Docstoc

Inna Iņaškina Cloning and biological activity of nuclear factor

Document Sample
Inna Iņaškina Cloning and biological activity of nuclear factor Powered By Docstoc
					           UNIVERSITY OF LATVIA
    BIOMEDICAL RESEARCH AND STUDY CENTRE




                 Inna Iņaškina


CLONING AND BIOLOGICAL ACTIVITY OF NUCLEAR
     FACTOR OF ACTIVATED T CELL (NFAT)


                  Abstract




                  Author:                Inna Iņaškina


                  Supervisors:   Professor Edgar Serfling
                                 Dr. biol. Ēriks Jankevics




                   Riga 2005
                                    SUMMARY
During the last decade when nuclear factor of activated T cells (NFAT) have been
intensively studied, is became clear that calcium-regulated NFAT proteins play a major
role in regulating inducible gene expression in the immune system, and that they also
have important biological functions in other cell types, both during development and in
the adult mammal.
        In the present study we have cloned and investigated multiply NFATc1 and
NFATc3 isoforms in lymphoid cells. The transcription factor NFATc1 is synthesised in
three prominent isoforms. These differ in the length of their C terminal peptide and
mode of synthesis. NFATc1/A, the shortest of three isoforms, is only one inducibly
expressed in T cells. In contrast, the two longer isoforms NFATc1/B and NFATc1/C
span extra C-terminal peptides of 128 and 246 aa, respectively, and are constitutively
synthesised in many T cells. We also tested cloned cDNA of corresponding isoforms in
transient transfection assays for promoter induction. NFATc1/A and NFATc1/C showed
a very similar, if not identical, strong stimulatory effect on the IL-2 promoter, while
NFATc1/B appeared to be the weakest transactivator. We also isolated three novel types
of murine NFATc3 cDNA, designated as NFATc3-37, NFATc3-20, and NFATc3-l,
and determined as isoforms alternatively spliced at the C-part. Using a number of
specific antisera in Western Blotting assay, we found that NFATc3-37 is an exclusive
form of NFATc3, constitutively expressed in the lymphoid tissues. An endogenous
NFATc3-37 from thymocytes or EL4 cells, but not from Jurkat cells, bound to the distal
NFAT site from the murine IL-2 promoter. This binding did not require the involvement
of AP-1 related proteins. None of the isoforms overexpressed in 293 or Jurkat cells were
able to activate the transiently transfected reporter gene driven by NFAT site from the
IL-2 promoter. However, both NFATc3-37 and NFATc3-20 activated transcription via
the NFAT I site from the Lck proximal promoter even without induction. Thus, albeit
NFATc3-37 is widely expressed in lymphoid tissues, it might be involved in the
regulation of the lck gene expression and, possibly, in the regulation of other genes
during thymocyte development.
        The second part of the work is devoted to the investigation of localisation and
conditions of nuclear translocation of NFATc1, NFATc2, and NFATc3 in various T and
B cell lines. The intracellular translocation of NFAT transcription factors is mediated by
the activity of calcium-dependent serine/threonine phosphatase calcineurin and
therefore is considered to be specifically inhibited by cyclosporin A (CsA). Our results
suggest distinct behaviour of NFAT factors in response to treatment with CsA. Whereas
the nuclear residence of NFATc2 and NFATc3 is always sensitive to treatment with
CsA, i.e. these proteins are exported back to the cytoplasm, NFATc1 is considerably
less affected by CsA and does not leave the cell nucleus. NFATc1 in high concentration
forms granular structures within the cell nuclei. The treatment of cells with CsA leads to
a decrease in the mobility of NFAT proteins and DNA-protein complex during
electrophoresis due to hyperphosphorylation. These observations are validated by
western blots, EMSA, immunostaining, and confocal microscopy of chimeric NFAT -
green fluorescent protein. In addition, functional studies show that CsA inhibits
transcription activity of endogenous NFATc1, but it has no effect on the overexpressed
protein.
        The transcriptional activation of NFAT factors is generally regulated by
dynamic interplay between calcineurin and Ras/Raf/Erk protein kinase cascade on one
hand and active serine/threonine protein kinases counteracting activity of calcineurin,
on the other hand. In the last part of the work we investigated the effect of glycogen
syntase kinase-3 (GSK-3) and protein kinase A (PKA) on the transcriptional activity of
NFAT family proteins - NFATc1/A, NFATc1/C, NFATc2 and NFATc3 using full-
length expression vectors, NFATc1/Gal4 chimeras and different reporters. We observed
that overexpression of GSK-3 dramatically inhibited transcriptional activity of all
coexpressed proteins, while overexpression of PKA significantly increased the level of
NFATc-mediated reporter gene production. We suggest that positive effect of PKA is a
sum of several distinct effects caused by different mechanisms. The functional assay
using NFATc1/Gal4 chimera with serine 269 mutated to alanine suppose that direct
effect of PKA on the transcription activity of NFATc1 is accomplishing through
phosphorylation of Ser269 and it is negative.

The work was carried out at the Biomedical Research and Study Centre, University of
Latvia and the Institute of Pathology, University of Würzburg, Germany.
The study was supported by grants 96.0733, 01.0246, and 04.1145 of the Latvian
Council of Science, the Graduirtenkollegs Regulation des Zellwachstums" (Würzburg
DFG-2.22-10/96-II 13), DFG grant (DFG Wuerzburg; SE 469/12-1) and the NATO
Collaborative Linkage Grant (HITECH.LG973289).
Results of this work are reflected in four papers and one manuscript and have been
presented in three international conferences and meetings.
                                 CONTENTS

ORIGINAL PAPERS                                                             5
ABBREVIATIONS                                                               6
INTRODUCTION                                                                7
            Structure and evolution of NFAT proteins                        7
            Activation of NFAT proteins                                     8
            NFAT-binding partners                                           9
            Biological function                                             10
RESULTS AND DISCUSSION                                                      11
The identification and characterisation of novel isoforms of
transcription factors NFATc1 and NFATc3 in lymphoid cells                   11
Constitutive and inducible synthesis of NFATc1 isoforms in lymphocytes      11
The NFATc1 isoforms differ in their C and N terminal peptides               11
NFATc1 isoforms differ in their transcriptional capacity                    12
Molecular cloning of murine NFATc3 cDNA                                     13
The distribution of NFATc3 isoforms in lymphoid cell lines                  15
The binding activity of endogenous nuclear NFATc3 depends from cell type    17
The transcription activity of NFATc3 isoforms                               18
Concluding remarks                                                          19
The different effect of immunosupressor cyclosporin A on the intracellular
trafficking and functional activity of NFAT transcription factors           20
Nuclear export of NFAT proteins is differentially affected by CsA           20
Cyclosporin A has no inhibitory effect on the translocation of NFATc1/A-GFP  23
The transcription activity of the overexpressed NFATc1 is not
CsA-sensitive in Jurkat cells                                               23
The immuno fluorescence staining of endogenous NFATc1 in the
Jurkat and EL4 cells                                                        25
Concluding remarks                                                          26
The complex effect of Protein Kinase A on the transcriptional
activity of NFAT family proteins                                            28
Concluding remarks                                                          30
CONCLUSIONS                                                                 33
REFERENCES                                                                  34
                               ORIGINAL PAPERS

This thesis is based on the following papers, which are referred by their Roman
numerals in the text:

I.     Iņaškina I., Serfling E. and Jankevics Ē. The Positive Effect of Protein Kinase
       A on the Transcriptional Activity of NFAT Family Proteins Occurs Via
       Different Signal Transduction Mechanism, (in manuscript)

II.    Iņaškina I., Serfling E. and Jankevics Ē. Intracellular Trafficking and
       Functional Activity of Nuclear Factors of Activated T Cells Are Differently
       Affected by Cyclosporine A. Proc Latvian Acad Sci, 2004, 58 (l):l-8.

III.   Iņaškina I., Jackevica L., Zajakins P., Serfling E. and Jankevics Ē.
       Transcription Factor NFATc3 Isoforms in Lymphoid Cells. Proc Latvian Acad
       Sci, 2003, 57 (1/2):11-16.

IV.    Chuvpilo S., Avots A., Berberich-Siebelt F., Glockner J., Fischer C, Kerstan A.,
       Escher C, Inashkina I., Hlubek F., Jankevics E., Brabletz T., Serfling E.
       Multiple NFATc isoforms with individual transcriptional properties are
       synthesized in T lymphocytes. J Immunol 1999,162 (12):7294-301.

V.     Chuvpilo S., Zimmer M., Kerstan A., Glockner J., Avots A., Escher C, Fischer
       C, Inashkina I., Jankevics E., Berberich-Siebelt F., Schmitt E., Serfling E.
       Alternative polyadenylation events contribute to the induction of NFATc in
       effector T cells. Immunity, 1999,10 (2):261-9.

Results of this work have been presents in the following international conferences and
scientific meetings:

I.     Inashkina I., Jankevics E. and Serfling E. Nuclear translocation of NFAT
       factors. NATO/FEBS International Summer School on Molecular Mechanism of
       signal Transduetion. Island of Spetsai, Greece, August 16-28, 1999, Abstracts,
       p29.

II.    Chuvpilo S., Zimmer M., Kerstan A., Glockner J., Avots A., Escher C. f Fischer
       C, Inashkina I., Jankevics E., Berberich-Siebelt F., Schmitt E., Serfling E.
       Alternative splice/polyadenylation events contribute to the inducible synthesis of
       NFATc in T effector cells. 15. Fruhjahrstagung Deutsche Gesellschaft fur
       Immunologie, Stuttgart, March 03-06,1999, Abstracts, p106.

III.   Chuvpilo S., Kerstan A., Glockner J. , Avots A., Escher C, Fischer C,
       Inashkina I., Jankevics E., Siebelt F., and Serfling E. Coordinated synthesis and
       nuclear translocation of NFATc isoforms duting T cell activation. 28 th Annual
       Meeting of the Deutsche Gesellschaft fur Immunologie, Würzburg, September
       25-27,1997. Immunobiol 1997,197:133-427, E 6.
                                  ABBREVIATIONS

aa              amino acid
Ab              antibodies
AICD            activation-induced cell death
AP-1            activator protein 1
bp              base pair
CDK             cycline-dependent kinase
CKI, II         casein kinase I, II
Cn              calcineurin
CsA             cyclosporin A
EMSA            electrophoretic mobility shift assay
Erk             extracellular signal-regulated kinase
FasL            Fas ligand
GFP             green fluorescent protein
GM-CSF          granulocyte/macrophage colony-stimulating factor
GSK-3           glycogen syntase kinase-3
GTP             guanosine triphosphate
HDAC            histone deacetylase
IL-2, 3, 4...    interleukin-2, 3, 4...
INF-           interferon 
JNK             c-Jun N-terminal kinase
MAP/SAP          mitogen-activated protein/ stress activated protein
MEF2            myocyte enhancer factor-2
NFAT            nuclear factor of activated T cells
NFKB            nuclear factor KB
PAAG            polyacrylamide gel
PBL             peripheral blood leukocytes
PKA             protein kinase A
PKC             protein kinase C
PTKs            protein tyrosine kinases
SAP             shrimp alkaline phosphatase
SD              standard deviation
TCR             T cell receptor
TNF-           tumour necrosis factor 
TonEBP          tonicity enhancer binding protein
TPA             12-O-tetradecanoylphorbol 13-acetate
WB              western blot
                                 INTRODUCTION
The NFAT (nuclear factor of activated T cells) family of transcription factors
encompasses five proteins functionally and evolutionary related to the Rel/NFkB family
of transcriptional activators [1, 2]. These include NFATc1 (NFATc/NFAT2), NFATc2
(NFATp/NFATl), NFATc3 (NFAT4/NFATx), NFATc4 (NFAT3), and NFAT5
(TonEBP: tonicity element binding protein or OREBP: osmotic response element
binding protein) (reviewed in [3]). The NFATc1-c4 proteins are regulated by
calcium/calcineurin dependent signalling - a rise in intracellular calcium activates the
serine/threonine phosphatase calcineurin [4], which dephosphorylates NFATc1 -c4
proteins [5]. NFATs are expressed in numerous cell type but they nevertheless play a
particularly important role of activation, proliferation and activation-induced cell death
(AICD) of lymphoid cells [4, 6-8].

