 5′ end of the element; however, the spacing between theNFAT and AP-1 sites is not optimal for cooperation (Table 6). The functionalsequence in the CD28RE is thought to be the GGAA sequence on the noncoding

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strand (TTCC in Table 6), which could serve as the nucleating sequence forbinding of NFAT proteins to aκB-like site (see discussion above): Mutation ofthis site diminishes or eliminates IL-2 promoter function in response to anti-CD3 (112, 158). Indeed, NFAT proteins appear to be capable of binding to theCD28RE independently of AP-1, as observed in nuclear extracts from Jurkat Tcells stably expressing HTLV-1 Tax (113). Given the available evidence, it isplausible that NFAT proteins could bind in either mode, to one or both ends ofthe CD28REs. It remains to be determined, however, whether either mode ofNFAT binding is functional in terms of CD28 costimulation.

Interactions Among NFAT and Rel Proteins onκB-Like SitesAn interesting question is the degree to which NFAT and Rel proteins contributeto transactivation atκB-like sites or CD28REs. Mixed dimers of NFAT and NF-κB proteins have not been observed, and so any givenκB-like site is expectedto bind either NFAT or NF-κB, and not a combination of both proteins. Therelative contributions of NFAT and Rel proteins to transactivation from the sitewill depend in part on the concentrations of NFAT, AP-1, and Rel proteins inthe nucleus at any given time of stimulation, and in part on relative affinitiesof these proteins for the site in question. Since NFAT proteins are activatedprimarily by calcium-mobilizing stimuli, whereas Rel proteins are activatedby many types of stimuli (159, 160), the cell type and mode of stimulationemployed will be crucial. Bound NFAT and Rel proteins may have similar orantagonistic effects on promoter function.

An interesting example of an interaction between NFAT and NF-κB is pro-vided by their competition on the P sequence of the human IL-4 promoter(TGGAAATTTTC, Table 6), which binds both NFAT and NF-κB (161). Thecorresponding P1 sequence in the mouse IL-4 promoter (TGGAAAATTTT,Table 6) differs by two nucleotides from the human sequence, and hence bindsNFAT with fourfold higher affinity and NF-κB with fourfold lower affinity thandoes the human sequence. In consequence, the activity of the human IL-4 pro-moter was repressed by PMA stimulation and by coexpression of RelA, whilethat of the mouse IL-4 promoter was not. Moreover, substitution of the mousesequence for the human P element eliminated the sensitivity of the human IL-4promoter to PMA stimulation and to Rel A (161), consistent with the proposedcompetition between NFAT and NF-κB for binding to the P site.

ROLE OF NFAT PROTEINS IN GENE TRANSCRIPTION

Multiple NFAT Binding Sites in Gene Regulatory RegionsA notable feature of NFAT-dependent promoters and enhancers is the presenceof multiple NFAT binding sites. This theme, first recognized in the IL-2 (162,

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163) and IL-4 (164, 165) promoters, has since been extended to the GM-CSFenhancer (141), two more sites in the IL-2 promoter (112), the TNF-α promoter(137), and a newly identified enhancer located 14-kb upstream of the IL-3 gene(P Cockerill, personal communication). Each of these regulatory regions con-tains three to five sites for NFAT binding, within a total length of 200 to 300base pairs. The implication is that higher-order, synergistic interactions amongNFAT-containing complexes are required for effective transcription. A similarhypothesis was proposed based on studies of reporter expression, using con-structs driven by multiple NFAT:AP-1 sites (71, 166). T cells stably expressingsuch constructs showed reporter expression in only a proportion of stimulatedcells, and changes in the strength or effectiveness of the stimulus changed thenumber of expressing cells rather than the level of reporter expression, suggest-ing that NFAT-dependent transcription was initiated in the stimulated cells onlywhen they attained a threshold level of nuclear NFAT proteins that permittedtandem occupancy of multiple sites. In vivo footprinting of the IL-2 promotersuggested that these long-range interactions were not limited to NFAT, but in-volved the synergistic assembly of all the relevant transcription factors into acoordinate promoter complex (167). Indeed, transcriptional activation in manysystems requires the ordered assembly of a cooperative transcription complex,containing transcription factors, coactivators, and the core transcriptional ma-chinery (168).

Cell Specificity of Cytokine Gene ExpressionEach cell type in the immune system produces a characteristic pattern of cy-tokines during an immune response (169). For instance, IL-2 is produced by Tcells, IL-4 by T cells and mast cells, IFN-γ by T cells and NK cells, and TNF-αby virtually every cell type. Other inducible genes such as CD40L, CTLA-4,and FasL are also selectively expressed. The cytokine profiles of T cells dif-ferentiating down the Th1 and Th2 pathways have been well documented; Th1cells preferentially produce IL-2 and IFN-γ , whereas Th2 cells produce IL-4,IL-5, IL-10, and IL-13 (169–171). In many pathological situations, the balancebetween Th1 and Th2 patterns of cytokine production determines the clinicaloutcome of the immune response (103, 170).

The proto-oncogene c-Maf, a member of the bZIP family, is held to beresponsible for the selective expression of IL-4 in Th2 cells (152). Within theT cell lineage, c-Maf is expressed in Th2 but not in Th1 cells. Coexpressionof c-Maf and NFAT1 in Th1 cells and B cells results in strong synergisticactivation of the IL-4 promoter, reflecting their interaction with the P0 NFATsite and an adjacent c-Maf binding site. As discussed above, the orientation andspacing of the NFAT and Maf-binding sites within this region suggest that theNFAT:Maf interaction differs substantially from the conventional NFAT:AP-1

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interaction, and protein-protein interactions between NFAT and c-Maf remainto be demonstrated. If the interactions are in fact cooperative, it is likely that theprotein-protein contacts involved will differ from those observed on compositeNFAT:AP-1 sites.

What determines the cell-specific expression of inducible genes? As notedabove, the sequences of the NFAT sites and their arrangement relative to sitesfor other transcription factors show considerable variability. For instance, theinteraction of NFAT and Oct proteins differs on the IL-2 and IL-4 promoters,being cooperative in the former case and competitive in the latter (172); bothNF-κB and NFAT:AP-1 are required for the function of the human GM-CSFpromoter (59); and GATA as well as NFAT proteins have been implicated inIL-5 gene induction (173–175). Selective gene expression is likely to be de-termined by the particular NFAT proteins and cooperating transcription factorsexpressed in individual cell types, or by cell-specific nuclear coactivators (orcorepressors) that mediate interactions between these transcription factors andthe basal transcription complex.

Target Genes for NFAT Proteins: Lessonsfrom NFAT1-Deficient MiceThe specialized functions of NFAT1 in the immune response have been exploredby targeted disruption of the NFAT1 gene (73, 176). In both cases the targetedexon was in the DNA-binding domain, and its disruption resulted either in theexpression of a deleted version of the protein without DNA-binding activity(176), or in no protein expression (null phenotype) (73). Except for a moderatedegree of splenomegaly, the NFAT1-deficient mice developed normally andwere immunocompetent, as they showed no impairment in IL-2, IL-4, TNF-α,and IFN-γ production by stimulated spleen cells. Surprisingly, however, certainprimary and secondary immune responses were markedly enhanced, with atendency toward the development of a late Th2-type response in at least threeexperimental situations: increased intrapleural accumulation of eosinophils andincreased serum IgE levels in an in vivo model of allergic inflammation (73);increased serum IgE levels in response to immunization with TNP-ovalbumin(176); and more efficient differentiation toward a Th2 phenotype in spleen cellsstimulated in vitro with IL-4 and anti-CD3 (176).

