(Received for publication, September 14, 1995; and in revised form, November 9, 1995)
From the
In T lymphocytes, the calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin, plays a pivotal role in transducing membrane-associated signals to the nucleus. One of the putative targets of calcineurin is the pre-existing, cytosolic component of the nuclear factor of activated T cells (NFATp; also referred to as NFAT1), which is one of several transcription factors required for the expression of interleukin 2. Inhibition of calcineurin by the immunosuppressive drugs cyclosporin A and FK506 prevents dephosphorylation of NFATp and its translocation to the nucleus. However, a physical interaction between calcineurin and NFATp has not been demonstrated. Here we demonstrate the binding of NFATp from lysates of T cells to immobilized calcineurin. Stimulation of T cells with calcium ionophore induced a shift in the molecular weight of NFATp that is due to its dephosphorylation. This dephosphorylation was inhibited by treatment of T cells with cyclosporin A or FK506 prior to stimulation. Of note, both the phosphorylated and the dephosphorylated form of NFATp bound to calcineurin. Furthermore, the binding of both forms of NFATp to calcineurin was inhibited by pretreatment of calcineurin with a complex of FK506 and its ligand FKBP12. Taken together these data strongly suggest a direct interaction of calcineurin with NFATp and that this interaction does not depend upon the phosphorylation sites of NFATp affected by activation.
The calcium/calmodulin-dependent serine/threonine phosphatase
calcineurin (PP2B) plays a crucial role in T cell activation.
Calcineurin (CN) ()is a ubiquitously expressed heterodimer,
composed of a 59-kDa calmodulin-binding catalytic subunit and a 19-kDa
Ca
-binding regulatory subunit that is required for
enzymatic activity. The central role of calcineurin in T cell signaling
was appreciated by its identification as the target of the
immunosuppressive drugs cyclosporin A (CsA) and
FK506(1, 2) . The phosphatase activity of CN is
inhibited by either drug when complexed to intracellular binding
proteins (immunophilins), i.e. CsA to cyclophilin and FK506 to
the FK506-binding protein 12 (FKBP12), respectively. Neither drug nor
immunophilin alone bind to or affect the activity of CN(2) . In
T lymphocytes both CsA and FK506 inhibit a variety of T cell responses.
A well characterized example of the effect of CsA and FK506 is the
inhibition of T cell receptor-mediated expression of cytokine
genes(3, 4, 5) .
Among the putative
targets of calcineurin is the nuclear factor of activated T cells
(NFAT)(6) , which regulates, in part, the expression of a
number of cytokine genes including IL-2, IL-3(7) ,
IL-4(8, 9) , granulocyte-macrophage colony-stimulating
factor(7) , and tumor necrosis factor (10) .
Within the IL-2 promoter, NFAT consists of a pre-existing, cytoplasmic
component and an ubiquitously expressed nuclear component, which
contains members of the AP1 family of transcription
factors(11, 12) . NFATp is one of several isoforms of
the cytosolic component of
NFAT(13, 14, 15, 16) . Increases in
intracellular calcium induce the translocation of the cytosolic
component of NFAT to the nucleus, where NFATp interacts with the AP1
elements Fos and Jun and binds to its cognate DNA-binding site. The
calcium-dependent dephosphorylation and nuclear translocation of NFAT
cytoplasmic components are inhibited by CsA and FK506 whereas the
generation of the nuclear components is unaffected (11, 17, 18, 19, 20) . It
has been further demonstrated that CN dephosphorylates NFATp in
vitro and that the dephosphorylated form of NFATp binds with a
higher affinity to an NFAT oligonucleotide(20, 21) .
Moreover, it has been shown that CN phosphatase activity correlates
with T cell receptor-induced IL-2 expression in vitro and in vivo(22, 23, 24) . Thus it
appears that CN plays a central role in transducing T cell receptor-
mediated signals to the nucleus. However, it has not been determined
whether the influence of CN on the nuclear translocation of the
cytosolic forms of NFAT is due to a direct or indirect interaction.
In this report we show that purified bovine CN immobilized to
calmodulin Sepharose beads precipitates NFATp from lysates of a murine
T cell line. Of note, CN associated with both the phosphorylated as
well as the dephosphorylated form of NFATp. The interaction of CN with
NFATp was inhibited by the complex of FK506FKBP12 but not by
either FK506 or FKBP12 alone. The finding of a physical association of
CN with NFATp supports a model whereby CN is directly involved in
dephosphorylation and nuclear translocation of NFATp.
Figure 1:
Increase in intracellular
Ca induces the dephosphorylation of NFATp. A, T cells (BYDP) were left untreated (control) or stimulated
for 5 min with 2 µM ionomycin (Iono), 5 ng/ml
PMA, or the combination of both. B, T cells were stimulated
for the indicated amount of time with 2 µM ionomycin.
Whole cell lysates were resolved by SDS-PAGE, transferred to
polyvinylidene difluoride membrane, and immunoblotted with NFATp
antiserum. Arrows indicate the putative phosphorylated and
dephosphorylated forms of NFATp. The band between both arrows was detected in whole cell lysates only but not in
precipitations (see Fig. 2and Fig. 3). Molecular size
markers are shown on the left of all
figures.