Structure and evolution of NFAT proteins
The structures of NFAT proteins are schematically compiled in Figure 1A. All NFAT
proteins are characterised by the presence of a highly conserved DNA binding domain,
often designated Rel similarity domain (RSD) due to sequence similarities with the
DNA binding (Rel) domain of Rel/NFicB factors, which harbours the sequences motifs
for DNA binding, interaction with AP-1 and, as shown for NFATc1, one nuclear
localisation signal (NLS) [9], Moreover, they contain a regulatory domain in front of the
RSD and at least one transactivation domain (TAD) near the N-terminus. The regulatory
domain harbours numerous phosphorylation sites which are organised in the conserved
serine-rich region (SRR) and three so-called SP motifs. These sites are substrates for
several serine/threonine protein kinases and of the protein phosphatase calcineurine,
which binds to this region (see Activation of NFAT proteins). In addition one NLS and
one nuclear export signal (NES) have been identified within this domain of NFATc1 [9,
10].
        All NFAT factors appear to be synthesised in several isoforms (Figure 1A)
which can differ in both their N- and C-terminal peptides [11-16], The C-terminal
sequences of the longest isoform NFATc1/C and NFATc2 harbour a second, albeit
weak TAD [17, 18]. One isoform of NFATc3 (early designated NFATxl) revealed a
strong TAD within 15 aa near its C-terminus which are highly conserved among all
NFAT factors. Deletion of this C-terminal peptide led to a strong decrease in NFATxl
activity after transfection into 293 and T cells. [14, 19].
        Among NFAT proteins, NFAT5 is unique and unlike the calcium-regulated
NFAT proteins. NFAT5 is constitutively dimeric, and dimerisation is essential for DNA
binding and transcriptional activity [20-22]. The RSD of NFAT5 (Figure 1A) shares
only 41-45% sequence similarity with the RSD of other NFATs [23]. Also NFAT5 is
not regulated by calcium/calcineurin but rather works as a tonicity-responsive
transcription factor required for certain aspects of T cell function as well as for kidney
homeostasis and function [20-22]. Unlike genuine NFATs, NFAT5 does not interact
with AP-1 factors, although it binds to the NFAT core binding motif TGGAAA.
Moreover, NFAT5 lacks clear sequence homology to NFAT peptides outside the RSD,
including the "regulatory region" [2].
        NFAT5 seems to represent an evolutionary link between NFKB and genuine
NFAT factors [2, 22]. In Drosophila, several NFkB-like proteins, such as Dorsal, Dif
and Relish, have been detected which act downstream of signalling cascades similar to
the NFicB-inducing cascades in mammalian cells. In analogy to mammalian RelA/p50
and RelB/pl05 NFKB complexes, Dif and Relish play important roles in the control of
Figure 1. Scheme of structure (A) and activation in T cells (B) of NFAT factors (from Serfling
et al., 2004).
A. DNA binding domains (RSD) are shown in yellow, transactivating domains (TAD), in red,
and the regulatory domains of NFATs in blue. NLS, nuclear localisation signals; NES, nuclear
export signal; SP, serine-proline rich motifs; SRR, serine-rich motifs. CBP, CN indicates the
binding of transcriptional co-factor CBP/p300 and of phosphatase calcineurin (CN) to the N-
termini of NFATs. NFATc1 was first cloned by Northrop et al. [24], NFATc2 by McCaffrey et
al. [25], NFATc3 by Masuda et al. [26] and Hoey et al. [27], NFATc4 by Hoey et al. [27],
respectively.
B. NFAT activation: the central position of the calcium/calmodulin-dependent phosphatase
calcineurin in control of nuclear NFAT translocation and activation is indicated. It is thought
that calcineurin-mediated dephosphorylation of regulatory region (RR) unmasks NLSs and
leads to their nuclear translocation. The immunosuppressants CsA and FK506 which bind to
calcineurin in concert with immunophilins are efficient inhibitors of calcineurin activity. Several
kinases, in particular GSK3, have been described to phophorylate the SRR and SP motifs in
the regulatory region of NFATs. The classicalRas/Raf/Erkcascade (but also other signaling
molecules, e.g. PKC members) was described to contribute to transacting capacity of NFATc1,
but also in control of AP-1 activity.

innate immune responses by activating anti-microbial genes (reviewed in [28]). There is
one NFAT-like gene of unknown function in the Drosophila genome, whose protein
product has approximately 56% sequence identity with the RSD of NFAT5 and 42-43%
identity with the RSDs of genuine NFATs [29]. Similarly, in the genome of the
ancestral chordate Ciona intestinalis there is only one NFAT-like gene which harbours
a RSD displaying 56% sequence identity at the protein level with the RSD of human
NFAT5 and 38-40% identity with those of other human NFATs [30]. For teleost fish,
NFAT-like transcription complexes controlled by a calcineurin-like activity have been
described, and there are five NFAT genes in the genome of the teleost fish Fugu
rubripes [31] corresponding to the five mammalian NFATs. This suggests that genuine
NFATs appeared with the emergence of the adaptive immune system and lymphocytes
approximately 500 million years ago, before the diversification of jawed fish.

Activation of NFAT proteins
The efficient transcriptional activation of NFAT factors in T cells needs at least two
signals, which are provided by activation of the TCR. These TCR-mediated signals lead
to (1) a rise in intracellular free calcium and calcineurin activation, and (2) the
stimulation of several PTKs, e.g. p56lck, and p21ras and other small GTP bounding
proteins, which activate a number of serine/threonine protein kinase cascades. While the
activation of calcineurin mediates the nuclear translocation of NFAT factors [32], [33],
activation of classical Ras/Raf/Erk and further protein kinase cascades controls the
transcriptional activation of NFATs [34] and the induction of AP-1 [35].
        In resting cells, NFAT proteins are phosphorylated, reside in the cytoplasm, and
show low affinity for DNA in vitro. NFAT activation is initiated by dephosphorylation
of the NFAT regulatory domain, a conserved ~300-aa region (Figure 1A). This domain
is encoded in a single exon in all four NFAT proteins from all vertebrate species for
which sequences are available [2]. Serine/threonine phosphatase calcineurin directly
dephosphorylates serines of SRR and SP repeats located within regulatory domain, thus
exposing NLS (Figure 1 A, B) and therefore leading to NFAT rapid nuclear import and
increase in the affinity of protein for its target sites in DNA [36-42]. Inhibition of the
phosphatase activity of calcineurin by FK506 or CsA results in the relocalisaion of
NFAT to the cytosol and loss of its DNA-binding ability [43].
        Although several serine/threonine protein kinases have been identified to
phosphorylate NFAT proteins, thereby counteracting the activity of calcineurin, an
integrated picture of NFAT phosphorylation has not yet emerged. CKI and GSK-3 are
constitutive NFAT kinases that promote NFAT nuclear export [44, 45]; phosphorylation
by GSK-3 requires prior phosphorylation by a priming kinase such as PKA [46, 47].
Consistent with its known sequence preference [48], GSK-3 phosphorylates the SPxx
motifs of NFATc1 [44]. The MAP kinases p38 and JNK are inducible kinases that
promote NFAT nuclear export, by selectively phosphorylating NFAT proteins at the SP
sequences at the beginning of their SRR-1 regions: JNK1 phosphorylates NFATc1 and
NFATc3 [49], whereas p38 selectively targets NFATc2 and NFATc4 [50, 51]. For
JNK1, a proposed mechanism is that phospholylation of the calcineurin-docking site of
NFATc1 blocks the interaction of NFATc1 with calcineurin [52].

NFAT-binding partners
Synergistic cooperation between NFAT and unrelated transcription factors AP-1 (Fos-
Jun proteins) on composite DNA elements which contain adjacent NFAT
(T/AGGAAAA/T) and AP-1 (TGAGTCA) binding sites regulates the expression of
diverse inducible genes (reviewed in [53]). The outcome of cooperative interaction
between NFAT and AP-1 is essential for a productive immune response, by stimulating
the expression of a growing number of genes encoding cytokines, chemokines, and cell
surface receptors (reviewed in [4]). The paradigm of NFAT-AP-1 cooperation has been
recognised in a number of different cytokine promoters/enhancer regions (reviewed in
[4, 6]). These include IL-2, IL-4, IL-5, GM-CSF, CD40L, IFN-y, IL-13, CTLA4, human
TNF-a, human IL-8, and E-selectin. The strong cooperative binding of NFAT-AP-1 on
specific DNA composite sites forms significantly more stable and higher affinity
complexes than binding of the individual proteins alone [4].
         In addition to its interaction with AP-1, NFAT engages in direct protein-protein
interactions and influences transcription synergistically with several families of
transcription factors: proteins such as Maf ICER that belongs to the same basic region-
leucine zipper family as AP-1 [54, 55]; the zinc finger proteins GAT A (see below), and
EGR [56]; the helix-turn-helix domain proteins Oct, HNF3, and IRE-4 [57-60]; the
MADS-box protein MEF2 [8, 61, 62]; and the nuclear receptor PPAR-y [63].
Cooperation of NFAT with GATA family members has been observed in many systems
such as functional assays using reporter constructs [64-66], yeast two-hybrid assay [64],
and co-immunoprecipitation experiments [67]. However, sequence inspection of
regulatory regions has not led to unambiguous identification of a composite NFAT-
GATA element with specific spacing and orientation, and cooperative NFAT-GATA
binding on DN A has not been reported.

Biological function
NFATc1, NFATc2 and NFATc3 are expressed in cells of the immune system where
they play a key role in regulating a large number of inducible genes during the immune
response (reviewed in [4]). These include IL-2, IL-S, GM-CSF, IL-4, IL-5, IL-10 [68],
IL-13, IFN-y, TNF-a, CD40L, FasL, CD5, IgK, CD25, IL-8, MIP4a and Cox 2. The
transcriptional activity of NFATs may be either activating or deactivating depending on
their binding partners. When NFATs cooperatively interact with activation partners
such as AP-1, MEF2, and GATA, they activate a set of NFAT-regulated genes that
mediate lymphocyte activation. On the other hand, interaction of NFAT with silencing
complexes (e.g., HDACs) on specific gene loci (e.g., CDK4 promoter) turns off gene
expression [69]. In addition, NFAT can induce expression of certain genes without the
need for cooperative recruitment of Fos and Jun [70]. In this situation NFAT may turn
on a completely new set of "anergy-associated genes" that mediates T cell anergy [71].
         Mice that are deficient for both NFATc2 and NFATc3 show a striking allergic
phenotype, suggesting a role for these proteins in suppressing production of the specific
cytokines that control the development of allergy [72]. In contrast, T cells deficient in
both NFATc1 and NFATc2 producing almost no cytokines upon stimulation, indicating
that NFAT is essential for activating transcription of most T cell cytokine genes. At the
same time, cells from these mice show an unprecedented phenotype of
hyperproliferation, suggesting a role for NFAT in suppressing B cell responses [73].
         All five NFAT proteins are also expressed in various non-lymphoid tissues,
where they are involved in the regulation of diverse cellular functions in organs other
than the immune system. The functions of NFAT proteins in extra-immune tissues have
been largely inferred from the phenotypes of gene-disrupted mice lacking individual or
multiple family members, and the identity of only a few target genes is known. Thus
targeted disruption of the NFATc1 gene results in intrauterine death of the NFATc1-
deflcient embryos due to a defect in cardiac valve formation [74, 75]. In muscle tissue
NFATc1 is thought to cooperate with GATA2 to induce myocyte hypertrophy [67].
NFATc4 is expressed in hippocampal neurones where it induces transcription of NFAT
dependent genes in response to depolarisation [76]. A calcineurin-dependent pathway
that induces cardiac hypertrophy and involves a possible NFATc4-GATA4 cooperation
has also been described [64]. Finally, NFAT proteins have been implicated in the
regulation of chondrogenesis and adipogenesis [77, 78]. Whether these extra-immune
NFAT-regulated processes require NFAT-AP-1 cooperation, or whether new
transcriptional partners such as GATA proteins are required for NFAT activity outside
the immune system, remains to be elucidated.
         The aim of this work is investigation of biological functions of transcription
factors NFATc1 and NFATc3 multiply isoforms.
         In the present study we have focused on the three aspects of the investigation of
NFAT family proteins: cloning and analysis of multiply NFATc1 and NFATc3 isoforms
in lymphoid cells; localisation and conditions of nuclear translocation of NFATc1,
NFATc2, and NFATc3 particularly in response to treatment with CsA in various cells;
the effect of kinases GSK-3 and PKA on the NFAT proteins-mediated transcriptional
activity.
                         RESULTS AND DISCUSSION
      THE IDENTIFICATION AND CHARACTERISATION OF NOVEL
   ISOFORMS OF TRANSCRIPTION FACTORS NFATC1 AND NFATC3 IN
                       LYMPHOID CELLS

Although several common structural features among the NFATc proteins are known,
but inactivation of the different NFATc genes in mice resulting in contrasting effects on
cell differentiation and activation. One mechanism contributing to the pleiotropic effect
of NFAT factors could be the mode of their expression. All NFAT factors appear to be
synthesised in several isoforms that can differ in both their N- and C-terminal peptides
[13, 14, 17]. In this chapter we report isolation and identification of human NFATc1
and murine NFATc3 isoforms. The properties of these proteins in expression manner
and transcriptional activity were also investigated in different cells.