Given the evidence that NFAT proteins are essential transcription factors forthe expression of cytokine genes, the overall immunocompetence of NFAT1-deficient mice suggests that many genes that are targets for NFAT1 may beredundantly controlled by another NFAT protein. However, the unusual pheno-type of enhanced immune responsiveness is consistent with the possibility thatsome genes are unique or preferred targets for NFAT1. In normal mice, activa-tion of NFAT1 may elevate the expression of inhibitory cytokines or cell-surface

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receptors in certain cells, or decrease the expression of activating effector pro-teins, thus dampening the overall magnitude of the immune response and per-haps limiting the late expression of Th2-type cytokines. By analogy, proteinkinase C (PKC), which is activated early in stimulated T cells, has both positiveeffects and feedback inhibitory effects on the subsequent response: It modifiesand activates the positive regulators Ras and Raf but also decreases [Ca2+]i byinhibiting phospholipase C-γ and activating plasma membrane calcium pumps(131). Alternative explanations for the phenotype of NFAT1-deficient mice arethat changes in early cytokine production (176) alter the balance of cytokinesproduced at later times, thus skewing the overall pattern of the response; or thatunless counterbalanced by the presence of NFAT1, the other NFAT proteinswill favor transcription of the cytokine genes characteristic of Th2 cells.

Given the fact that NFAT proteins or their mRNAs have been detected outsidethe immune system, some target genes for NFAT1 and other NFAT proteins arelikely to be expressed in nonlymphoid cells. For instance, mRNAs encodingall four NFAT proteins are expressed in testis and/or ovary and in skeletalor cardiac muscle, suggesting a possible involvement in reproduction and inmuscle development or function. New members of the NFAT family may alsobe discovered, with presently unsuspected roles.

FUTURE DIRECTIONS

The molecular characterization of NFAT proteins has greatly advanced ourunderstanding of inducible gene transcription in the immune system. How-ever, the basis for the cell-specific expression of genes induced via the recep-tor/calcium/calcineurin pathway remains to be understood. This will involvedefining the functions of individual NFAT proteins, both biochemically andby targeted gene disruption; exploring the mechanisms by which calcineurin,kinases, and other signaling molecules regulate the phosphorylation status,subcellular localization and transactivation functions of NFAT proteins; anddetermining whether NFAT-mediated transcription involves cooperation withcell-specific transcription factors or coactivator proteins. Other questions arewhether NFAT proteins play a role in other biological and immune processes,such as lymphocyte development and programmed cell death; or in diseaseprocesses such as immunodeficiency or autoimmune diseases or oncogenesis.

ACKNOWLEDGMENTS

We thank the many colleagues who generously provided us with unpublishedinformation and discussed their research with us, and we apologize sincerelyto those whose work we have overlooked or omitted to cite because of spacelimitations. We are grateful to all the members of our laboratories for their

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contributions to this review, particularly Dr. J Aramburu for help with Figure 2.Work in the A.R. and P.G.H. laboratories was supported by grants from theNational Institutes of Health and Hoffmann-La Roche, Inc.

Visit the Annual Reviews home pageathttp://www.annurev.org.

Literature Cited

1. Rao A. 1994. NF-ATp: a transcription fac-tor required for the co-ordinate inductionof several cytokine genes.Immunol. To-day15:274–81

2. Crabtree GR, Clipstone NA. 1994. Signaltransmission between the plasma mem-brane and nucleus of T-lymphocytes.Annu. Rev. Biochem.63:1045–83

3. Jain J, Loh C, Rao A. 1995. Transcrip-tional regulation of the interleukin 2 gene.Curr. Opin. Immunol.7:333–42

4. Serfling E, Avots A, Neumann M. 1995.The architecture of the interleukin-2promoter: a reflection of T lympho-cyte activation.Biochem. Biophys. Acta1263:181–200

5. Weiss A, Littman DR. 1994. Signal trans-duction by lymphocyte antigen receptors.Cell 76:263–74

6. Cantrell D. 1996. T cell antigen receptorsignal transduction pathways.Annu. Rev.Immunol.14:259–74

7. Crabtree GR. 1989. Contingent geneticregulatory events in T lymphocyte acti-vation.Science243:355–61

8. Kelly K, Siebenlist U. 1995. Immediate-early genes induced by antigen receptorstimulation.Curr. Opin. Immunol.7:327–32

9. Cockerill GW, Bert AG, Ryan GR, Gam-ble JR, Vadas MA, Cockerill PN. 1995.Regulation of granulocyte-macrophagecolony-stimulating factor and E-selectinexpression in endothelial cells by cy-closporin A and the T-cell transcriptionfactor NFAT.Blood86:2689–98

10. Leonard DG, Ziff EB, Greene LA. 1987.Identification and characterization of m-RNAs regulated by nerve growth factor inPC12 cells.Mol. Cell. Biol.7:3156–67

11. Schreiber SL, Crabtree GR. 1992. Themechanism of action of cyclosporin A andFK506.Immunol. Today13:136–42

12. Sigal NH, Dumont FJ. 1992. CyclosporinA, FK-506, and rapamycin: pharmaco-logic probes of lymphocyte signal trans-duction.Annu. Rev. Immunol.10:519–60

13. Sigal NH, Dumont F, Durette P, Siekierka

JJ, Peterson L, Rich DH, Dunlap BE,Staruch MJ, Melino MR, Koprak SL,Williams D, Witzel B, Pisano JM. 1991.Is cyclophilin involved in the immuno-suppressive and nephrotoxic mechanismof action of cyclosporin A?J. Exp. Med.173:619–28

14. Dumont FJ, Staruch MJ, Koprak SL,Siekierka JJ, Lin CS, Harrison R, SewellT, Kindt VM, Beattie TR, Wyvratt M,Sigal NH. 1992. The immunosuppressiveand toxic effects of FK-506 are mechanis-tically related: pharmacology of a novelantagonist of FK-506 and rapamycin.J.Exp. Med.176:751–60

15. Liu J. 1993. FK506 and cyclosporin,molecular probes for studying intracellu-lar signal transduction.Immunol. Today14:290–9

16. Kincaid RL, O’Keefe SJ. 1993. Cal-cineurin and immunosuppression: acalmodulin-dependent protein phos-phatase acts as the “gatekeeper” tointerleukin-2 gene transcription.Adv.Protein Phosphatases7:543–83

17. Bierer BE, Hollander G, Fruman D,Burakoff SJ. 1993. Cyclosporin A andFK506: molecular mechanisms of im-munosuppression and probes for trans-plantation biology.Curr. Opin. Immunol.5:763–73

18. Wiederrecht G, Etzkorn F. 1994. The im-munophilins. Perspec. Drug DiscoveryDesign2:57–84

19. Masuda ES, Naito Y, Arai K-I, Arai N.1993. Expression of lymphokine genes inT cells.The Immunologist1:198–203

20. Shaw J-P, Utz PJ, Durand DB, Toole JJ,Emmel EA, Crabtree GR. 1988. Identifi-cation of a putative regulator of early Tcell activation genes.Science241:202–5

21. Mattila PS, Ullman KS, Fiering S,Emmel EA, McCutcheon M, CrabtreeGR, Herzenberg LA. 1990. The action ofcyclosporin A and FK506 suggest a novelstep in the activation of T lymphocytes.EMBO J.9:4425–33

22. Flanagan MW, Corth´esy B, Bram RJ,

Ann

u. R

ev. I

mm

unol

. 199

7.15

:707

-747

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y U

nive

rsity

of

Wis

cons

in -

Milw

auke

e on

12/

04/1

7. F

or p

erso

nal u

se o

nly.