Constitutive and inducible synthesis of NFATc1 isoforms in lymphocytes
The nuclei of untreated Jurkat cells contain relatively low amounts of NFATc1 proteins
larger than 100 kDa. After treatment with TPA plus ionomycin (TPA/Iono), the
concentrations of these nuclear proteins increase steadily over a period of 3-4 hr (Figure
2A, lanes 1-5). However, 3-4 hr after treatment, a shorter prominent NFATc1 protein of
about 90 kDa appears (Figure 2A, lane 5) that corresponds in size to NFATc cloned by
Northrop et al. [24]. Similar patterns of nuclear NFATc1 proteins appear to be typical
for many T and B cell lines, e.g., A3.01 T and Daudi B cells (Figure 2B and C, lanes 1-
2). Other lymphoid cells express the 90 kDa NFATc1 protein in very different amounts.
Thus, Raji B lymphoma cells synthesise it in low amounts (Figure 2C, lanes 3-4), EL-4
(Figure 9 A, lines 7-8) and L-5178Y T lymphoma cells in considerable, but H9 T cells
not at all (Figure 2B, lanes 3-4).
       To elucidate whether this heterogeneity in nuclear NFATc1 proteins is a
peculiarity of lymphoid tumour cells, we investigated nuclear NFATc1 proteins from
naive murine CD4+ T cells and Th1 and Th2 effector cells after differentiation in vitro.
As shown in Figure 2D (line 1), naive T cells do not contain any detectable amount of
nuclear NFATc1. TPA/Iono treatment for 6-12 hr leads to the predominant expression
of two NFATc1 proteins larger than 100 kDa (Figure 2D, lane 2). TCR stimulation of
these cells and differentiation for 8-10 days to Th1 or Th2 cells results in a nuclear
accumulation of NFATc1, in particular of proteins larger than 100 kDa (Figure 2D,
lines 2 and 5). However, stimulation of effector Th1 and Th2 cells with TPA/Iono gives
rise to a predominant accumulation of the 90 kDa NFATc1 protein (Figure 2D, lines 4
and 6). The similar results were obtained in WB assay using nuclear proteins from PBL
T cells. Figure 2E (line 1) shows that untreated PBL T cells did not contain any
detectable nuclear NFATc1. Stimulation of PBL T cells with TPA/Iono resulted in the
detection of NFATc1 isoforms B and C after 2 h (Figure 2E, line 2). After 6 h, NFATc1
isoform A of about 90 kDa became detectable (Figure 2E, line 3).

The NFATc1 isoforms differ in their C and N terminal peptides
The occurrence of multiple nuclear NFATc1 proteins prompted us to clone NFATc1
cDNAs from a human Namalwa B cell cDNA library. Six out often positive NFATc1
cDNA clones isolated correspond to NFATc1 described previously [24], which we will
designate as NFATc1/A. The residual four cDNAs were considerably longer. Two,
designated as NFATc 1/B, lack the last C-terminal 19 aa residues of NFATc1/A and,
Figure 2. Three nuclear NFATc1 isoforms are expressed in lymphoid cells.
 A, B, C. 10 ng nuclear proteins from uninduced and induced for different time Jurkat T cells
(A), uninduced and induced for 4 hr A3.01 and H9 T cells (B) or Raji and Daudi B cells (C)
were fractionated and immunodetected using the 7A6 Ab. Proteins from 293 cells transfected
with NFATc1/A, B, or C cDNA expression vectors were fractionated in the same gels (three
right lines of each panel).
D. WB of nuclear proteins isolated from the differentiated murine Th1 and Th2 effector cells.
Naive CD4+CD62L+ T cells were isolated from the spleens of BALB/c mice, stimulated in vitro
with anti-TCRp Ab and anti-CD28 Ab, incubated in the presence of either IL-12 for Th1 or IL-4
for Th2 differentiation, and stimulated with TPA and Iono for 5 hr as described previously [79].
E. PBL-Ts were either left uninduced or were induced for 2-12 h with TPA+Iono and obtained
nuclear proteins were electrophoretically separated and immunoblotted. The NFATc1 isoforms
A, B, and C are indicated.

instead, contain a C-terminal stretch of 128 aa. A third cDNA, designated as
NFATc1/C, encodes in addition to the 128 aa stretch an extra C terminal peptide of 118
aa and an N terminal peptide of 29 aa, instead of the 42 aa N terminal stretch of
NFATc1/A. Both NFATc1/B and C cDNAs carry a 3’ untranslated mRNA sequence of
1844 or 1796 nucleotides, respectively, instead of the 360 nucleotides 3' tail found in
NFATc1/A (Figure 3A).
        The extra C terminal peptides in NFATc1/B and C show sequence homologies
of 30.6% to those in NFATc2 (Figure 3B) [11]. The more proximal region (aa 685 to
813 in NFATc1/C) exhibits 36.7% homology while the proline-rich NFATc1/C-
specific peptide shows a poor homology to NFATc2, except an identical decapeptide
spanning the aa 911-920 inNFATc1/C (Figure 3B).
        When the three NFATc1 cDNAs were expressed in human 293 cells, the
proteins synthesised were identical in size to the NFATc1 proteins in Jurkat cells and
other lymphoid tumour cells (Figures 3 A-C).

NFATc1 isoforms differ in their transcriptional capacity
To test the transcriptional activity of individual NFATc1 isoforms we cotransfected
expression vectors for NFATc1/A, -B, or -C together with a variety of
promoter/reporter gene constructs into 293 cells, which express only minor amounts of
endogenous NFATc1 (data not shown). Figure 4A shows that all three NFATc1
isoforms were properly expressed and translocated into the nuclei as proteins of
approximately 90, 110, and 140 kDa, respectively. Dephosphorylation of nuclear
Figure 3. Organisation of human NFATc1 isoforms.
A. Scheme of NFATc1 cDNAs isolated from a human Namalwa B cell cDNA library. The
transactivation domains of NFATc1 isoforms, TAD-A and TAD-B, and the Rel similarity
domain (RSD) are indicated. The 3* untranslated mRNA segments of 360 bp (in NFATc1/A),
1844 bp (NFATc1/B), and 1796 bp (NFATc1/C) are shown as solid brown lines. No. 1 indicates
the translational start codon and the numbers after the protein coding parts indicate the numbers
of aa residues for each NFATc protein. The 5' untranslated mRNA segments are not shown.
B. Sequence comparison between the extra C terminal peptides in NFATc 1/B and /C with those
in NFATc2 [11]. Identical aa residues between the C terminal peptides are indicated by long
vertical dashes, similar residues by short dashes. Gaps are indicated by horizontal dashes. Note
the identical decapeptide near the C termini of NFATc1/C and NFATc2.

proteins led to an increase in their electrophoretic mobility, indicating phosphorylation
of all three isoforms in these cells (Figure 4B). All three NFATc1 proteins stimulated
the inducible activation of luciferase reporter genes driven by four copies of the distal
NFAT site (Pu-bd) from the murine IL-2 promoter [80] or three copies of Pu-be from
the murine IL-4 promoter (Figure 4C and D) [81]. In all transfection assays, NFATc 1/B
was the weakest trans-activator (Figure 4C), whereas NFATc1/C appeared to be the
strongest NFATc1, being 2-fold stronger than NFATc1/A in the activation of Pu-ba and
3-fold stronger hi PU-DB activation (Figure 4D).
        We also tested the activities of individual NFATc1 isoforms on the TPA-
mediated induction of the IL-2 promoters after transfection into 293 cells. Luciferase
reporter gene construct governed by full-length murine IL-2 promoter was cotransfected
with NFATc1 expression vectors. All three isoforms increased the induction of IL-2
promoters (Figure 4E), in particular after stimulation with TPA. In this assay,
NFATc 1/B again appeared to be the weakest transactivator, leading to a 2- to 5-fold
weaker activation of the IL-2 promoters than NFATc1/A. Surprisingly, NFATc1/A
exerted the same or even a stronger effect on the induction of both promoters as the
longest isoform NFATc1/C carrying two TADs (Figure 4E).

Molecular cloning of murine NFATc3 cDNA
Full-length cDNA of human NFATc3 was used to screen a murine thymus cDNA
library. High stringency hybridisation resulted in 41 positive plaques. The obtained
Figure 4. Activation of NFAT site-driven reporter genes and IL-2 promoter by NFATc1
isoforms A, B, and C in 293 cells.
A. Expression of NFATc1 cDNAs after transfection into 293 cells analyses by WB using 7A6
Ab. 10 g of expression vectors RSV-NFATc1/A, B, or C were transfected into 293 cells, 20 hr
later the cells were induced by TPA for 3 h and used for the preparation of nuclear and
cytoplasmic proteins. 2 g of proteins were fractionated on 10% SDS PAAG and
immunodetected; C, Cytosolic extracts; N, Nuclear extracts.
B. Dephosphorylation of NFATc1 isoforms. 2 g of nuclear proteins from 293 cells transfected
with RSV-NFATc1/A, /B, or /C were incubated in the absence (-) or the presence of 1 U of SAP
(Ph) at 37°C for 30 min followed by electrophoresis and WB. Note the increase in mobility of
NFATc1 proteins, indicating their previous phosphorylation by an unknown protein kinase.
C. and D. Transactivation of luciferase reporter genes controlled by four copies of the distal
NFAT site of murine IL-2 promoter (Pu-bd) or three copies of an NFAT site from the murine
IL-4 promoter (Pu-bs) by NFATc1 isoforms. DNA, 0.5 g (left panel) or 0.25 g (right panel),
of luciferase reporter genes was cotransfected into 293 cells as indicated with 5 g of RSV-
NFATc1/A or B expression plasmids (left) or RSV-NFATc1/A or C plasmids (right) and the
empty vector control as indicated. The SDs of three experiments are shown.
E. Activation of lymphokine promoter by NFATc1/A, B, and C in 293 cells. 0.25 g of
luciferase reporter constructs driven by the murine IL-2 promoter (up to -293) was
cotransfected into 293 cells as indicated together with 10 g of RSV-NFATc1/A and B (upper)
or NFATc1/A and C constructs (lower). Time of induction is indicated, v, Cotransfection with
an empty expression plasmid.

positive clones were characterised and found to represent three types of a common
cDNA differing in their 3' parts (Figure 5A). The longest of these clones, designated 20,
had 3357 nucleotides and a predicted open reading frame of 903 amino acids with
deduced molecular weight of 98 kDa.
          There were a 92, 98, and 70% ammo acid identity with hNFATc3 hi the N-
terminal, Rel homology domain, and C-terminal portions, respectively. Within the Rel
homology domain, isolated molecule displayed a 67-70% homology to sequences of
other members of the NFATc family that is characteristic feature of the family. The N-
terminal part demonstrated another region containing a conserved motif characterised
by a serine/proline repeat consensus sequence SPxxSPxxSPrxsxt[D/E][D/E]swl, which
is itself repeated three times (Fig. 4B).
         Two other isolated clones, designated 37 and 1, lacked the in-frame initiation
codon and part of the 5’ coding region; therefore they represented a partial cDNA of
alternatively spliced isoforms. The clone 1 had a 111-amino acid deletion of the C-part
of the Rel homology domain replaced with a 13-amino acid unrelated sequence. Clone
37 showed a difference in the 3 ‘-coding region: replacement of 30-amino acids of clone
20 with a distinct 203-amino acid sequence. 3'untranslated parts of all clones also were
different. Although N-terminus heterogeneity of NFATc factors has been reported, we
determined the isolated cDNA clones as alternatively spliced forms differing in their C-
part only. We reconstituted the full cDNA sequences of NFATc3 clones 37 and 1 by
assembling the 5' end of clone 20 containing the initiation codon and the cDNA
sequence of the other two clones respectively. The reconstituted clone 37 (NFATc3-37)




with a predicted length of 1080 amino acids was similar to human NFAT4 [27] and
NFATX [26]. Therefore, NFATc3-37 represented the most abundant form, but the other
two were novel isolated clones of NFATc3 that possibly exist in the cells.

The distribution ofNFATc3 isoforms in lymphoid cell lines
To investigate the distribution of NFATc3 in different cell types and to distinguish the
various isoforms, we raised antisera directed against unique isoforms (Figure 5A). In
addition, we created an antiserum against a conserved motif in the N-terminus of
NFATc3, recognising all of the NFATc3 isoforms. To test the ability of each serum to
recognise its target protein in a specific manner, we prepared immunoblot assay using
nuclear extracts from human 293 cells that were transiently transfected with pNFATc3-
20ex and pNFATc3-37ex. As expected, the antiserum 20d recognised both isoforms, 20
and 37 (Figure 6A). Antisera 20b and 37b specifically reacted with the corresponding
isoforms (data not shown).
        To analyse the distribution of identified isoforms of NFATc3 and to investigate
whether any of them had a predominant expression, we tested several lymphoid cell
lines representing different types and developmental stages - EL4, L-5178Y, Jurkat
TAg, A20J, and 70Z. Cells were treated (or not) with TPA and Iono for different
periods of time, cytoplasmic and nuclear extracts were prepared, and WB experiments
were performed. The same cell extracts were tested in parallel with 20b, 20d, and 37b
antibodies. The 37b antibodies reacted with one single protein of about 155 kDa (Figure
6B, lower panel), the size of which corresponds to that predicted by the reconstituted
clone 37 cDNA. The WB assay with 20d antibodies, recognising all isoforms of
Figure 6. A. The obtained antisera against all NFATc3 recognise isoforms 20 and 37
overexpressed in 293 cells. The 5 g of total protein per sample were separated and
immunodetected.
B. NFATc3-37 is only one isoform expressed in T and B cells. The 20 g of nuclear (even
lines) and cytoplasm (odd lines) proteins from uninduced (1-2, 5-6) or induced with TPA/Iono
for 3h (3-4, 7-8) cells were fractionated and immunodetected using Ab20d recognising all
NFATc3 proteins, or Ab37b recognising only isoform 37.
C and D. NFATc3-37 is constitutively expressed, noninducible, calcium-dependent and CsA-
sensitive. 20 g of fractionated extracts from the A20J (C) and L-5178Y (D) cells, untreated
and treated were separated on the 8% SDS PAGE and immunodetected using Ab37b. C,
Cytosolic extract; N, Nuclear extract.