Crabtree GR. 1991. Nuclear associationof a T-cell transcription factor blockedby FK-506 and cyclosporin A.Nature352:803–7

23. Jain J, McCaffrey PG, Miner Z, KerppolaTK, Lambert JN, Verdine GL, Curran T,Rao A. 1993. The T-cell transcription fac-tor NFATp is a substrate for calcineurinand interacts with Fos and Jun.Nature365:352–55

24. McCaffrey PG, Luo C, Kerppola TK, JainJ, Badalian TM, Ho AM, Burgeon E, LaneWS, Lambert J, N., Curran T, Verdine GL,Rao A, Hogan PG. 1993. Isolation of thecyclosporin-sensitive T cell transcriptionfactor NFATp.Science262:750–54

25. Northrop JP, Ho SN, Chen L, ThomasDJ, Timmerman LA, Nolan GP, AdmonA, Crabtree GR. 1994. NF-AT compo-nents define a family of transcription fac-tors targeted in T-cell activation.Nature369:497–502

26. Tokumitsu H, Masuda ES, Tsuboi A, AraiK-I, Arai N. 1993. Purification of the 120kDa component of the human nuclear fac-tor of activated T cells (NF-AT).Biochem.Biophys. Res. Comm.196:737–44

27. Jain J, Miner Z, Rao A. 1993. Analysisof the preexisting and nuclear forms ofnuclear factor of activated T cells.J. Im-munol.151:837–48

28. McCaffrey PG, Perrino BA, SoderlingTR, Rao A. 1993. NF-ATp, a T lympho-cyte DNA-binding protein that is a tar-get for calcineurin and immunosuppres-sive drugs.J. Biol. Chem.268:3747–52

29. Northrop JP, Ullman KS, Crabtree GR.1993. Characterization of the nuclearand cytoplasmic components of thelymphoid-specific nuclear factor of acti-vated T cells (NF-AT) complex.J. Biol.Chem.268:2917–23

30. Luo C, Burgeon E, Carew JA, BadalianTM, McCaffrey PG, Lane WS, HoganPG, Rao A. 1996. Recombinant NFAT1(NFATp) is regulated by calcineurin inT cells and mediates the transcription ofseveral cytokine genes.Mol. Cell. Biol.16:3955–66

31. Hoey T, Sun Y-L, Williamson K, XuX. 1995. Isolation of two new membersof the NFAT gene family and functionalcharacterization of the NFAT proteins.Immunity2:461–72

32. Masuda E, Naito Y, Tokumitsu H, Camp-bell D, Saito F, Hannum C, Arai K-I, AraiN. 1995. NFATx, a novel member of theNFAT family that is expressed predom-inantly in the thymus.Mol. Cell. Biol.15:2697–706

33. Ho SN, Thomas DJ, Timmerman LA,

Li X, Francke U, Crabtree GR. 1995.NFATc3, a lymphoid-specific NFATcfamily member that is calcium-regulatedand exhibits distinct DNA binding speci-ficity. J. Biol. Chem.270:19898–907

34. Park J, Takeuchi A, Sharma S. 1996.Characterization of a new isoform of theNFAT (nuclear factor of activated T cells)gene family member NFATc.J. Biol.Chem.34:29014–21

35. Weiss MJ, Orkin SH. 1995. GATA tran-scription factors: key regulators of hema-topoiesis.Exp. Hematol.23:99–107

36. Schindler C, Darnell JE, Jr. 1995.Transcriptional responses to polypeptideligands: the JAK-STAT pathway.Annu.Rev. Biochem.64:621–51

37. Jain J, Burgeon E, Badalian TM, HoganPG, Rao A. 1995. A similar DNA-bindingmotif in NFAT family proteins and theRel homology region.J. Biol. Chem.270:4138–45

38. Nolan GP. 1994. NF-AT-AP-1 and Rel-bZIP: hybrid vigor and binding under theinfluence.Cell 77:795–98

39. Chytil M, Verdine GL. 1996. The Relfamily of eukaryotic transcription factors.Curr. Opin. Struct. Biol.6:91–100

40. Luo C, Shaw KTY, Raghavan A, Aram-buru J, Garcia-Cozar F, Perrino BA,Hogan PG, Rao A. 1996. Interaction ofcalcineurin with a domain of the tran-scription factor NFAT1 that controls nu-clear import.Proc. Natl. Acad. Sci. USA93:8907–12

41. Luo C, Burgeon E, Rao A. 1996. Mech-anisms of transactivation by nuclear fac-tor of activated T cells-1.J. Exp. Med.184:141–47

42. Wang DZ, McCaffrey PG, Rao A. 1995.The cyclosporin-sensitive transcriptionfactor NFATp is expressed in severalclasses of cells in the immune system.Ann. N. Y. Acad. Sci.766:182–94

43. Ruff VA, Leach KL. 1995. Direct demon-stration of NFATp dephosphorylation andnuclear localization in activated HT-2cells using a specific NFATp polyclonalantibody.J. Biol. Chem.270:22602–7

44. Aramburu J, Azzoni L, Rao A, PerussiaB. 1995. Activation and expression of thenuclear factor of activated T cells, NFATpand NFATc, in human natural killer cells:regulation upon CD16 ligand binding.J.Exp. Med.182:801–10

45. Ho AM, Jain J, Rao A, Hogan PG.1994. Expression of the transcription fac-tor NFATp in a neuronal cell line and inthe murine nervous system.J. Biol. Chem.269:28,181–86

46. Rutherford SL, Zuker CS. 1994. Protein

Ann

u. R

ev. I

mm

unol

. 199

7.15

:707

-747

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y U

nive

rsity

of

Wis

cons

in -

Milw

auke

e on

12/

04/1

7. F

or p

erso

nal u

se o

nly.




folding and the regulation of signalingpathways.Cell 79:1129–32

47. Woronicz JD, Lina A, Calnan BJ, Szy-chowski S, Cheng L, Winoto A. 1995.Regulation of thr Nur77 orphan steroidreceptor in activation-induced apoptosis.Mol. Cell. Biol.15:6364–76

48. Wesselborg S, Fruman DA, Sagoo JK,Bierer BE, Burakoff SJ. 1996. Identifica-tion of a physical interaction between cal-cineurin and nuclear factor of activated Tcells (NFATp).J. Biol. Chem.271:1274–77

49. Park J, Yaseen NR, Hogan PG, RaoA, Sharma S. 1995. Phosphorylation ofthe transcription factor NFATp inhibitsits DNA binding activity in cyclosporinA-treated human B and T cells.J. Biol.Chem.270:20653–59

50. Shaw KT-Y, Ho AM, Raghavan A, KimJ, Jain J, Park J, Sharma S, Rao A, HoganPG. 1995. Immunosuppressive drugs pre-vent a rapid dephosphorylation of thetranscription factor NFAT1 in stimulatedimmune cells.Proc. Natl. Acad. Sci. USA92:11205–9

51. Loh C, Shaw KT-Y, Carew J, Viola JPB,Luo C, Perrino BA, Rao A. 1996. Cal-cineurin binds the transcription factorNFAT1 and reversibly regulates its activ-ity. J. Biol. Chem.271:10884–91

52. Loh C, Carew JA, Kim J, Hogan PG, RaoA. 1996. T-cell receptor stimulation elic-its an early phase of activation and a laterphase of deactivation of the transcriptionfactor NFAT1.Mol. Cell. Biol. 16:3945–54

53. Shibasaki F, Price ER, Milan D, McKeonF. 1996. Role of kinases and the phos-phatase calcineurin in the nuclear shut-tling of transcription factor NF-AT4.Na-ture382:370–73

54. O’Keefe SJ, Tamura J, Kincaid RL,Tocci MJ, O’Neill EA. 1992. FK-506- and CsA-sensitive activation of theinterleukin-2 promoter by calcineurin.Nature357:692–94

55. Clipstone NA, Crabtree GR. 1992. Iden-tification of calcineurin as a key signalingenzyme in T-lymphocyte activation.Na-ture357:695–97

56. Kubo M, Kincaid RL, Webb DR, Ran-som JT. 1994. The Ca2+/calmodulin-activated, phosphoprotein phosphatasecalcineurin is sufficient for positive tran-scriptional regulation of the mouse IL-4gene.Int. Immunol.6:179–88

57. Goldfeld AE, Tsai E, Kincaid R, BelshawPJ, Schreiber SL, Strominger JL, Rao A.1994. Calcineurin mediates human tumor

necrosis factorα gene induction in stimu-lated T and B cells.J. Exp. Med.180:763–68

58. Tsuboi A, Masuda ES, Naito Y, Tokumi-tsu H, Arai K-I, Arai N. 1994. Calcineurinpotentiates activation of the granulocyte-macrophage colony-stimulating factorgene in T cells: involvement of the con-served lymphokine element 0.Mol. Biol.Cell 5: 119–28

59. Jenkins F, Cockerill PN, Bohmann D,Shannon MF. 1995. Multiple signalsare required for function of the hu-man granulocyte-macrophage colony-stimulating factor gene promoter in Tcells.J. Immunol.155:1240–51

60. Fruman DA, Pai S-Y, Burakoff SJ, BiererBE. 1995. Characterization of a mu-tant calcineurin Aα gene expressed byEL4 lymphoma cells.Mol. Cell. Biol.15:3857–63

61. Muramatsu T, Kincaid RL. 1996. In-hibition of NF-AT signal transductionevents by a dominant-negative form ofcalcineurin.Biochem. Biophys. Res. Com-mun.218:466–72

62. Zhang W, Zimmer G, Chen J, Ladd D,Li E, Alt FW, Wiederrecht G, Cryan J,O’Neill EA, Seidman CE, Abbas AK, Sei-dman JG. 1996. T cell responses in cal-cineurin Aα-deficient mice.J. Exp. Med.183:413–20

63. Putney JWJ. 1990. Capacitative calciumentry revisited.Cell Calcium11:611–24

64. Clapham DE. 1995. Calcium signaling.Cell 80:259–68

65. Lewis RS, Cahalan MD. 1995. Potas-sium and calcium channels in lympho-cytes.Annu. Rev. Immunol.13:623–53

66. Boss V, Talpade DJ, Murphy TJ. 1996.Induction of NFAT-mediated transcrip-tion by Gq-coupled receptors in lymphoidand non-lymphoid cells.J. Biol. Chem.271:10429–32

67. Mason MJ, Grinstein G. 1993. Ionomycinactivates electrogenic Ca2+ influx in ratthymic lymphocytes.Biochem. J.296:33–39

68. Morgan AJ, Jacob R. 1994. Ionomcyin en-hances Ca2+ influx by stimulating store-regulated cation entry and not by a directaction at the plasma membrane.Biochem.J. 300:665–72

69. Serafini AT, Lewis RS, Clipstone NA,Bram RJ, Fanger C, Fiering S, Herzen-berg LA, Crabtree GR. 1995. Isolation ofmutant T lymphocytes with defects in ca-pacitative calcium entry.Immunity3:239–50

70. Fanger CM, Hoth M, Crabtree GR, Lewis

Ann

u. R

ev. I

mm

unol

. 199

7.15

:707

-747

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y U

nive

rsity

of

Wis

cons

in -

Milw

auke

e on

12/

04/1

7. F

or p

erso

nal u

se o

nly.




RS. 1995. Characterization of T cell mu-tants with defects in capacitative calciumentry: genetic evidence for the physiolog-ical roles of CRAC channels.J. Cell Biol.131:655–67

71. Negulescu PA, Shastri N, Cahalan MD.1994. Intracellular calcium dependenceof gene expression in single T lympho-cytes. Proc. Natl. Acad. Sci. USA91:2873–77

72. Mulkey RM, Endo S, Shenolikar S,Malenka RC. 1994. Involvement of acalcineurin/inhibitor-1 phosphatase cas-cade in hippocampal long-term depres-sion.Nature369:486–88

73. Xanthoudakis S, Viola JPB, Shaw KTY,Luo C, Wallace JD, Bozza PT, Luk DC,Curran T, Rao A. 1996. An enhanced im-mune response in mice lacking the trans-cription factor NFAT1.Science272:892–95

74. Ghosh P, Sica A, Cippitelli M, Subleski J,Lahesmaa R, Young HA, Rice NR. 1996.Activation of nuclear factor of activated Tcells in a cyclosporin A-resistant pathway.J. Biol. Chem.271:7700–4

75. Hubbard MJ, Cohen P. 1993. On targetwith a new mechanism for the regula-tion of protein phosphorylation.TrendsBiochem. Sci.18:172–77

76. Cameron AM, Steiner JP, Roskams AJ,Ali SM, Ronnett GV, Snyder SH. 1995.Calcineurin associated with the inosi-tol 1,4,5-trisphophate receptor-FKBP12complex modulates Ca2+ flux. Cell 83:463–72

77. Coghlan VM, Perrino BA, Howard M,Langeberg LK, Hicks JB, Gallatin WM,Scott JD. 1995. Association of proteinkinase A and protein phosphatase 2Bwith a common anchoring protein.Sci-ence267:108–11

78. Faux MC, Scott JD. 1996. Molecularglue: kinase anchoring and scaffold pro-teins.Cell 85:9–12

79. James P, Vorherr T, Carafoli E. 1995.Calmodulin-binding domains: just twofaced or multi-faceted?Trends Biochem.Sci.20:38–42

80. Kissinger CR, Parge HE, Knighton DR,Lewis CT, Pelletier LA, Tempczyk A,Kalish VJ, Tucker KD, Showalter RE,Moomaw EW, Gastinel LN, HabukaN, Chen X, Maldonado F, Barker JE,Bacquet R, Villafranca JE. 1995. Crys-tal structures of human calcineurin andthe human FKPB12-FK506-calcineurincomplex.Nature378:641–44

81. Bolen JB. 1995. Protein tyrosine ki-nases in the initiation of antigen receptor

signaling.Curr. Opin. Immunol.7:306–11

82. Howe LR, Weiss A. 1995. Multiple ki-nases mediate T-cell-receptor signaling.Trends Biochem. Sci.20:59–64

83. Carrera AC, Paradis H, Borlado LR,Roberts TM, Martinez-A C. 1995. Lckunique domain influences Lck specificityand biological function.J. Biol. Chem.270:3385–91

84. Qian D, Mollenauer MN, Weiss A. 1996.Dominant-negative Zeta-associated pro-tein 70 inhibits T cell antigen receptor sig-naling.J. Exp. Med.183:611–20

85. Katzav S, Sutherland M, Packham G, YiT, Weiss A. 1994. The protein tyrosinekinase ZAP-70 can associate with theSH2 domain of proto-Vav.J. Biol. Chem.269:32579–85

85a. Wu J, Motto DG, Koretzky GA, Weiss A.1996. Vav and SLP-76 interact and func-tionally cooperate in IL-2 gene activation.Immunity4:593–602

86. Wu J, Katzav S, Weiss A. 1995. A func-tional T-cell receptor signaling pathwayis required for p95vav activity.Mol. Cell.Biol. 15:4337–46

87. Woodrow M, Clipstone NA, Cantrell D.1993. p21ras and calcineurin synergize toregulate the nuclear factor of activated Tcells.J. Exp. Med.178:1517–22

88. Genot E, Cleverley S, Henning S, CantrellD. 1996. Multiple p21ras effector path-ways regulate nuclear factor of activatedT cells.EMBO J.15:3912–22

89. Coso OA, Chiariello M, Yu J-C, TeramotoH, Crespo P, Xu N, Miki T, Gutkind JS.1995. The small GTP-binding proteinsRac1 and Cdc42 regulate the activity ofthe JNK/SAPK signaling pathway.Cell81:1137–46

90. Coso OA, Teramoto H, Simonds WF,Gutkind JS. 1996. Signaling from Gprotein-coupled receptors to c-Jun ki-nase involves βγ subunits of het-erotrimeric G proteins acting on a Ras andRac1-dependent pathway.J. Biol. Chem.271:3963–66

91. Hill CS, Wynne J, Treisman R. 1995. TheRho family GTPases RhoA, Rac1, andCDC42Hs regulate trnscriptional activityby SRF.Cell 81:1159–70

92. Minden A, Lin A, Claret F-X, Abo A,Karin M. 1995. Selective activation of theJNK signaling cascade and c-Jun tran-scriptional activity by the small GTPasesRac and Cdc42Hs.Cell 81:1147–57

93. Olson MF, Ashworth A, Hall A. 1995. Anessential role for Rho, Rac, and Cdc42GTPases in cell cycle progression throughG1.Science269:1270–72

Ann

u. R

ev. I

mm

unol

. 199

7.15

:707

-747

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y U

nive

rsity

of

Wis

cons

in -

Milw

auke

e on

12/

04/1

7. F

or p

erso

nal u

se o

nly.