NFATc3, resulted in one single band with a molecular weight of about 150-160 kDa
(Figure 6B, upper panel), independently of cell type. It was obvious that these two
bands were nearly identical. Surprisingly, there were no bands corresponding to proteins
predicted by clones 1, 20 or other isoforms of NFATc3 (comparing the pattern in the
lines 1 to 8 on the lower panel to the same lines on the upper). The absence of the
NFATc3-20 isoform was also confirmed by the feet that 20b antibodies did not react
with the cell extracts (data not shown).
       To investigate the features of NFATc3-37, we performed experiments using
chemical agents affecting inducibility (TPA) and subcellular translocations (Iono and
CsA) of the NFAT proteins. The same T and B cells were either left untreated, or were
stimulated with Iono or/and TPA in the presence or absence of CsA for 15 min, 3, and
24 hours. Figure 6C and D shows an example of expression pattern and localisation of
protein in two types of cells - A20J (C) and L-5178Y (D). NFATc3-37 is constitutively
expressed in lymphoid lineage cells and its relative amount varies from weak expression
(in Jurkat cells) to high (in EL4 and 70Z, data not shown), which may reflect different
roles of this transcription factor in different types of cells. The ratio between nuclear
and cytoplasmatic protein is different among cell lines (Figure 6C and D, lanes 1-2),
which can be explained by individual features of the cells. Treatment of the cells with
Iono for 15 min leads to rapid translocation of protein into the nucleus, which can be
inhibited partially or completely by CsA (Figure 6C and D, lines 3-6). Prolonged
treatment of cells with TPA or TPA/Iono does not lead to a significant increase of
nuclear nor total NFATc3-37 (lines 7-10), confirming the constitutive noninducible
expression of this protein. Nuclear localisation remains sensitive to CsA (lines 11-12).

The binding activity of endogenous nuclear NFATc3 depends from cell type
Since the published data regarding the binding properties of overexpressed NFATc3 are
very discrepant [82] ,[83] and since transcription factors are often overexpressed to a
level that may permit non-physiological binding in such studies, we investigated the
binding activity of endogenous NFATc3.
         Several bands were detected in EMSA with the distal NFAT site of the murine
IL-2 promoter using nuclear extracts from stimulated and unstimulated Jurkat cells (data
not shown), but the presence of NFATc3 within the complexes could not be proven by
super shift. While the sensitivity of EMSA method is much higher than sensitivity of
WB analysis, and since a weak band corresponding to NFATc3-37 was obtained in WB
with Jurkat cells, we suggest that only a minimal part of nuclear NFATc3 is able to bind
to this site. The one possible reason of that could be the inability of NFATc3-37 to bind
to the NFAT site in context of the IL-2 promoter. Although NFATc3-37 is expressed in
many types of cells, including PBL, its maximal expression has been detected in the
thymus and its main function must lie within this area. From this point of view, the IL-2
promoter specific for PBL (Jurkat cells generally belong to this type) can not be the
appropriate partner for NFATc3 in DNA-protein interactions. Secondly, efficient
binding of NFAT family proteins to this DNA site may obligatory require the presence
of AP-1-related proteins in the binding complex, as it has been observed for some of
them (reviewed in [4]; [70]). In this context, the inability of NFATc3 to form a complex
with AP-1 proteins leads to the inability to bind efficiently to the IL-2 NFAT site.
         To elucidate these suggestions, nuclear extracts from induced or noninduced
EL4 cells and thymocytes were examined in EMSA using the same NFAT site. Two
inducible DNA-protein complexes were observed in the nuclear extracts from EL4 cells
(Figure 7 A, lines 1,3,5). The upper complex gave a clear shift with Ab37b (Figure 7 A,
lines 2,4,6) and therefore represented the NFATc3 complex with a distal NFAT site
from murine IL-2 promoter. TPA/Iono induced thymocytes found several binding
complexes (Figure 7B, line 1). One band, corresponding to the heaviest complex, was
shifted conspicuously by antibodies against NFATc3 (line 2). Additional super shift
experiments using Ab against AP-1 proteins (cFos, FosB, JunB, and JunD) did not
detect a contribution of these proteins in the upper complex nor in the others (lines 3-6),
Additional proof of this is data suggesting that the thymocyte-specific NFAT binding
site of the Lck proximal promoter is a composite element of NFAT and Myb sites, but
not the AP-1 site (A. Avots, C. Stibbe, personal communications).
        Taken together, the source of NFATc3 (the type of lymphoid cell) determines
the binding activity of this protein with respect to different binding sites, while the
origin of the promoters plays only a secondary role. This can be explained by specific
conditions under which NFATc3 binds to the NFAT site with maximum efficiency.
Possibly the involvement of a cofactor that interacts with NFATc3 is required for the
sufficient binding of the complex to the DNA, or perhaps NFATc3 needs to become
activated through specific modification (e.g. phosphorylation) that is not required in the
case of NFATc1 or NFATc2. In any case, these specific conditions occur in the
thymocytes and other lymphoid cells, but not in PBL or related cells (Jurkat).

The transcription activity ofNFATc3 isoforms
To determine whether NFATc3, in addition to binding to DNA in vitro, could also
activate transcription from NFAT site in vivo, the NFATc3 expression constructs were
overexpressed into 293 cells. Neither pNFATc3-20ex, nor pNFATc3-37ex, was able to
induce expression of reporter gene driven by three copies of the distal NFAT binding
site from murine IL-2 promoter into 293 cells, over those in cells transfected with mock
plasmid DNA as a control, independently on stimulation (data not shown). WB and
EMSA experiments using extracts from transiently transfected 293 cells confirmed a
very high level of expression, but detected only weak binding of NFATc1 and did not




Figure 8. Transcription activity of the NFATc3 isoforms 37 and 20.
A. Neither NFATc3-37 nor NFATc3-20 expression construct is able to transactivate the distal
NFAT site from IL-2 promoter. 3 g of expression vectors pRSV-NFATc1/A,
pNFATc2,
pNFATc3-37ex, pNFATc3-20ex or mock plasmid were cotransfected into Jurkat cells with 1 g
of a luciferase reporter gene construct driven by three copies of the distal NFAT site from the
IL-2 promoter.
B. Both NFATc3-20 and NFATc3-37 are able to activate transcription via an NFAT binding site
within the Lck proximal promoter. 3 g of expression plasmids were cotransfected into Jurkat
cells with 1 g of luciferase reporter construct driven by four copies of NFAT I site from the
Lck proximal promoter.
detect any notable binding of overexpressed NFATc3 (data not shown). Similar results
were observed in the transfections of Jurkat cells (Figure 8A). Transfection of the
NFATc1 expression construct resulted in an increase of the inducible level of reporter
gene expression. Despite this, there was no effect observed on the level of reporter gene
production in cells transfected with the pNFATc3-37ex or pNFATc3-20ex plasmids.

         Next, the NFATc3, NFATc2 and NFATc1 cDNA expression constructs were
cotransfected into Jurkat cells with the reporter gene construct containing four copies of
the NFATI binding site from the Lck proximal promoter. The Lck proximal promoter is
active in the thymus only at an early developmental stage of T lymphopoesis, however
it is essentially silent in peripheral T cells ([84]). Transfection of each of the NFATc
family members resulted in an increase in the basal level of reporter gene expression in
unstimulated Jurkat cells (Figure 8B), as compared to that in the cells transfected with
mock plasmid as a control. NFATc3-37 appeared the strongest trans-activator within the
family in the context of Lck promoter, activating reporter gene production by tenfold
under the nonstimulating conditions. Upon stimulation, cells transfected with
pNFATc3-20ex and pNFATc3-37ex did not show a significant increase in the level of
reporter gene product in comparison to unstimulated cells. The transcription activity of
pNFATc3-20ex and pNFATc3-37ex, in contrast to that of NFATc1 and NFATc2 was
completely abolished by CsA.

Concluding remarks
All members of NFATc transcription factor family are expressed in multiple isoforms.
This has been shown in detail for NFATc2 and NFATc3, which are expressed in several
isoforms in T lymphocytes and other cells [11, 14]. The NFATc1 isoforms A, B and C
described here are the most prominent NFATc1 proteins in lymphoid cells. In addition
to alternative splicing at the 3'end, the detection of a different 5'peptide in NFATc1/C
cDNA (Figure 3A) and published data [12, 13] suggest the existence of 5'heterogeneity
in NFATc1 proteins. All three major NFATc1 proteins contain a strong transactivation
domain, TAD-A, near the N terminus (Figure 3A, [34]).
        Contrary to NFATc2 that is constitutively synthesised in numerous lymphoid
cells, NFATc1 was reported to be inducibly expressed in T lymphocytes [13, 24].
However, this only is true for NFATc1/A, the shortest of three isoforms. In contrast, the
two longer isoforms NFATc1/B and C are constitutively synthesised in many T cells,
similar to NFATc2. All three NFATc2 isoforms are similar in length or even longer
than NFATc1/C, the longest NFATc1 isoform. A short isoform lacking an extra C -
terminal peptide, as does NFATc1/A, has also been described for NFATc3 [27], but it
never have been shown whether it is inducibly synthesised like NFATc1/A. We also
isolated three types of murine NFATc3 cDNA, one of them was short and lacked C-
terminal part, therefore reminded NFATc1/A. However, further experiments identified
long isoform NFATc3-37 as a prominent NFATc3 protein constitutively expressed in
the lymphoid cells and did not found any protein corresponded to others cDNAs.
NFATc3-37 protein is similar NFATc3 proteins, identified by other groups - human
NFAT4 [27] and NFATX [26].
        The C-terminal peptide of NFATc1/C shows >30% homology to a C-terminal
QP-rich stretch of approximately 220 aa shared by NFATc2 isoforms and is able to act
as a TAD [18]. Such a second TAD has also been identified within the C-terminal
portion of the longest isoform of NFATc3, (early designated as NFATxl [14]). Thus, the
existence of a C-terminal TAD is not a peculiarity of the long NFATc1 isoform C but
is a typical component of numerous NFAT proteins.
        The identification of TAD-B in NFATc1/C raises the question of which roles
these isoforms play in gene control in T lymphocytes and other cells where they are
expressed at different relative levels. When tested in transient transfections assays for
promoter induction, NFATc 1/A and C showed a very similar, if not identical, strong
stimulatory effect on the IL-2 (Figure 4E) and IL-4 [17] promoters while NFATc 1/B
reached about 50% of this effect. Although NFATc factors alone are poor activators of
the IL-5 promoter, in cooperation with other transcription factors, such us GATA-3 and
Ets-1, they strongly stimulate the protein kinase A-mediated IL-5 promoter induction
[66]. No marked differences among the three NFATc1 isoforms in respect to IL-5
promoter-driven reporter gene expression were detected when they were cotransfected
with GATA-3 or/and Ets-1 [17]. The conspicuous differences between the NFATc1
isoforms in activation of the IL-2 and IL-4 promoters, on the one hand, and of the IL-5
promoter, on the other, suggest important functional roles of individual isoforms in
promoter control. Several lines of evidence indicate that threshold levels of NFATc play
a crucial role in the induction of promoters in T cells. In addition, due to the different
transcriptional capacities of NFATc isoforms, changes in isoform composition will
result in marked differences in specific transcriptional activity of nuclear NFATc1.
        Unlike other NFATs, NFATc3 did not show efficient binding to the distal NFAT
binding site from murine IL-2 promoter in the Jurkat cells (Figura 7A), and both
NFATc3-20 and NFATc3-37, were not able to activate significantly transcription from
the distal IL-2 NFAT DNA binding site. At the same time both could activate
transcription via the NFAT binding site within the Lck proximal promoter (Figure 8).
These results, together with the data on Lck promoter footprinting (A. Avots, personal
communications), our EMSA results, and the expression of NFATc3 and NFATc2 in
the thymocytes suggest that the main biological role of the NFATc3-37 lies in the
regulation of the genes during the development of the thymocytes, especially lck gene.


 THE DIFFERENT EFFECT OF IMMUNOSUPRESSOR CYCLOSPORIN A ON
 THE INTRACELLULAR TRAFFICKING AND FUNCTIONAL ACTIVITY OF
                NFAT TRANSCRIPTION FACTORS

How it has been described in details in Literature Review, all NFAT femily members
have been reported as calcium/calcineurin-pathway-dependent [4, 33] and sensitive to
the immunosupressive drugs CsA and FK506 [43]; reviewed in [85]. After induction of
cells, NFAT factors rapidly translocate from cytoplasm to nucleus. The treatment of
cells with immunosuppressant drugs blocks this translocation. However, our previous
studies indicated that CsA might not affect nuclear localisation of NFATc1. In the
presented work we investigated the cytoplasmic/nuclear localisation and conditions of
nuclear translocation of NFATc1 in comparison with NFATc2 and NFATc3 in various
human and murine T- and B- cell lines at distinct differentiation stages.