94. Qiu R-G, Chen J, Kirn D, McCormickF, Symons M. 1995. An essential rolefor Rac in Ras transformation.Nature374:457–59

95. Su B, Karin M. 1996. Mitogen-activatedprotein kinase cascades and regulation ofgene expression.Curr. Opin. Immunol.8:402–11

96. Whitehurst CE, Geppert TD. 1996.MEK1 and the extracellular signal-regulated kinases are required for thestimulation of IL-2 gene transcription inT cells.J. Immunol.156:1020–29

97. Nghiem P, Ollick T, Gardner P, Schul-man H. 1994. Interleukin-2 trans-criptional block by multifunctionalCa2+/calmodulin kinase.Nature 371:347–50

98. Hama N, Paliogianni F, Fessler BJ,Boumpas DT. 1995. Calcium/calmo-dulin-dependent protein kinase II down-regulates both calcineurin and protein ki-nase C-mediated pathways for cytokinegene transcription in human T cells.J.Exp. Med.181:1217–22

99. Ho N, Gullberg M, Chatila T. 1996. Ac-tivation protein 1-dependent transcrip-tional activation of interleukin 2 gene byCa2+/calmodulin kinase type IV/Gr.J.Exp. Med.184:101–12

100. Miranti CK, Ginty DD, Huang G, ChatilaT, Greenberg ME. 1995. Calcium ac-tivates serum response factor-dependenttranscription by a Ras- and Elk-1-independent mechanism that involves aCa2+/calmodulin-dependent kinase.Mol.Cell. Biol.15:3672–84

101. Schulman H. 1993. The multifunctionalCa2+/calmodulin-dependent protein ki-nases.Curr. Opin. Cell Biol.5:247–53

102. Bram RJ, Crabtree GR. 1994. Calciumsignalling in T cells stimulated by acyclophilin B-binding protein. Nature371:355–58

103. Lenschow DJ, Walunas TL, Bluestone JA.1996. CD28/B7 system of T cell costim-ulation.Annu. Rev. Immunol.14:233–58

104. Thompson CB, Lindsten T, LedbetterJA, Kunkel SL, Young HA, EmersonSG, Leiden JM, June CH. 1989. CD28activation pathway regulates the pro-duction of multiple T-cell-derived lym-phokines/cytokines.Proc. Natl. Acad.Sci. USA86:1333–37

105. Lindsten T, June CH, Ledbetter JA, StellaG, Thompson CB. 1989. Regulation oflymphokine messenger RNA stability bya surface-mediated T cell activation path-way.Science244:339–43

106. Umlauf SW, Beverly B, Lantz O,

Schwartz RH. 1995. Regulation of inter-leukin 2 gene expression by CD28 cos-timulation in mouse T-cell clones: bothnuclear and cytoplasmic RNAs are reg-ulated with complex kinetics.Mol. Cell.Biol. 15:3197–205

107. Fraser JD, Irving BA, Crabtree GR, WeissA. 1991. Regulation of interleukin-2 geneenhancer activity by the T cell accessorymolecule CD28.Science251:313–16

108. Fraser JD, Weiss A. 1992. Regulation ofT-cell lymphokine gene transcription bythe accessory molecule CD28.Mol. Cell.Biol. 12:4357–63

109. Ghosh P, Tan T-H, Rice NR, Sica A,Young HA. 1993. The interleukin 2CD28-responsive complex contains atleast three members of the NFκB family:c-Rel, p50, and p65.Proc. Natl. Acad. Sci.USA90:1696–700

110. Bryan RG, Li Y, Lai J-H, Van M, RiceNR, Rich RR, Tan T-H. 1994. Effect ofCD28 signal transduction on c-Rel in hu-man peripheral blood T cells.Mol. Cell.Biol. 14:7933–42

111. Lai J-H, Horvath G, Subleski J, Bruder J,Ghosh P, Tan T-H. 1995. RelA is a po-tent transcriptional activator of the CD28response element within the interleukin 2promoter.Mol. Cell. Biol.15:4260–71

112. Rooney JW, Sun Y-L, Glimcher LH, HoeyT. 1995. Novel NFAT sites that mediateactivation of the interleukin-2 promoterin response to T-cell receptor stimulation.Mol. Cell. Biol.15:6299–310

113. Good L, Maggirwar SB, Sun S-C. 1996.Activation of the IL-2 gene promoter byHTLV-1 Tax involves induction of NF-ATcomplexes bound to the CD28-responsiveelement.EMBO J.15:3744–50

114. Civil A, Bakker A, Rensink I, Doerre S,Aarden LA, Verweij CL. 1996. Nuclearappearance of a factor that binds the CD28response element within the interleukin-2 enhancer correlates with interleukin-2production.J. Biol. Chem.271:8321–27

115. Himes SR, Katsikeros R, Shannon MF.1996. Constimulation of cytokine geneexpression in T cells by the human Tleukemia/lymphotropic virus type 1 transactivator Tax.J. Virol. 70:4001–8

116. June CH, Bluestone JA, Nadler LM,Thompson CB. 1994. The B7 andCD28 receptor families.Immunol. Today15:321–31

117. Nunes JA, Collette Y, Truneh A, OliveD, Cantrell DA. 1994. The role of p21ras

in CD28 signal transduction: triggeringof CD28 with antibodies, but not the lig-and B7–1, activates p21ras. J. Exp. Med.180:1067–76

Ann

u. R

ev. I

mm

unol

. 199

7.15

:707

-747

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y U

nive

rsity

of

Wis

cons

in -

Milw

auke

e on

12/

04/1

7. F

or p

erso

nal u

se o

nly.




118. June CH, Ledbetter JA, Gillespie MM,Lindsten T, Thompson CB. 1987. T-cellproliferation involving the CD28 pathwayis associated with cyclosporine-resistantinterleukin 2 gene expression.Mol. Cell.Biol. 7:4472–81

119. Chen D, Rothenberg EV. 1994. Inter-leukin-2 transcription factors as molec-ular targets of cAMP inhibition: de-layed inhibition kinetics and combina-torial transcription roles.J. Exp. Med.179:931–42

120. Lacour M, Arrighi J-F, M¨uller KM, Carl-berg C, Saurat J-H, Hauser C. 1994.cAMP up-regulates IL-4 and IL-5 produc-tion from activated CD4+ T cells whiledecreasing IL-2 release and NF-AT induc-tion. Int. Immunol.6:1333–43

121. Neumann M, Grieshammer T, ChuvpiloS, Kneitz B, Lohoff M, Schimpl A, FranzaBR, Jr., Serfling E. 1995. RelA/p65 is amolecular target for the immunosuppres-sive action of protein kinase A.EMBO J.14:1991–2004

122. Tsuruta L, Lee H-J, Masuda ES, Koyano-Nakagawa N, Arai N, Arai K-I, Yokota T.1995. Cyclic AMP inhibits expression ofthe IL-2 gene through the nuclear factor ofactivated T cells (NF-AT) site, and trans-fection of NF-AT cDNAs abrogates thesensitivity of EL-4 cells to cyclic AMP.J.Immunol.154:5255–64

123. Paliogianni F, Boumpas DT. 1996.Prostaglandin E2 inhibits the nuclear tran-scription of the human interleukin 2, butnot the IL-4, gene in human T cells bytargeting transcription factors AP-1 andNF-AT. Cell. Immunol.171:95–101

124. Vacca A, Felli MP, Farina AR, MartinottiS, Maroder M, Screpanti I, Meco D,Petrangeli E, Frati L, Gulino A. 1992.Glucocorticoid receptor-mediated sup-pression of the interleukin 2 gene expres-sion through impairment of the coopera-tivity between nuclear factor of activatedT cells and AP-1 enhancer elements.J.Exp. Med.175:637–46

125. Northrop JP, Crabtree GR, Mattila PS.1992. Negative regulation of interleukin2 transcription by the glucocorticoid re-ceptor.J. Exp. Med.175:1235–45

126. Paliogianni F, Raptis A, Ahuja SS, NajjarSM, Boumpas DT. 1993. Negative tran-scriptional regulation of human inter-leukin 2 (IL-2) gene by glucocorticoidsthrough interference with nuclear tran-scription factors AP-1 and NF-AT.J. Clin.Invest.91:1481–89

127. Brabletz T, Pfeuffer I, Schorr E, Siebelt F,Wirth T, Serfling E. 1993. Transforminggrowth factorβ and cyclosporin A inhibit

the inducible activity of the interleukin-2 gene in T cells through a noncanoni-cal octamer-binding site.Mol. Cell. Biol.13:1155–62

128. Felli MP, Vacca A, Meco D, ScrepantiI, Farina AR, Maroder M, Martinotti S,Petrangeli E, Frati L, Gulino A. 1991.Retinoic acid-induced down-regulationof the interleukin-2 promoter viacis-regulatory sequences containing an oc-tamer motif. Mol. Cell. Biol. 11:4771–78

129. Alroy I, Towers TL, Freedman LP.1995. Transcriptional repression of theinterleukin-2 gene by vitamin D3: directinhibition of NFATp/AP-1 complex for-mation by a nuclear hormone receptor.Mol. Cell. Biol.15:5789–99

130. Novak TJ, Chen D, Rothenberg EV. 1990.Interleukin-1 synergy with phosphoinosi-tide pathway agonists for induction ofinterlekin-2 gene expression: molecularbasis of costimulation.Mol. Cell. Biol.10:6325–34

131. Rao A. 1991. Signaling mechanisms in Tcells.Critical Rev. Immunol.10:495–519

132. Barton K, Muthusamy N, ChanyangamM, Fischer C, Clendenin C, Leiden JM.1996. Defective thymocyte proliferationand IL-2 production in transgenic miceexpressing a dominant-negative form ofCREB.Nature379:81–85

133. Brindle F, Nakajima T, Montminy M.1995. Multiple protein kinase A-regulatedevents are required for transcriptional in-duction by cAMP.Proc. Natl. Acad. Sci.USA92:10,521–25

134. Muller CW, Rey FA, Sodeoka M, VerdineGL, Harrison SC. 1995. Structure of theNF-κB p50 homodimer bound to DNA.Nature373:311–17

135. Ghosh G, Van Duyne G, Ghosh S, SiglerPB. 1995. Structure of NF-κB p50 ho-modimer bound to aκB site. Nature373:303–10

136. McCaffrey PG, Goldfeld AE, Rao A.1994. The role of NFATp in cyclosporinA-sensitive tumor necrosis factor-α genetranscription.J. Biol. Chem.269:30445–50

137. Tsai EY, Yie J, Thanos D, Goldfeld AE.1996. Cell-type specific regulation of thehuman TNF-α gene in B cells and Tcells by NFATp and ATF-2/Jun.Mol. Cell.Biol. 16:459–67

138. Cress WD, Triezenberg SJ. 1991. Criticalstructural elements of the VP16 transcrip-tional activation domain.Science251:87–90

139. Schmitz ML, dos Santos Silva MA, Alt-mann H, Czisch M, Holak TA, Baeuerle

Ann

u. R

ev. I

mm

unol

. 199

7.15

:707

-747

. Dow

nloa

ded

from

ww

w.a

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cces

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ed b

y U

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of

Wis

cons

in -

Milw

auke

e on

12/

04/1

7. F

or p

erso

nal u

se o

nly.




PA. 1994. Structural and functional anal-ysis of the NF-κB p65 C terminus.J. Biol.Chem.269:25613–20

140. Chen L, Oakley MG, Glover JNM, JainJ, Dervan PB, Hogan PG, Rao A, Ver-dine GL. 1995. Only one of the two DNA-bound orientations of AP-1 found in solu-tion cooperates with NFATp.Current Bi-ology5:882–89

141. Cockerill PN, Bert AG, Jenkins F, RyanGR, Shannon MF, Vadas MA. 1995.Human granulocyte-macrophage colony-stimulating factor enhancer function is as-sociated with cooperative interactions be-tween AP-1 and NFATp/c.Mol. Cell. Biol.15:2071–79

142. Visvader JE, Crossley M, Hill J, OrkinSH, Adams JM. 1995. The C-terminalzinc finger of GATA-1 or GATA-2 is suf-ficient to induce megakaryocyte differen-tiation of an early myeloid cell line.Mol.Cell. Biol.15:634–41

143. Tapscott SJ, Davis RL, Thayer MJ, ChengP-F, Weintraub H, Lassar AB. 1988.MyoD1: a nuclear phosphoprotein re-quiring a Myc homology region to con-vert fibroblasts to myoblasts.Science242:405–11

144. Bassuk AG, Leiden JM. 1995. A directphysical association between ETS andAP-1 transcription factors in normal hu-man T cells.Immunity3:223–37

145. Stein B, Baldwin AS Jr, Ballard DW,Greene WC, Angel P, Herrlich P. 1993.Cross-coupling of the NF-κB p65 andFos/Jun transcription factors produces po-tentiated biological function.EMBO J.12:3879–91

146. Stein B, Cogswell PC, Baldwin AS, Jr.1993. Functional and physical associa-tions between NF-κB and C/EBP familymembers: a Rel domain-bZIP interaction.Mol. Cell. Biol.13:3964–74

147. Yang-Yen H-F, Chambard J-C, Sun Y-L,Smeal T, Schmidt TJ, Drouin J, KarinM. 1990. Transcriptional interference be-tween c-Jun and the glucocorticoid recep-tor: mutual inhibition of DNA bindingdue to direct protein-protein interaction.Cell 62:1205–15

148. Su B, Jacinto E, Hibi M, Kallunki T, KarinM, Ben-Neriah Y. 1994. JNK is involvedin signal integration during costimulationof T lymphocytes.Cell 77:727–36

149. Rincon M, Flavell RA. 1994. AP-1 tran-scriptional activity requires both T-cellreceptor-mediated and co-stimulatorysignals in primary T lymphocytes.EMBOJ. 13:4370–81

150. Tsytsykova AV, Tsitsikov EN, Geha RS.1996. The CD40L promoter contains nu-

clear factor of activated T cells-bindingmotifs which require AP-1 binding foractivation of transcription.J. Biol. Chem.271:3763–70

151. Schubert LA, King G, Cron RQ, LewisDB, Aruffo A, Hollenbaugh D. 1995. Thehuman gp39 promoter.J. Biol. Chem.270:29624–27

152. Ho I-C, Hodge MR, Rooney JW,Glimcher LH. 1996. The proto-oncogenec-maf is responsible for tissue-specific ex-pression of interleukin-4.Cell 85:973–83

153. Goldfeld AE, McCaffrey PG, StromingerJL, Rao A. 1993. Identification of a novelcyclosporin-sensitive element in the hu-man tumor necrosis factorα gene pro-moter.J. Exp. Med.178:1365–79

154. Tsai EY, Jain J, Pesavento PA, Rao A,Goldfeld AE. 1996. Tumor necrosis factorα gene regulation in activated T cells in-volves ATF-2/Jun and NFATp.Mol. Cell.Biol. 16:459–67

155. Okamoto S-I, Mukaida N, YasumotoK, Rice N, Ishikawa Y, Horiguchi H,Murakami S, Matsushima K. 1994. Theinterleukin-8 AP-1 andκB-like sites aregenetic end targets of FK506-sensitivepathway accompanied by calcium mobi-lization.J. Biol. Chem.269:8582–89

156. Nabel G, Baltimore D. 1987. An inducibletranscription factor activates expressionof human immunodeficiency virus in Tcells.Science326:711–13

157. Whitley MZ, Thanos D, Read MA,Maniatis T, Collins T. 1994. A strikingsimilarity in the organization of the E-selectin and beta interferon gene promot-ers.Mol. Cell. Biol.14:6464–75

158. Civil A, Geerts M, Aarden LA, VerweijCL. 1992. Evidence for a role of CD28REas a response element for distinct mito-genic T cell activation signals.Eur. J. Im-munol.22:3041–43

159. Siebenlist U, Franzoso G, Brown K. 1994.Structure, regulation and function of NF-κB. Annu. Rev. Cell. Biol.10:405–55

160. Baldwin AS, Jr. 1996. The NF-κB andIκB proteins: new discoveries and in-sights.Annu. Rev. Immunol.14:649–81

161. Casolaro V, Georas SN, Song Z, ZubkoffID, Abdulkadir SA, Thanos D, OnoSJ. 1995. Inhibition of NF-AT-dependenttranscription by NF-κB: implications fordifferential gene expression in T helpercell subsets.Proc. Natl. Acad. Sci. USA92:11623–27