Nuclear export of NFAT proteins is differentially affected by CsA
To investigate the cytoplasmic/nuclear localisation of NFATc proteins, several
lymphoid cell lines representing different types and developmental stages - EL4, L-
5178Y, Jurkat TAg, A20J, and 70Z were treated with TPA and Iono in presence or
absence of CsA for 15 min and 3 hours, cytoplasmic and nuclear extracts were prepared
and immunoblot assays were performed. The same cell extracts were run in parallel on
three gels and tested with antibodies against NFATc1, NFATc2, and NFATc3. Figure 9
shows the localisation of endogenous NFATc1 (A), NFATc2 (C), and NFATc3 (B) in
Figure 9. A, B, C. Cytoplasmic/nuclear localisation and translocation of NFATc proteins in
EL4 cells. 20 g of fractionated extracts from cells differentially treated in the presence or
absence of CsA were separated on the SDS PAAG and immunodetected using antibodies
against NFATc1 (A), NFATc2 (C) and NFATc3 (B).
D. Hyperphosphorylation of NFATc1 and NFATc3 in L-5178Y cells after treatment with CsA.
Green arrows indicate the dephosphorylated form; red arrows indicate the hyperphosphorylated
form of NFATs. C, cytosolic extract; N, nuclear extract; the absence of contamination of
extracts was proved using antibodies against cytoplasm-specific protein lactate dehydrogenase
and nuclear protein upstream stimulatory factor (data not shown).
E. Dephosphorylation of NFATc3 in A20J cells. 20 g of fractionated extracts from A20J cells
treated as indicated were incubated in the absence (-) or the presence of 1 U of SAP (Ph) at
37°C for 30 min followed by electrophoresis and WB. Note the increase in mobility of NFATc3
proteins from the cells treated with CsA after dephosphorylation by SAP, indicating then -
previous phosphorylation.

differentially treated EL4 cells. The cytoplasmic fraction of untreated cells indicated
very low amounts of NFAT proteins. This could be due to higher dilution of proteins
during preparation of extracts. The three major isoforms of endogenous NFATc1,
described in the first part of Results, similarly to NFATc2 and NFATc3, were localised
in the nuclear fraction of untreated EL4 cells (Figure 9 A, B, and C, lines 2). The
treatment of cells with Iono for 15 min led to additional accumulation of protein in the
nucleus (compare lines 4 to lines 2), as it could be expected. Prolonged treatment of
cells with TPA and Iono led to a considerable increase of nuclear protein (lines 8) due to
the synthesis de novo. Treatment of cells with CsA before stimulation inhibited nuclear
import of NFATc2 and NFATc3 and caused their partial accumulation in the cytoplasm.
Under the same conditions, the nuclear residence of NFATc1 was not affected (Figure
9A, lines 5-6, 9-10). Similar results were obtained when cells were treated with CsA
alone (Figure 9D).
        The WB assays using extracts from the other cell lines showed similar results
(data not shown) - while NFATc2 and NFATc3 always remained sensitive to CsA and
were exported back to cytoplasm, NFATc1 accumulated in the nucleus and was not
affected by CsA. An individual threshold level of calcineurin for each cell line may
Figure 10. The complex-formation between the distal NFAT site of the murine IL-2 promoter and
NFATc1 proteins in the lymphoid cells is not disturbed by CsA treatment. EMSA was carried out
with 2 or 2.5 g of nuclear proteins from EL4 (A) or Jurkat (B) cells treated as indicated. 1 l of
undiluted Ab 7A6 was used for super shift (ss). The specific complexes detected are indicated by
black arrows. The complexes shifted by Ab are indicated by green arrows. Note the shift of DNA-
protein complexes from the cells treated with CsA.

explain the minor differences in expression pattern within different cell lines. For
example, the NFATc1 pattern in resting EL4 cells is explained by the mutation in
calcineurin gene that leads to high constitutive activity of endogenous calcineurin [86]. Two
additional observations should be noted here. First, the relative concentrations of NFATc2
and NFATc3 in the cytoplasmic fraction from cells treated with CsA seemed to be
considerably higher in comparison to that in the untreated cells (Figure 9B and C, compare
lines 5-6 to 1-2). Such an increase in concentration could not be explained only by
disruption of equilibrium between import and export of NFAT proteins by CsA. We
therefore propose that additional NFAT was released from the subcellular compartment
where it was not accessible during regular preparation of cell extracts. The intracellular
membranes may be an example of such a compartment and CsA treatment may serve as a
signal for the release of attached NFAT. Second, the treatment of cells with CsA led to
hyperphosphorylation of all NFAT factors regardless to their localisation, which
resulted in a decrease in mobility in the PAAG and a shift in WB (Figure 9A, B, and C,
lines 5-6 and 9-10). Dephosphorylation of nuclear proteins by treatment with SAP led to an
increase in their electrophoretic mobility (Figure 9E). The treatment of resting cells with
CsA alone led to the same results (Figure 9D, lines 3-4 and 7-8), supporting the notion that
such hyperphosphorylation occurs independent of the calcium/calcineurin signal.
        The nuclear localisation of NFAT proteins itself does not guarantee its binding
activity. In addition, results obtained in EMSA experiments using the distal NFAT site of
the murine IL-2 promoter confirmed complex-formation between the NFAT binding site
and NFATc1 proteins (Figure 10). Several DNA-protein complexes, apparently shifted by
antibodies against NFATc1, were observed with extracts from EL4 (Figure 10A), Jurkat
(Figure 10B), and A20J cells (data not shown). The treatment of cells with CsA did not
disturb the complex-formation between NFAT binding site and NFATc1 protein,
independently of the cell type. Moreover, comparison between EMSA pattern
of EL4 cells, nonstimulated and treated with CsA alone (Figure 10A, compare lines 1-2
with 5-6) or differently stimulated in the presence or absence of CsA as indicated
(compare lines 3-4 with 7-8, 9-10 with 11-12 in Figure 10A, and also lines 9-10 with
11-12 in Figure 10B) revealed a slight shift of the bands, similar to the shift observed in
WB.

Cyclosporin A has no inhibitory effect on the transhcation of NFATc1/A-GFP
To consolidate the WB results, we constructed chimeric proteins consisting of NFATc1 or
NFATc3 and GFP. The chimeric constructs pF143-NFATc1 and pF143-NFATc3 were
overexpressed in 293 cells and the subcellular localisation and CsA-dependent nuclear
import/export of overexpressed proteins were examined by confocal microscopy.
       The overexpressed NFATc3-GFP was localised in the cytoplasm of
unstimulated 293 cells (Figure 11, image 4). The stimulation of cells with Iono resulted in
the nuclear import of NFATc3-GFP, while the same treatment in the presence of CsA
prevented it and allowed protein to remain in the cytoplasm (Figure 11, 5-6).




        The other protein, NFATc1-GFP was expressed at a very high level and in the
resting 293 cells was present in both compartments, but mostly in the nucleus (Figure
11, image 1). Similarly to NFATc3, stimulation of cells with Iono caused the
translocation of NFATc1-GFP to the nucleus (Figure 11, 2). However, the presence of
CsA during induction with Iono did not inhibit nuclear import of NFATc1-GFP (Figure
11, 3). Several cells with NFATc1 localised completely in the cytoplasm were found in
uninduced and CsA/Iono-treated samples (Figure 11, images 7-8).

The transcription activity of the overexpressed NFATc1 is not CsA-sensitive in
Jurkat cells
In order to test whether CsA inhibits transcription activity of NFATc1 factors, the
NFATc1/A and NFATc3 expression vectors were cotransfected into Jurkat cells with a
reporter gene construct driven by four copies of NFAT I site from the Lck proximal
promoter. Figure 12A shows that transfection of both pRSV-NFATc1/A and pNFATc3-
37ex plasmids resulted in an increase in the inducible level of reporter gene expression.
Either inducible or basic transcription activity of overexpressed NFATc3 was
completely abolished in the presence of CsA. There was no effect on the level of
reporter gene production after the same treatment in the cells transfected with pRSV-
NFATc1/A plasmid. However, reporter gene expression was clearly inhibited in the
presence of CsA in the cells transfected with mock plasmid DNA as a control. Thus, the
endogenous NFAT factors (NFATc1, NFATc2, and NFATc3) in toto are sensitive to
the CsA.
        Summarising data on functional studies we suppose that the difference of
NFATc1 versus NFATc2 and NFATc3 could be quantitative rather qualitative, which
would mean that translocation of NFATc1 is generally less sensitive to CsA than in case
of the other NFAT factors. Confirmation experiments were performed by inducing
Jurkat cells transfected with pRSV-NFATc1/A and pNFATc3-37ex plasmids with
TPA/Iono in the presence of increasing concentration of CsA. The use of a two-fold
higher concentration of CsA led to approximately a 30% decrease in the level of
NFATc1/A activation (Figure 12B) in comparison to the cells treated with the
conventional concentration of CsA. A further increase of the concentration of CsA did
not result in significant inhibition of inducible transcription activity of NFATc1.




Figure 12. Cyclosporin A has no inhibitory effect on reporter gene transactivation by
overexpressed NFATc1 in Jurkat cells. Standard conditions of stimulation (A) and increasing
concentrations of CsA (B) were used.
3 g of expression plasmids were cotransfected into Jurkat cells with 1 g of luciferase reporter
construct driven by four copies of the NFAT I site from the Lck proximal promoter. The chart
legends show the treatment of the cells: 2xCsA, two-times standard concentration of the CsA;
3xCsA, three-times standard concentration, et cetera. DMSO+TPA/Iono, control for the effect
of solvent (CsA is dissolved in DMSO).
        * not detected for mock transfection.

       In the next experiment Jurkat cells were transiently transfected with different
amount of pRSV-NFATc1/A expression plasmid and induced with TPA/Iono in the
presence of CsA in conventional concentration. The results of this experiment (not
shown) revealed that very low amounts of expression vector (up to 200 ng) did not
increase the reporter gene activity over those in mock sample and did not affect it's CsA
sensitivity. The further increase in amount (from 400 ng up to 1.5 g) resulted in
decrease of sensitivity to CsA. At the same time the level of reporter gene expression
was comparable to that in mock transfection or only slightly higher. Under the
concentration conveniently used in the functional assay (3-5  g/sample),
transcription activity of overexpressed NFATc1/A was maximal but there was no
inhibitory effect of CsA observed. Taken together, the transcription activity of
overexpessed NFATc1/A was not inhibited by CsA, while the pool of endogenous
NFATc factors was sensitive to CsA and transcription activity was abolished. In
the similar experiment using Jurkat cells transfected with different amount of
pNFATc3-37ex plasmid, reporter gene expression was completely inhibited in
the presence of CsA independently on expression plasmid concentration.

The immunofluorescence staining of endogenous NFATc1 in the Jurkat
and EL4 cells
Proteins of the NFAT family, overexpressed in the 293 cells, are not always
regulated according to physiological mechanisms in the lymphoid cells (see
previous part). The transfection efficiency in Jurkat cells was too low to use the
NFATc1-GFP and NFATc3-GFP constructs for fluorescent microscopy. Instead,
immunofluorescence staining using antibodies against endogenous NFATc1 was
performed with Jurkat and




       Therefore, CsA decreased nuclear import of endogenous NFATc1 in the
EL4 cells, but did not affect the NFATc1 overexpressed in the 293 cells. Similar
data were obtained in an experiment using Jurkat cells (data not shown).
Concluding remarks
 We investigated the cytoplasmic/nuclear localisation and conditions of nuclear
 translocation of NFATc1, NFATc2 and NFATc3 in various T- and B- cell lines. The
 WB studies showed a marked difference in the translocation manner between NFATc1
 and other NFAT factors. Whereas the nuclear residence of NFATc2 and NFATc3 was
 always sensitive to treatment with CsA and these factors demonstrated cytoplasmic
 distribution (Figure 9B and C), NFATc1 was not affected by CsA and accumulated in
 the nucleus (Figure 9A). The similar results were obtained in the EMSA experiments
 using the distal NFAT site of the murine IL-2 promoter. Independently of the cell type,
 the treatment of cells with CsA did not disturb the complex-formation between NFAT
 binding site and NFATc1 protein, but resulted in a slight shift of the bands (Figure 10),
 similar to the shift observed in WB.
         The examination of 293 cells, overexpressing chimeric proteins NFATc1/A-
GFP and NFATc3-GFP, by confocal microscopy showed that nuclear import of
NFATc3-GFP in stimulated cells was completely inhibited by CsA, while NFATc1/A-
GFP accumulated in the nucleus and was not affected by CsA (Figure 11).
Furthermore, functional studies using a reporter gene construct driven by four copies
of the NFAT I site from the Lck proximal promoter demonstrated a high level of
transcription activity of NFATc1 under conditions when transcription activity of
NFATc3 was abolished by CsA (Figure 12A).
         Thus, the observation of different responses of NFAT proteins to the treatment
with inhibitor CsA was validated by different methods, WB and EMSA in the case of
endogenous NFATc1 and confocal microscopy and transient transfection assay for the
overexpressed NFATc1. We suggest that translocation of NFATc1 is generally less
sensitive to CsA than in case of the other NFAT factors that is quantitative difference of
NFATc1 versus NFATc2 and NFATc3. The transient transfection experiments
performed with increasing concentration of CsA did not provide obvious supportive
evidence for that hypothesis. A five-fold increase of CsA did not significantly affect
NFATc1/A transcription activity (Figure 12B). Next, the titration of NFATc1/A
expression vector showed that level of reporter gene production, considerably exceeding
those in cells transfected with mock plasmid as a control, is not inhibited by CsA (data
not shown), while transcription activity comparable to that of endogenous NFATc in
control sample is abolished by CsA.
         The results of immunofluorescence staining using antibodies against
endogenous NFATc1 performed with Jurkat and EL4 cells showed that CsA was able to
inhibit nuclear translocation of endogenous NFATc1 (Figure 13). Nevertheless, slight
differences in the subcellular localisation in untreated and CsA treated cells could be
observed. In untreated cells, NFATc1 was evenly distributed throughout the cytoplasm
(Figure 13, image 3). After CsA treatment NFATc1 was mainly localised close to
nuclear surface (Figure 13, image 9). We suggest that localisation of NFATc1 near the
nuclear surface and its possible interaction with the nuclear membrane could be a cause
of the consequent presence of protein in the nuclear fraction of cell extracts. Evidence to
support this notion was a high concentration of NFAT proteins (data not shown) in WB
experiments using nuclear membrane fractions.
        Inhibition of the reporter gene production by CsA in the mock sample (Figure
12A), where transcription activity of transfected NFATc1/A did not decrease, can be
explained in two ways. First, the pool of endogenous NFAT proteins in the Jurkat cells
consists mainly not of NFATc1, but of NFATc2 and NFATc3, which are inhibited by
CsA. However, the WB experiment showed that NFATc2 and NFATc1 are the major
forms expressed in these cells and that NFATc3 was much less expressed. Second, the
Figure 14. The scheme of nuclear/cytoplasmic shuttling of transcriptions factors NFATc2/
NFATc3 (a-c) and NFATc1 (d-f).
(a; d) The NFAT transcription factors are continuously subjects of transport, (b; e) The
calcium-activated phosphatase calcineurin dephosphorylates NFAT's, resulting in their nuclear
import. Dephosphorylated NFATc1 can form granular structures, (c) The immunosuppressant
CsA inhibits calcineurin and therefore prevents nuclear import of NFATc2 and NFATc3, and
enhances their return to the cytoplasm, (f) After exposure to the CsA, the majority of NFATc1
remains in the nucleus and localises near the nuclear membrane. The arrows indicate the
direction of change in the balance between import and export of NFATc factors.