162. Randak C, Brabletz T, Hergenr¨other M,Sobotta I, Serfling E. 1990. CyclosporinA suppresses the expression of the inter-leukin 2 gene by inhibiting the binding

Ann

u. R

ev. I

mm

unol

. 199

7.15

:707

-747

. Dow

nloa

ded

from

ww

w.a

nnua

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s.or

g A

cces

s pr

ovid

ed b

y U

nive

rsity

of

Wis

cons

in -

Milw

auke

e on

12/

04/1

7. F

or p

erso

nal u

se o

nly.




of lymphocyte-specific factors to the IL-2enhancer.EMBO J.9:2529–36

163. Brabletz T, Pietrowski I, Serfling E. 1991.The immunosuppressives FK506 and cy-closporin A inhibit the generation of pro-tein factors binding to the two purineboxes of the interleukin 2 enhancer.Nu-cleic Acids Res.19:61–67

164. Szabo SJ, Gold JS, Murphy TL, Mur-phy KM. 1993. Identification ofcis-acting regulatory elements controllinginterleukin-4 gene expression in T cells:roles for NF-Y and NF-ATc.Mol. Cell.Biol. 13:4793–805

165. Chuvpilo S, Schomberg C, Gerwig R,Heinfling A, Reeves R, Grummt F, Ser-fling E. 1993. Multiple closely-linkedNFAT/octamer and HMG I(Y) bindingsites are part of the interleukin-4 pro-moter.Nucleic Acids Res.21:5694–704

166. Fiering S, Northrop JP, Nolan GP, MattilaPS, Crabtree GR, Herzenberg LA. 1990.Single cell assay of a transcription factorreveals a threshold in transcription acti-vated by signals emanating from the T-cellantigen receptor.Genes Dev.4:1823–34

167. Garrity PA, Chen D, Rothenberg EV,Wold BJ. 1994. Interleukin-2 transcrip-tion is regulated in vivo at the level ofcoordinated binding of both constitutiveand regulated factors.Mol. Cell. Biol.14:2159–69

168. Tjian R, Maniatis T. 1994. Transcriptionalactivation: a complex puzzle with feweasy pieces.Cell 77:5–8

169. Paul WE, Seder RA. 1994. Lymphocyteresponses and cytokines.Cell 76:241–51

170. Carter LL, Dutton RW. 1996. Type 1 andtype 2: a fundamental dichotomy for all T-cell subsets.Curr. Opin. Immunol.8:336–42

171. Bucy RP, Karr L, Huang G-Q, Li J,Carter D, Honjo K, Lemons JA, MurphyKM, Weaver CT. 1995. Single cell anal-ysis of cytokine gene coexpression dur-ing CD4+ T-cell phenotype development.Proc. Natl. Acad. Sci. USA92:7565–69

172. Pfeuffer I, Klein-Hebling S, HeinflingA, Chuvpilo S, Escher C, Brabletz T,Hentsch B, Schwarzenbach H, MatthiasP, Serfling E. 1994. Octamer factors exerta dual effect on the IL-2 and Il-4 promot-ers.J. Immunol.153:5572–85

173. Prieschl EE, Gouilleux-Gruart V, WalkerC, Harrer NE, Baumruker T. 1995. A nu-clear factor of activated T cell-like tran-scription factor in mast cells is involved inIL-5 gene regulation after IgE plus anti-gen stimulation.J. Immunol.154:6112–19

174. Yamagata T, Nishida J, Sakai R, TanakaT, Honda H, Hirano N, Mano H, Yazaki Y,Hirai H. 1995. Of the GATA-binding pro-teins, only GATA-4 selectively regulatesthe human interleukin-5 gene promoter ininterleukin-5 producing cells which ex-press multiple GATA-binding proteins.Mol. Cell. Biol.15:3830–39

175. Siegel MD, Zhang D-H, Ray P, Ray A.1995. Activation of the interleukin-5 pro-moter by cAMP in murine EL-4 cells re-quires the GATA-3 and CLE0 elements.J. Biol. Chem.270:24548–55

176. Hodge MR, Ranger AM, de la BrousseFC, Hoey T, Grusby M, Glimcher LH.1996. Hyperproliferation and dysregu-lation of IL-4 expression in NF-ATp-deficient mice.Immunity4:397–405

177. Venkataraman L, Francis DA, Wang Z,Liu J, Rothstein TL, Sen R. 1994. Cy-closporin A-sensitive induction of NF-ATin murine B cells.Immunity1:189–96

178. Choi MSK, Brines RD, Holman MJ,Klaus GGB. 1994. Induction of NF-AT in normal B lymphocytes by anti-immunoglobulin or CD40 ligand in con-junction with IL-4. Immunity1:179–87

179. Yaseen NR, Maizel AL, Wang F, SharmaS. 1993. Comparative analysis of NFAT(nuclear factor of activated T cells) com-plex in human T and B lymphocytes.J.Biol. Chem.268:14285–93

180. Weiss DL, Hural J, Tara D, TimmermanLA, Henkel G, Brown MA. 1996. Nuclearfactor of activated T cells is associatedwith a mast cell interleukin 4 transcrip-tion complex.Mol. Cell. Biol.16:228–35

181. Hutchinson LE, McCloskey MA. 1995.FcεRI-mediated induction of nuclear fac-tor of activated T-cells.J. Biol. Chem.270:16333–38

182. Desai DM, Newton ME, Kadlecek T,Weiss A. 1990. Stimulation of the phos-phatidylinositol pathway can induce T-cell activation.Nature348:66–69

183. Li X, Ho SN, Luna J, Giacalone J,Thomas DJ, Timmerman LA, CrabtreeGR, Francke U. 1995. Cloning and chro-mosomal localization of the human andmurine genes for the T-cell transcrip-tion factor NFATc and NFATp.Cytogenet.Cell Genet.68:185–91

184. Luo C, Edelhoff S, Disteche C, CopelandNG, Jenkins NA, Hogan PG, Rao A. 1996.Normal function of the transcription fac-tor NFAT1 in wasted mice: chromosomelocalization of NFAT1 gene.Gene. Inpress

185. Svensson U, Holst E, Sundler R. 1995.Cyclosporin-sensitive expression of cy-tokine mRNA in mouse macrophages

Ann

u. R

ev. I

mm

unol

. 199

7.15

:707

-747

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y U

nive

rsity

of

Wis

cons

in -

Milw

auke

e on

12/

04/1

7. F

or p

erso

nal u

se o

nly.




responding to bacteria.Mol. Immunol.32:157–65

186. Kaye RE, Fruman DA, Bierer BE, AlbersMW, Zydowsky LD, Ho SI, Jin Y-J,Castells MC, Schreiber SL, Walsh CT,Burakoff SJ, Austen KF, Katz HR. 1992.Effects of cyclosporin A and FK506 onFcε receptor type I-initiated increases incytokine mRNA in mouse bone marrow-derived progenitor mast cells: resistanceto FK506 is associated with a deficiencyin FK506-binding protein FKBP12.Proc.Natl. Acad. Sci. USA89:8542–46

187. Tocci MJ, Matkovich DA, Collier KA,Kwok P, Dumont F, Lin S, DegudicibusS, Siekierka JJ, Chin J, Hutchinson N.1989. The immunosuppressant FK506 se-lectively inhibits expression of early T cellactivation genes.J. Immunol.143:718–26

188. Stranick KS, Payvandi F, Zambas DN,Umland SP, Egan RW, Billah MM. 1995.Transcription of the murine interleukin 5gene is regulated by multiple promoter el-ements.J. Biol. Chem.270:20575–82

189. Kita H, Ohnishi T, Okubo Y, Weiler D,Abrams JS, Gleich GJ. 1991. Granulo-cyte/macrophage colony-stimulating fac-tor and interleukin 3 release from humanperipheral blood eosinophils and neu-trophils.J. Exp. Med.174:745–48