regulatory mechanism of the Jurkat cells, similarly to 293 cells, was not able to control
properly the transcription activity of NFATc1, if it was overexpressed at very high
level. NFATc 1-GFP when expressed in excess in 293 cells also formed granular
structures within the nuclei (Figure 11, images 1-3). We suggest that these structures
were formed by overexpressed protein itself or in interaction with other nuclear
elements, and once being formed, further disturbed nuclear export. Such granular
structures, therefore, could retain protein in the nucleus independently of the presence of
CsA.
        Our results suggest a differential effect of CsA on the endogenous and
overexpressed NFATc1 and distinct behaviour of NFAT factors in response to treatment
with CsA. While CsA inhibited transcription activity of endogenous NFATc1, it had no
effect on the overexpressed protein.
        Our observations are summarised in Figure 14. In the uninduced state NFAT's
are localised in both nucleus and cytoplasm (Figure 14, a; d). After induction via the
calcium pathway, the majority of the NFAT's rapidly translocates to the nucleus and
only a minor fraction of the transcription factor remains in the cytoplasm (Figure 14, b;
e). NFATc1 in a high concentration forms granular structures in the cell nuclei (Figure
14, e; f). The treatment of cells with CsA blocks nuclear translocation of NFAT's and
enhances nuclear export of NFATc2 or NFATc3 (Figure 14, c), but exerts considerably
less effect on NFATc1 (Figure 14, f). Despite the effect of CsA, the majority of
NFATc1 does not leave the cell nucleus.
         THE COMPLEX EFFECT OF PROTEIN KINASE A ON THE
        TRANSCRIPTIONAL ACTIVITY OF NFAT FAMILY PROTEINS

The relations between two kinases - GSK-3 and PKA and their involvement in the
regulation of NFATc1 lie in the focus of this chapter. G. Crabtree and collegues
described that GSK-3 phosphorylates NFATc1 only if it was first phosphorylated by
PKA. PKA phosphorylates the NFATc1 directly at serines in positions 245, 269 and
294. The phosphorylation of PKA sites at Ser245 and Ser294 creates a series of overlaping
GSK-3 substrate sites and therefore, PKA should be determinated as priming kinase for
GSK-3 [44, 46, 47]. Recent data suggesting that interaction between PKA, GSK-3 and
NFAT is not described by simple scheme: priming kinase - NFAT kinase - NFAT as a
substrate. Here we investigate the molecular effects of PKA and GSK-3 on the function
of NFATc1 in cells.




Figure 15. GSK-3 has an inhibitory, but PKA a stimulatory effect on the level of transactivation
of the distal NFAT site from IL-2 promoter by overexpressed NFATc1/A (A), NFATc1/C (B),
NFATc3 (C) and NFATc2 (D).
0.4 g (A and B) or 1 g (C and D) of expression vectors pRSV-NFATc1/A, pRSV-
NFATc1/C, pNFATc2, pNFATc3ex or corresponding amount of mock plasmid were
cotransfected into Jurkat cells with 1 g of a luciferase reporter gene construct driven by three
copies of the distal NFAT site from the IL-2 promoter alone or together with GSK-3 or PKA
expression vector, in following amount - 0, 0.4, 0.8, 1.2, and 1.6 g of each or as
combination of 1.2 g of both. Results of one representative experiment are shown here. Red
columns indicate nonstimulating, but green columns stimulating conditions. m, mock plasmid.
         In order to determine the effect of GSK-3 and PKA on the NFAT transcriptional
 activity, NFATc1/A was coexpressed in Jurkat cells either with increasing amount of
 GSK-3, or PKA, or both kinases together, and activities of reporter genes driven by
 three copies of the distal NFAT binding site from murine IL-2 promoter were measured.
 Figure 15A demonstrates that overexpression of GSK-3 led to a strong inhibition of
 NFAT-dependent reporter gene production, while overexpression of PKA resulted in
 significant increase in the level of NFATc1/A transcriptional activity. Parallel
 transfections, performed with pRSV-NFATc1/C, pNFATc2 and pNFATc3-37ex
 constructs, did not show as strong transactivation as NFATc1/A, but, in general,
 demonstrated similar pattern (Figure 15B - D).
         In order to clarify is this up-regulation of reporter gene production a
consequence of activation of NFAT or of AP-1 component, we repeated the same
experiment using different reporter gene driven by four copies of NFAT I site from the
Lck proximal promoter (data not shown). This site is a composite element of NFAT and
Myb sites (A. Avots, C. Stibbe, personal communications) and does not contain AP-1
component. Like in the first experiment, overexpression of GSK-3 resulted in
diminishing of transcriptional activity of all coexpressed proteins - NFATc1/A,
NFATc 1/C, NFATc2, and NFATc3 In contrast, the presence of PKA did not show any
significant effect on the level of reporter gene production in the cells expressing NFATc
proteins (data not shown). These results suggested that up-regulating effect of the PKA
on the reporter gene driven by three copies of the distal NFAT binding site from murine
IL-2 promoter is explained by acting prevalently at the level of AP-1 component, and
not NFATc component. Another explanation may be the presence of Myb binding site
within the structure of NFAT I site. The influence of PKA on the reporter activity via
Myb proteins is still unclear although both direct interaction between PKA and Myb and
involving of other proteins in such interaction have been studied [87-89].
         The strong inhibition of NFAT transcriptional activity by GSK-3 in the absence
of PKA controverts with previously published data suggesting that GSK-3 completely
opposes ionomycin-induced nuclear accumulation of NFATc1 only in synergism with
PKA [46]. A possible explanation is that the limited endogenous PKA activity is
sufficient to prime overexpressed GSK-3 or it may efficiently act with the aid of other
endogenous kinases.
         To create the conditions when any possibility of PKA to affect the level of
reporter gene production via activating AP-1 proteins is completely excluded we
constructed a Ga14/NFATc1 chimeric protein. This protein consists of the 2-400 aa of
NFATc1/A harbouring predicted binding sites for the PKA and GSK-3 kinases, fused to
the 1-147 aa of the yeast transcription factor Gal4 containing the DNA binding and
dimerization domain. The use of such chimeric protein in cotransfection with luciferase
reporter gene construct driven by five copies of Gal4 binding site avoids activation of
reporter gene by endogenous NFATc proteins and influence of other transcription
factors, particularly AP-1. The chimeric constructs pGal4/2-400NFATc1 and pGal4/2-
400NFATc1m269, where serine 269 has mutated to alanine, were coexpressed in Jurkat
cells with the PKA in different concentrations and activity of reporter gene was
detected. Figure 16 shows that PKA enhanced NFATc-dependent production of reporter
gene in cells transfected with both constructs - normal and mutated. This is similar to
our results obtained in experiments using reporter driven by three copies of the distal
NFAT binding site from murine IL-2 promoter, therefore proves that PKA affects the
level of reporter gene production by enhancing of transcriptional activity of NFATc
proteins, but without involving AP-1 proteins. Next, the reporter gene production in the
Figure 16. PKA enhances the transcription activity of both Gal4/NFATc1 chimeric proteins -
wild type and those where serine 269 has mutated to alanine. 1 g of pGal4/2-400NFATc1,
pGal4/2-400NFATc1m269 or mock plasmid were cotransfected into Jurkat cells with 1 g of
luciferase reporter construct driven by five copies of Gal4 binding site and PKA expression
vector in amount 0, 0.4, 0.8, and 1.6 fig. Results of one representative experiment are shown
here.

cells transfected with mutated construct pGal4/2-400NFATc1m269 was significantly
higher in comparison to cells transfected with wild type pGal4/2-400NFATc1. This
means that inactivation of one of PKA phosphorylation sites results in up-regulation of
NFATc1-mediated transcriptional activity.

Concluding remarks
Taking together, the results of our experiments using full-length expression vectors as
well Gal4/NFATc chimeras demonstrated that overexpression of GSK-3 dramatically
inhibited transcriptional activity of all coexpressed proteins - NFATc1/A, NFATc1/C,
NFATc2 and NFATc3 (Figure 15), while overexpression of PKA significantly
increased the level of NFATc-mediated reporter gene production (Figure 15, 16).

         Generally, the effect of PKA on the NFAT mediated reporter gene production
does not have a single meaning, but is a result of several different mechanisms (Figure
17).
        (1) The PKA works as the priming kinase for GSK-3. It has been shown that
GSK-3 phosphorylates NFATc1 only if it is first phosphorylated by PKA at Ser245 and
Ser294 that creates a series of overlaping GSK-3 substrate sites [44, 46, 47]. Then GSK-3
phosphorylates NFATc1 (Figure 17, pathway 1) and, similar to several other kinases,
exports it to the cytoplasm and therefore decreases transcriptional activity of NFATc1.
        (2) PKA phosphorylates GSK-3 itself and inactivates it [90] that eventually
inhibit the export of NFAT and could result in increase of transcriptional activity
(Figure 17, pathway 2).
        (3) PKA phosphorylates NFATc1 directly at serines in positions 245, 269 and
294. The Ser245 and Ser294 are necessary for further phosphorylation by GSK-3 [46], but
Ser269 might be responsible for the direct effect of PKA on NFATc1 (Figure 17,
pathway 3).
Figure 17. The scheme explaining the effect of PKA on the NFAT mediated reporter gene
production as a sum result of several different mechanisms: 1) PKA works as the priming kinase
for GSK-3; 2) PKA inactivates GSK-3 by phosphorylation; 3) direct effect of PKA on the
NFAT transcriptional activity; 4) PKA activates transcription via AP-1. See Concluding
remarks for the details.

        (4) At last, the distal NFAT site from murine IL-2 promoter, often used as a part
of a reporter construct in the transcriptional activity assays, can bind AP-1 proteins [36,
91]. At the same time, PKA can increase AP-1 activity [92, 93], therefore it is possible
that up-regulation of reporter gene production occurs via activation of AP-1 component
(Figure 17, pathway 4) and this effect may mask the real effect of PKA on the
transcriptional activity of NFATc1. The use of Gal4/NFATc1 chimeric proteins instead
of full-length NFATc1 avoids the involvement of AP-1 proteins and prevents the
distortion of the results.
        The first two mechanisms (Figure 17, pathways 1 and 2), GSK-3 priming by
PKA and GSK-3 inactivation by PKA, start at the same point of pathway, carry on
simultaneously and lead to the opposite results. Because of this it is very difficult to
distinguish these two mechanisms and their effects in the frames of our experiment. The
outcome of the third mechanism may be distinguished from the previous two. We
assume that direct effect of PKA on the NFATc1 is accomplishing through its
phosphorylation at Ser269 (Figure 17, pathway 3) and replacement of serine to alanine in
this position will disrupt this effect. Mutation of Ser 26 led to increase in the level of
NFAT-dependent reporter gene production (Figure 16) and therefore suggested that
phosphorylation of this site negatively affects NFATc1 transcriptional activity.
        Altogether, we propose that in the different experiments under the diverse
conditions, various mechanisms play a prevalent role. The NFATc proteins,
overexpressed alone in the cells (Figure 15) are regulated by limited amounts of
endogenous GSK-3 and PKA that resulted in moderate level of transcriptional activity.
The coexpression of GSK-3 (Figure 15, data not shown) causes running of first
mechanism and abolishing of NFATc-mediated transcriptional activity, while results of
others two are masked, because the endogenous PKA is not sufficient to inactivate
overexpressed GSK-3. The coexpression of PKA with NFATc1 (Figure 15, 16) causes
inhibition of GSK-3 over usual level, deficiency of its active form and, as a result, arrest
of the first mechanism. Therefore, in that case, NFATc1 transcriptional activity is a
cumulative effect of the second and the third pathways.
        The cells transfected with wild type or mutated Gal4/NFATc1 chimeric
constructs are equally processing first two mechanisms while differ in respect to third
pathway, which is inactivated in the cells expressing mutated fusion protein. This allow
us to conclude that direct effect of PKA on the transcriptional activity of NFATc1 really
exists, otherwise, there would not be difference between cells expressing wild type and
mutant fusion proteins, and identify it as negative.
                                 CONCLUSIONS

> Three isoforms of human transcription factor NFATc1, designated NFATc1/A,
  NFATc1/B and NFATc1/C, were cloned and characterised. The shortest isoform
  NFATc1/A corresponded to NFATc1 described previously, the two longer isoforms
  NFATc1/B and NFATc1/C spanned extra C-terminal peptides. The shortest isoform
  NFATc1/A is only one inducibly expressed in many T and B cell lines, and in
  Th1/Th2 cells as well.

> Three novel types of murine NFATc3 cDNA, designated an NFATc3-37, NFATc3-
  20 and NFATc3-l were isolated and determinated as isoforms alternatively spliced
  at the C-part. Using a number of raised specific antisera, NFATc3-37 isoform was
  identified as a prominent form of NFATc3, constitutively expressed in the lymphoid
  cells.

> An endogenous NFATc3-37 from thymocytes or EL4 cells, but not from Jurkat
  cells, was able to bind to the distal NFAT site from the murine IL-2 promoter. This
  binding did not require the involvement of AP-1 related proteins. None of the
  isoforms were able to activate the transiently transfected reporter gene driven by
  NFAT site from the IL-2 promoter. However, both NFATc3-37 and NFATc3-20
  activated transcription via the NFAT I site from the Lck proximal promoter even
  without induction.

> Distinct behaviour of NFATc factors in response to treatment with
  immunosuppressant cyclosporin A (CsA) was observed. Whereas the nuclear
  residence of NFATc2 and NFATc3 was always sensitive to treatment with CsA, i.e.
  these proteins were exported back to the cytoplasm, NFATc1 was considerably less
  affected by CsA and did not leave the cell nucleus. NFATc1 in high concentration
  forms granular structures within the cell nuclei. The treatment of cells with CsA led
  to a decrease in the mobility of NFAT proteins and DNA-protein complex during
  electrophoresis due to hyperphosphorylation. These observations were validated by
  western blots, EMSA, immunostaining, and confocal microscopy of chimeric
  NFAT-GFP. In addition, functional studies showed that CsA inhibits transcription
  activity of endogenous NFATc1, but it has no effect on the overexpressed protein.

> Overexpression of GSK-3 had dramatic inhibitory effect on the transcriptional
  activity of all coexpressed proteins - NFATc1/A, NFATc1/C, NFATc2
  and
  NFATc3, while overexpression of PKA significantly increased the level of NFATc-
  mediated reporter gene production.

> The functional assay using Gal4/NFATc1 chimera suggested that effect of PKA is
  not mediated via activation of AP-1 proteins. Replacement of serine to alanine in
  position 269 of Gal4/NFATc1 construct led to increase in NFATc1 -dependent
  reporter gene production. This may suggest that direct effect of PKA on the
  transcription activity of NFATc1 is accomplishing through phosphorylation of
  Ser269 and it is negative.
                                     REFERENCES
1. Chytil M. and Verdine G.L. (1996) The Rel family of euk aryotic transcription factors.
   Curr.Opin.Stuct.Biol. 6,91-100.

2. Graef I.A., Gastier J.M., Francke U. and Crabtree G.R. (2001) Evolutionary relationships among
   Rel domains indicate functional diversification by recombination. Proc.Natl.Acad.Sci.U.S.A 98,
   5740-5745.

3. Feske S., Okamura R, Hogan P.G. and Rao A. (2003) Ca2+/calcineurin signalling in cells of the
   immune system. Biochem.Biophys.Res.Commun, 311,1117-1132.

4. Rao A., Luo C. and Hogan P.G. (1997) Transcription factors of the NFAT family: regulation and
   function. Annu.Rev.Immunol. 15, 707-747.

5. Clipstone N.A and Crabtree G.R. (1992) Identification of calcineurin as a key signalling enzyme
   in T- lymphocyte activation. Nature 357, 695-697.

6. Hogan P.G., Chen L., Nardone J. and Rao A. (2003) Transcriptional regul ation by calcium,
   calcineurin, and NFAT. Genes Dev. 17,2205-2232.

7. Serfling R, Berberich-Siebelt F., Chuvpilo S., Jankevics E., Klein-Hessling S., Twardzik T. and
   Avots A. (2000) The role of NF-AT transcription factors in T cell activation and
   differentiation(l) [In Process Citation], Biochim.Biophys.Acta 1498, 1-18.

8. Crabtree G.R. and Olson E.N. (2002) NFAT signaling: choreographing the social lives of cells.
   Cell 109 Suppl, S67-S79.

9. Beals C.R., Clipstone N.A, Ho S.N. and Crabiree G.R. (1997) Nuclear localization of NF-ATc
   by a calcineurin-dependent, cyclosporin- sensitive intramolecular interaction. Genes Dev. 11,
   824-834.

10. Klemm J.D., Beals C.R. and Crabtree G.R. (1997) Rapid targeting of nuclear proteins to the
    cytoplasm. Curr.Biol. 7, 638-644.

11. Luo C, Burgeon E., Carew J.A., McCaffrey P.G., Badalian T.M., Lane W.S., Hogan P.G. and
    Rao A. (1996) Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and
    mediates transcription of several cytokine genes. Mol.Cell Biol. 16, 3955-3966.

12. Park J., Takeuchi A. and Sharma S. (1996) Characterization of a new isoform of the NFAT
    (nuclear factor of activated T cells) gene family member NFATc [published erratum appears in J
    Biol Chem 1996 Dec 27; 271(52):33705]. J.Biol.Chem. 271, 20914-20921.

13. Lyakh L., Ghosh P. and Rice N.R. (1997) Expression of NFAT-family proteins in normal human
    T cells. Mol.Cell Biol. 17, 2475-2484.

14. Imamura R., Masuda E.S., Naito Y., Imai S., Fujino T., Takano T., Arai K. and Arai N. (1998)
    Carboxyl-terminal 15-amino acid sequence of NFATxl is possibly created by tissue-specific
    splicing and is essential for transactivation activity in T cells. J.Immunol. 161,3455-3463.

15. Chuvpilo S., Zimmer M., Kerstan A, Glockner J., Avots A., Escher C, Fischer C, Inashkina I.,
    Jankevics E., Berberich-Siebelt F., Schmitt E. and Serfling E. (1999) Alternative
    polyadenylation events contribute to the induction of NF- ATc in effector T cells. Immunity. 10,
    261-269.

16. Sherman M.A., Powell D.R., Weiss DX. and Brown M.A. (1999) NF -ATc isoforms are
    differentially expressed and regulated in murine T and mast cells. J.Immunol. 162, 2820-2828.
17. Chuvpilo S., Avots A., Berberich-Siebelt F., Glockner J., Fischer C, Kerstan A., Escher C,
    Inashkina I., Hlubek F., Jankevics E., Brabletz T. and Serfling E. (1999) Multiple NF-ATc
    isoforms with individual transcriptional properties are synthesized in T lymphocytes. J.Immunol.
    162,7294-7301.

18. Luo C, Burgeon E. and Rao A. (1996) Mechanisms of transactivation by nuclear factor of
    activated T cells-1. J.Exp.Med 184,141-147.

19. Masuda E.S., Liu J., Imamura R., Imai S.I., Arai K.I. and Arai N. (1997) Control of NFATxl
    nuclear translocation by a calcineurin-regulated inhibitory domain. Mol.Cell Biol. 17, 2066-
    2075.

20. Lopez-Rodriguez C, Aramburu J., Rakeman A.S. and Rao A. (1999) NFAT5, a constitutively
    nuclear NFAT protein that does not cooperate with Fos and Jun. Proc.Natl.Acad.Sci.U.S.A 96,
    7214-7219.

21. Lopez-Rodriguez C, Antos C.L., Shelton J.M., Richardson J.A., Lin F., Novobrantseva T.I.,
    Bronson R.T., Igarashi P., Rao A. and Olson E.N. (2004) Loss of NFAT5 results in renal atrophy
    and lack of tonicity-responsive gene expression. Proc.Natl.Acad.Sci.U.S.A 101,2392-2397.

22. Lopez-Rodriguez C, Aramburu J., Jin L., Rakeman A.S., Michino M. and Rao A. (2001)
    Bridging the NFAT and NF-kappaB families: NFAT5 dimerization regulates cytokine gene
    transcription in response to osmotic stress. Immunity. 15,47-58.

23. Miyakawa H, Woo S.K., Dahl S.C., Handler J.S. and Kwon H.M. (1999) Tonicity-responsive
    enhancer binding protein, a rel-like protein that stimulates transcription in response to
    hypertonicity. Proc.Natl.Acad.Sci.U.S.A 96, 2538-2542.

24. Northrop J.P., Ho S.N., Chen L., Thomas D.J., Timmerman L.A., Nolan G.P., Admon A. and
    Crabtree G.R. (1994) NF-AT components define a family of transcription factors targeted in T-
    cell activation [see comments]. Nature 369,497-502.

25. McCaffrey P.G., Luo C, Kerppola T.K., Jain J., Badalian T.M., Ho A.M., Burgeon E., Lane
    W.S., Lambert J.N. and Curran T. (1993) Isolation of the cyclosporin -sensitive T cell
    transcription factor NFATp. Science 262, 750-754.

26. Masuda E.S., Naito Y., Tokumitsu H., Campbell D., Saito F., Hannum G, Arai K. and Arai N.
    (1995) NFATx, a novel member of the nuclear factor of activated T cells family that is expressed
    predominantly in the thymus. Mol.Cell Biol. 15,2 697-2706.

27. Hoey T., Sun Y.L., Williamson K. and Xu X. (1995) Isolation of two new members of the NF
    AT gene family and functional characterization of the NF-AT proteins. Immunity. 2, 461-472.

28. Serfling E., Berberich-Siebelt F., Avots A., Chuvpilo S., Klein-Hessling S., Jha M.K., Kondo E.,
    Pagel P., Schulze-Luehrmann J. and Palmetshofer A. (2004) NFAT and NF-kappaB factors-the
    distant relatives. IntJ.Biochem.Cell Biol. 36, 1166-1170.

29. Adams M.D. and others (2000) The genome sequence of Drosophila melanogaster. Science 287,
    2185-2195.

30. Dehal P. and others (2002) The draft genome of Ciona intestinalis: insights into chordate and
    vertebrate origins. Science 298,2157-2167.

31. Aparicio S. and others (2002) Whole-genome shotgun assembly and analysis of the genome of
    Fugu rubripes. Science 297, 1301-1310.

32. Liu J. (1993) FK506 and ciclosporin: molecular probes for studying intracellular signal
    transduction. Trends Pharmacol. Sci. 14,182-188.
33. Crabtree G.R. (1999) Generic signals and specific outcomes: signaling through Ca2+,
    calcineurin, and NF-AT. Celt 96, 611-614.

34. Avots A., Buttmann M, Chuvpilo S., Escher C, Smola U., Bannister A.J., Rapp U.R.,
    Kouzarides T. and Serfling E. (1999) CBP/p300 integrates Rat7Rac-signaling pathways in the
    transcriptional induction of NF-ATc during T cell activation. Immunity. 10, 515-524.

35. Treisman R. (1996) Regulation of transcription by MAP kinase cascades. Curr.Opin..Cell Biol. 8,
    205-215.

36. Jain J., McCaffrey P.G., Miner Z., Kerppola T.K., Lambert J.N., Verdine G.L., Curran T. and
    Rao A. (1993) The T-cell transcription factor NFATp is a substrate for calcineurin and interacts
    with Fos and Jun. Nature 365, 352-355.

37. Luo C, Shaw K.T., Raghavan A., Aramburu J., Garcia-Cozar F., Perrino B.A., Hogan P.G. and
    Rao A. (1996) Interaction of calcineurin with a domain of the transcription factor NFAT1 that
    controls nuclear import. Proc.Natl.AcadSci.U.S.A 93, 8907-8912.

38. Ruff V.A. and Leach K.L. (1995) Direct demonstration of NFATp dephosphorylation and
    nuclear localization in activated HT-2 cells using a specific NFATp polyclonal antibody.
    J.Biol.Chem. 270,22602-22607.

39. Park J., Yaseen N.R., Hogan P.G., Rao A. and Sharma S. (1995) Phosphorylation of the
    transcription factor NFATp inhibits its DNA binding activity in cyclosporin A-treated human B
    and T cells. J.Biol.Chem. 270,20653-20659.