190. Casolaro V, Georas SN, Marone G, OnoSJ. 1996. Molecular mechanisms control-ling interleukin-4 synthesis.Curr. Opin.Immunol.8:6–11

191. Andersson J, Nagy S, Groth C-G, Ander-sson U. 1992. Effects of FK506 and cy-closporin A on cytokine production stud-ied in vitro at a single-cell level.Immunol-ogy75:136–42

192. Bourke PF, van Leeuwen BH, Camp-bell HD, Young IG. 1995. Localizationof the inducible enhancer in the mouseinterleukin-5 gene that is responsive to T-cell receptor stimulation.Blood85:2069–77

193. Karlen S, D’Ercole M, Sanderson CJ.1996. Two pathways can activate theinterleukin-5 gene and induce bindingto the conserved lymphokine element 0.Blood88:211–21

194. Lee HJ, Masuda ES, Arai N, Arai K-I, Yokota T. 1995. Definition ofcis-regulatory elements of the mouse inter-leukin-5 gene promoter.J. Biol. Chem.270:17541–50

195. Prieschl EE, Pendl GG, Harrer NE,Baumruker T. 1995. p21ras links FcεRI toNF-AT family member in mast cells: theAP3-like factor in this cell type is anNF-AT family member.J. Immunol.155:4963–70

196. Fruman DA, Bierer BE, Benes JE, Bura-koff SJ, Austen KF, Katz HR. 1995.The complex of FK506-binding pro-tein 12 and FK506 inhibits calcineurinphosphatase activity and IgE activation-induced cytokine transcripts, but not exo-cytosis, in mouse mast cells.J. Immunol.154:1846–51

197. Durez P, Abramowicz D, G´erard C, vanMechelen M, Amraoui Z, Dubois D,Leo O, Velu T, Goldman M. 1993. Invivo induction of interleukin 10 by anti-CD3 monoclonal antibody or bacteriallipopolysaccharide: differential modu-lation by cyclosporin A.J. Exp. Med.177:551–55

198. Dolganov G, Bort S, Lovett M, Burr J,Schubert L, Short D, McGurn M, Gib-son C, Lewis DB. 1996. Coexpression ofthe interleukin-13 and interleukin-4 genescorrelates with their physical linkage inthe cytokine gene cluster on human chro-mosome 5q23–31.Blood87:3316–26

199. van der Pouw Kraan TCTM, Boeije LCM,Troon JTM, Rutschmann SK, WijdenesJ, Aarden LA. 1996. Human IL-13 pro-duction is negatively influenced by CD3engagement: enhancement of IL-13 pro-duction by cyclosporin A.J. Immunol.156:1818–23

200. Masuda ES, Tokumitsu H, Tsuboi A,Shlomai J, Hung P, Arai K-I, AraiN. 1993. The granulocyte-macrophagecolony-stimulating factor promotercis-acting element CLE0 mediates inductionsignals in T cells and is recognized by fac-tors related to AP1 and NFAT.Mol. Cell.Biol. 13:7399–407

201. McCaffrey PG, Jain J, Jamieson C, SenR, Rao A. 1992. A T cell nuclear fac-tor resembling NF-AT binds to an NF-κB site and to the conserved lymphokinepromoter sequence “Cytokine-1”.J. Biol.Chem.267:1864–71

202. Cockerill PN, Shannon MF, BertAG, Ryan GR, Vadas MA. 1993.The granulocyte-macrophage colony-stimulating factor/interleukin 3 locus isregulated by an inducible cyclosporinA-sensitive enhancer.Proc. Natl. Acad.Sci. USA90:2466–70

203. Granelli-Piperno A. 1990. Lymphokinegene expression in vivo is inhibited by cy-closporin A.J. Exp. Med.171:533–44

204. Penix L, Weaver WM, Pang Y, YoungHA, Wilson CB. 1993. Two essentialregulatory elements in the human iner-feron γ promoter confer activation spe-cific expression in T cells.J. Exp. Med.178:1483–96

205. Campbell PM, Pimm J, Ramassar V,

Ann

u. R

ev. I

mm

unol

. 199

7.15

:707

-747

. Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y U

nive

rsity

of

Wis

cons

in -

Milw

auke

e on

12/

04/1

7. F

or p

erso

nal u

se o

nly.




Halloran PF. 1996. Identification ofa calcium-inducible, cyclosporine-sensi-tive element in the IFN-γ promoter thatis a potential NFAT binding site.Trans-plantation61:933–39

206. Brown DA, Nelson FB, Reinherz EL,Diamond DJ. 1991. The human inter-feron-γ gene contains an inducible pro-moter that can be transactivated by tax Iand II.Eur. J. Immunol.21:1879–85

207. Ahuja SS, Shrivastav S, Danielpour D,Balow JE, Boumpas DT. 1995. Regula-tion of transforming growth factor-β1 andits receptor by cyclosporine in human Tlymphocytes.Transplantation60:718–23

208. Li B, Sehajpal PK, Khanna A, VlassaraH, Cerami A, Stenzel KH, SuthanthiranM. 1991. Differential regulation of trans-forming growth factorβ and interleukin2 genes in human T cells: demonstra-tion by usage of novel competitor DNAconstructs in the quantitative polymerasechain reaction.J. Exp. Med.174:1259–62

209. Splawski JB, Nishioka J, Nishioka Y, Lip-sky PE. 1996. CD40 ligand is expressedand functional on activated neonatal Tcells.J. Immunol.156:119–27

210. McCaffrey PG, Kim PK, Valge-ArcherVE, Sen R, Rao A. 1994. CyclosporinA sensitivity of the NF-κB site of theIL2Rα promoter in untransformed murineT cells.Nucleic Acid. Res.22:2134–42

211. Taylor-Fishwick DA, Siegel JN. 1995.Raf-1 provides a dominant but not ex-clusive signal for the induction of CD69expression on T cells.Eur. J. Immunol25:3215–21

212. Brunner T, Yoo NJ, LaFace D, Ware CF,Green DR. 1996. Activation-induced celldeath in murine T cell hybridomas. Dif-ferential regulation of Fas (CD95) versusFas ligand expression by cyclosporin A

and FK506.Int. Immunol.8:1017–26213. Venkataraman L, Wang W, Sen R. 1996.

Differential regulation of c-Rel transloca-tion in activated B and T cells.J. Immunol.159:1149–55

214. Kang S-M, Tsang W, Doll S, Scherle P, KoH-S, Tran A-C, Lenardo MJ, Staudt LM.1992. Induction of the POU domain tran-scription factor Oct-2 during T-cell acti-vation by cognate antigen.Mol. Cell. Biol.12:3149–54

215. Yazbanbakhsh K, Choi J-W, Li Y, Lau LF,Choi Y. 1995. Cyclosporin A blocks apop-tosis by inhibiting the DNA binding activ-ity of the transcription factor Nur77.Proc.Natl. Acad. Sci. USA92:437–41

216. Woronicz JD, Calnan B, Ngo V, WinotoA. 1994. Requirement for the orphansteroid receptor Nur77 in apoptosis of T-cell hybridomas.Nature367:277–81

217. Wang C-Y, Bassuk AG, Boise LH,Thompson CB, Bravo R, Leiden JM.1994. Activation of the granulocyte-macrophage colony-stimulating factorpromoter in T cells requires cooperativebinding of Elf-1 and AP-1 transcriptionfactors.Mol. Cell. Biol.14:1153–59

218. Ullman KS, Flanagan WM, Edwards CA,Crabtree GR. 1991. Activation of earlygene expression in T lymphocytes by Oct-1 and an inducible protein, OAP40.Sci-ence254:558–62

219. Rooney JW, Hoey T, Glimcher LH. 1995.Coordinate and cooperative roles forNFAT and AP-1 in the regulation of themurine IL-4 gene.Immunity2:473–83

220. Perkins D, Wang Z, Donovan C, He H,Mark D, Guan G, Wang Y, Walunas T,Bluestone J, Listman J, Finn PW. 1996.Regulation of CTLA-4 expression duringT cell activation.J. Immunol.156:4154–59

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ev. I

mm

unol

. 199

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:707

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. Dow

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from

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transcription factors of the nfat family:regulation...

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