40. Okamura H., Aramburu J., Garcia-Rodriguez C, Viola J.P., Raghavan A., Tahiliani M., Zhang
    X., Qin J., Hogan P.G. and Rao A. (2000) Concerted dephosphorylation of the transcription
    factor NFAT1 induces a con format ional switch that regulates transcriptional activity [In Process
    Citation]. Mol.Cell 6, 539-550.

41. Porter CM., Havens M.A. and Clipstone N.A. (2000) Identification of amino acid residues and
    protein kinases involved in the regulation of NFATc subcellular localization. J.BiolChem. 275,
    3543-3551.

42. Neal J.W. and Clipstone N.A. (2000) Glycogen synthase kinase-3 inhibits the DNA binding
    activity of NFATc. J.Biol.Chem.

43. Liu J., Farmer J.D., Jr., Lane W.S., Friedman J., Weissman I. and Schreiber S.L. (1991)
    Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP- FK506 complexes. Cell
    66, 807-815.

44. Beals C.R., Sheridan C.M, Turck C.W., Gardner P. and Crabtree G.R. (1997) Nuclear export of
    NF-ATc enhanced by glycogen synthase kinase-3. Science 275,1930-1934.

45. Zhu J., Shibasaki F-, Price R., Guillemot J.C, Yano T., Dotsch V., Wagner G., Ferrara P. and
    McKeon F. (1998) Intramolecular masking of nuclear import signal on NF-AT4 by casein kinase
    I and MEKK1. Cell 93, 851-861.

46. Neilson J., Stankunas K. and Crabtree G.R. (2001) Monitoring the duration of antigen-receptor
    occupancy by calcineurin/glycogen-synthase-kinase-3 control of NF-AT nuclear
    shuttling.
    Curr.Opin.Immunol. 13,346-350.

47. Sheridan CM., Heist E.K., Beals C.R., Crabtree G.R. and Gardner P. (2002) Protein kinase A
    negatively modulates the nuclear accumulation of NF -ATc1 by priming for subsequent
    phosphorylation by glycogen synthase kinase-3. J.Biol.Chem. 277, 48664-48676.

48. Harwood A.J. (2001) Regulation of GSK-3: a cellular multiprocessor. Cell 105, 821-824.
49. Chow C.W., Rincon M., Cavanagh X, Dickens M. and Davis R.J. (1997) Nuclear accumulation
    of NFAT4 opposed by the JNK signal transduction pathway. Science 278,1638-1641.

50. Gomez d.A., Martinez-Martinez S., Maldonado J.L., Ortega-Perez I. and Redondo J.M. (2000) A
    role for the p38 MAP kinase pathway in the nuclear shuttling of NFATp. J.Biol.Chem. 275,
    13872-13878.

51. Yang T.T., Xiong Q., Enslen H., Davis R.J. and Chow C.W. (2002) Phosphorylation of NFATc4
    by p38 mitogen-activated protein kinases. Mol.Cell Biol. 22,3892-3904.

52. Chow C.W. and Davis R.J. (2000) Integration of calcium and cyclic AMP signaling pathways by
    14-3-3. MolCellBiol 20, 702-712.

53. Macian F., Lopez-Rodriguez C. and Rao A. (2001) Partners in transcription: NFAT and AP-1.
    Oncogene 20, 2476-2489.

54. Ho I.C., Hodge M.R., Rooney J.W. and Glimcher L.H. (1996) The proto -oncogene c-maf is
    responsible for tissue-specific expression of interleukin-4. Cell 85,973-983.

55. Bodor J., Bodorova J. and Gress R.E. (2000) Suppression of T cell function: a potential role for
    transcriptional repressor ICER. J.Leukoc.Biol 67,.774-779.

56. Decker E.L., Skerka C. and Zipfel P.F. (1998) The early growth response protein (EGR -1)
    regulates interleukin-2 transcription by synergistic interaction with the nuclear factor of activated
    T cells. J.Biol.Chem. 273, 26923-26930.

57. Furstenau U., Schwaninger M., Blume R., Jendrusch E.M. and Knepel W. (1999)
    Characterization of a novel calcium response element in the glucagon gene. J.BioLChem. 274,
    5851-5860.

58. Bert A.G., Burrows J., Hawwari A., Vadas M.A. and Cockerill P.N. (2000) Reconstitution of T
    cell-specific transcription directed by composite NFAT/Oct elements [In Process Citation].
    J.Immunol. 165, 5646-5655.

59. Rengarajan J., Mowen K.A., McBride K.D., Smith E.D., Singh H. and Glimcher L.H. (2002)
    Interferon regulatory factor 4 (IRF4) interacts with NFATc2 to modulate interleukin 4 gene
    expression. J.Exp.Med. 195,1003-1012.

60. Hu CM., Jang S.Y., Fanzo J.C. and Pernis A.B. (2002) Modulation of T cell cytokine
    production by interferon regulatory factor-4. J.BioLChem. 277, 9238-49246.

61. McKinsey T.A., Zhang C.L. and Olson E.N. (2002) MEF2: a calcium-dependent regulator of
    cell division, differentiation and death. Trends BiochemSci. 27, 40-47.

62. Olson E.N. and Williams R.S. (2000) Remodeling muscles with calcineurin. Bioessays 22, 510-
    519.

63. Yang X.Y., Wang L.H., Chen T., Hodge D.R., Resau J.H., DaSilva L. and Farrar W.L. (2000)
     Activation of human T lymphocytes is inhibited by peroxisome proliferator-activated receptor
     gamma (PPARgamma) agonists. PPARgamma co-association with transcription factor NFAT.
     J.Biol.Chem. 275, 4541-4544.

64. Molkentin J.D., Lu J.R., Antos C.L., Markham B., Richardson J., Robbins J., Grant S.R. and
    Olson E.N. (1998) A calcineurin-dependent transcriptional pathway for cardiac hypertrophy.
    Cell 93, 215-228.

65. Avni O., Lee D., Macian F., Szabo S.J., Glimcher L.H. and Rao A. (2002) T(H) cell
    differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes.
    Nat.Immunol. 3, 643-651.
66. Klein-Hessling S., Jha M.K., Santner-Nanan B., Berberich-Siebelt F., Baumruker T., Schimpl A.
    and Serfling E. (2003) Protein kinase A regulates GATA-3-dependent activation of IL-5 gene
    expression in Th2 cells. J.Immunol. 170,2956-2961.

67. Musaro A., McCullagh K.J., Naya F.J., Olson E.N. and Rosenthal N. (1999) IGF -1 induces
    skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1.
    Nature 400,581-585.

68. Im S.H., Hueber A., Monticelli S., Kang K.R and Rao A. (2004) Chromatin-level regulation of
    the IL10 gene in T cells. J.Biol.Chem. 279, 46818-46825.

69. Baksh S., Widlund H.R., Frazer-Abel A.A., Du J., Fosmire S., Fisher D.E., DeCaprio J.A.,
    Modiano J.F. and Burakoff S.J. (2002) NFATc2-mediated repression of cyclin-dependent kinase
    4 expression. Mol.Cell 10,1071-1081.

70. Marian F., Garcia-Rodriguez C. and Rao A. (2000) Gene expression elicited by NFAT in the
    presence or absence of cooperative recruitment of fos and Jun [In Process Citation]. EMBO J.
    19,4783-4795.

71. Marian F., Garcia-Cozar F., 1m S.H., Horton H.F., Byrne M.C. and Rao A. (2002)
    Transcriptional mechanisms underlying lymphocyte tolerance. Cell 109, 719-731.

72. Ranger AM., Oukka M., Rengarajan J. and Glimcher L.R (1998) Inhibitory function of two
    NFAT family members in lymphoid homeostasis and Th2 development. Immunity. 9, 627-635.

73. Peng S.L., Gerth A.J., Ranger A.M. and Glimcher L.R (2001) NFATc1 and NFATc2 together
    control both T and B cell activation and differentiation. Immunity. 14,13-20.

74. Ranger A.M., Grusby M.J., Hodge M.R., Gravallese E.M., de la Brousse F.C., Hoey T.,
    Mickanin C, Baldwin H.S. and Glimcher L.H. (1998) The transcription factor NF -ATc is
    essential for cardiac valve formation [see comments]. Nature 392, 186-190.

75. de la Pompa J.L., Timmerman L.A., Takimoto H., Yoshida R, Elia A.J., Samper E., Potter J.,
    Wakeham A., Marengere L., Langille B.L., Crabtree G.R. and Mak T.W. (1998) Role of the NF-
    ATc transcription factor in morphogenesis of cardiac valves and septum [see comments]. Nature
    392, 182-186.

76. Graef I.A., Mermelstein P.G., Stankunas K., Neilson J.R., Deisseroth K., Tsien R.W. and
    Crabtree G.R. (1999) L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in
    hippocampal neurons. Nature 401, 703-708.

77. Ranger A.M., Gerstenfeld L.C., Wang J., Kon T., Bae R, Gravallese E.M., Glimcher M.J. and
    Glimcher L.H. (2000) The nuclear factor of activated T cells (NFAT) transcription factor
    NFATp (NFATc2) is arepressor of chondrogenesis [see comments]. J.Exp.Med 191,9-22.

78. Ho I.C., Kim J.H., Rooney J.W., Spiegelman B.M. and Glimcher L.R (1998) A potential role
    for the nuclear factor of activated T cells family of transcriptional regulatory proteins in
    adipogenesis. Proc.Natl.Acad.SciU.S.A 95, 15537-15541.

79. Schmitt E., Hoehn P., Germann T. and Rude E. (1994) Differential effects of interleukin-12 on
    the development of naive mouse CD4+ T cells. Eur.J.Immunol. 24, 343-347.

80. Randak C, Brabletz T., Hergenrother M., Sobotta I. and Serfling E. (1990) Cyclosporin A
    suppresses the expression of the interleukin 2 gene by inhibiting the binding of lymphocyte-
    specific factors to the IL-2 enhancer. EMBOJ. 9, 2529-2536.

81. Chuvpilo S., Schomberg C, Gerwig R., Heinfling A., Reeves R., Grummt F. and Serfling E.
    (1993) Multiple closely-linked NFAT/octamer and HMG I(Y) binding sites are part of the
    interleukin-4 promoter. Nucleic Acids Res. 21,5694-5704.
82. Liu J., Koyano-Nakagawa N., Amasaki Y., Saito-Ohara F., Ikeuchi T., Imai S., Takano T., Arai
    N., Yokota T. and Arai K. (1997) Calcineurin-dependent nuclear translocation of a murine
    transcription factor NFATx: molecular cloning and functional characterization. Mol.Biol.Cell 8,
    157-170.

83. Ho S.N., Thomas D.J., Timmerman L.A., Li X., Francke U. and Crabtree G.R. (1995) NFATc3,
    a lymphoid-specific NFATc family member that is calcium- regulated and exhibits distinct DNA
    binding specificity. J.Biol.Chem. 270, 19898-19907.

84. Reynolds P.J., Lesley J., Trotter J., Schulte R., Hyman R. and Sefton B.M. (1990) Changes in
    the relative abundance of type I and type II lck mRNA transcripts suggest differential promoter
    usage during T-cell development. MolCell Biol. 10, 4266-4270.

85. Hemenway C.S. and Heitman J. (1999) Calcineurin. Structure, function, and inhibition. Cell
    Biochem.Biophys. 30,115-151.

86. Fruman D.A., Pai S.Y., Burakoff S.J. and Bierer B.E. (1995) Characterization of a mutant
    calcineurin A alpha gene expressed by EL4 lymphoma cells. MolCell Biol. 15, 3857-3863.

87. Dai P., Akimaru H., Tanaka Y., Hou D.X., Yasukawa T., Kanei-Ishii C, Takahashi T. and Ishii
    S. (1996) CBP as a transcriptional coactivator of c-Myb. Genes Dev. 10,528-540.

88. Bergholtz S., Andersen T.O., Andersson K.B., Borrebaek J., Luscher B. and Gabrielsen O.S.
    (2001) The highly conserved DNA-binding domains of A-, B- and c-Myb differ with respect to
    DNA-binding, phosphorylation and redox properties. Nucleic Acids Res. 29, 3546-3556.

89. Xu H., Inouye M., Hines E.R., Collins J.F. and Ghishan F.K. (2003) Transcriptional regulation
    of the human NaPi-IIb cotransporter by EGF in Caco-2 cells involves c-myb. AmJ.Physiol Cell
    Physiol 284, C1262-C1271.

90. Fang X., Yu S.X., Lu Y., Bast R.C., Jr., Woodgett J.R. and Mills G.B. (2000) Phosphorylation
    and inactivation of glycogen synthase kinase 3 by protein kinase A. Proc.Natl.Acad.Sci.U.S.A
    97,11960-11965.

91. Jain J., McCaffrey P.G., Valge-Archer V.E. and Rao A. (1992) Nuclear factor of activated T
    cells contains Fos and Jun. Nature 356, 801-804.

92. Barton K., Muthusamy N., Chanyangam M., Fischer C, Clendenin C. and Leiden J.M. (1996)
    Defective thymocyte proliferation and IL-2 production in transgenic mice expressing a
    dominant-negative form of CREB. Nature 379, 81-85.

93. de Groot R.P. and Sassone-Corsi P. (1992) Activation of Jun/AP -1 by protein kinase A,
    Oncogene 7, 2281-2286.

				
DOCUMENT INFO