Figure 2:
Calcineurin associates with phosphorylated
and dephosphorylated NFATp and FK506FKBP12 inhibits this
interaction. T cells were incubated with the immunosuppressants FK506 (F, 0.1 µM), 0.1 µM FK506 plus 10
µM rapamycin (R+F), or in medium with
ethanol (0.2%) as diluent control for 30 min at 37 °C. Subsequently
cells were activated with ionomycin (2 µM) and PMA (5
ng/ml) or ethanol control (0.2%) for 5 min at 37 °C. Activated
(+) or nonactivated(-) cells were lysed. 500 µl of
lysates were added to calmodulin-Sepharose beads (CaM) that
had been preincubated for 30 min with or without purified CN (0.2
µM) in the presence of FK506 (F, 40
µM) and purified FKBP12 (BP, 4 µM)
or ethanol control (4%) in a volume of 250 µl. After an additional
60-min incubation, calmodulin-Sepharose beads and associated proteins
were washed and incubated with FK506 plus FKBP12 (F+BP)
or ethanol in the same concentrations and volume as during
preincubation. After additional washes precipitated proteins were
resolved by SDS-PAGE and immunoblotted with NFATp antiserum. The arrows indicate the phosphorylated and dephosphorylated forms
of precipitated NFATp.
Figure 3:
Rapamycin restores the
FK506FKBP12-inhibited association of CN with NFATp. A,
500-µl lysates of activated (+) or nonactivated(-) T
cells (stimulated as in Fig. 2) were added to
calmodulin-Sepharose beads that had been preincubated for 60 min with
or without purified CN (0.2 µM) in the presence of FK506
(4 µM), rapamycin (Rapa, 400 µM),
ethanol (4%) as control, and/or purified GST-FKBP12 (4 µM)
in a volume of 250 µl. After incubation for an additional 60 min,
calmodulin-Sepharose beads were washed, and precipitated proteins were
immunoblotted with NFATp antiserum or FKBP12 antiserum (B).
In
order to assess the specificity of the FK506FKBP12-mediated
inhibition of NFATp to CN, rapamycin was added in 100-fold excess to
FK506 during preincubation of CN/CaM beads and GST-FKBP12 with or
without FK506. After preincubation, lysates of activated or
nonactivated T cells were added, and proteins associated with CN/CaM
beads were resolved by SDS-PAGE and blotted with anti-NFATp, anti-CN
(data not shown), or anti-FKBP12 antibodies (Fig. 3).
Preincubation of CN/CaM beads with FK506 and FKBP12 (GST-FKBP12) led to
the binding of FK506
FKBP12 to CN and the inhibition of the
interaction of CN with NFATp. Only the combination of both FKBP12 plus
FK506 blocked the binding of NFATp to CN; neither drug nor immunophilin
alone had any effect on the association nor did rapamycin
FKBP12.
Inhibition by FK506
FKBP12 was mediated by blocking the binding of
CN to NFATp and not by disturbing the interaction of CN with CaM, since
immunoblots for CN revealed no loss of CN (precipitated via CaM beads)
in the presence of FK506
FKBP12 (data not shown). Addition of
rapamycin in 100-fold excess to FK506 restored the association of NFATp
to CN, thus reversing the effect of FK506
FKBP12 on the CN/NFATp
interaction (Fig. 3). Although the presence of FK506 is
necessary for optimal binding of FKBP12 to CN some binding of FKBP12
alone to CN/CaM beads was noted.
It has been reported that
FK506FKBP12 inhibits the phosphatase activity of CN on
protein/peptide substrates but not on small organic substrates such as para-nitrophenyl phosphate(2) . This finding suggests
that the complex of FK506
FKBP12 inhibits substrate binding to CN
rather than affecting the phosphatase activity of CN. It further
implies that the catalytic domain of CN consists of at least two sites:
a substrate-binding site and a catalytic site. Our observation that (i)
CN binds both the phosphorylated and dephosphorylated forms of NFATp
and (ii) these interactions are inhibited by FK506
FKBP12 supports
the notion that the major interaction of CN with NFATp occurs via the
substrate-binding site rather than the catalytic site. The recently
solved x-ray crystallographic structure of FK506
FKBP12 bound to
CN revealed that the FK506
FKBP12 complex binds to the A-chain of
CN at a site approximately 10 Å removed from the catalytic
site(27) . This structural information is consistent with our
data, suggesting that the interaction of NFATp with the catalytic site
is not a requirement for binding.
Though we were able to demonstrate
the binding of NFATp to CN, we could not exclude the possibility of a
third protein involved in this interaction. While it has been shown
that CsA and FK506 inhibit the dephosphorylation and nuclear
translocation of NFATp, it has not yet been demonstrated whether the
influence of CN on the nuclear translocation of the cytosolic form of
NFAT is due to a direct or indirect interaction. In the direct model,
CN dephosphorylates NFAT directly, thereby enabling it to cross the
nuclear membrane. Thus, the nuclear translocation of the cytoplasmic
component of NFAT would be regulated by dephosphorylation, as has been
shown for the regulation of the yeast transcription factor
SWI5(28, 29) . Alternatively, the indirect model takes
into account that all four isoforms of the cytosolic component of NFAT
contain a region similar to the Rel homology domain of the NFB
family(14, 15, 16) . It has, therefore, been
suggested that NFAT in nonactivated T cells might be retained by a
cytosolic anchoring protein, in a similar manner as NF
B is
retained by I
B(30) . Dephosphorylation of the putative
anchoring protein by CN would then result in the release of NFAT and
its subsequent translocation to the nucleus(31) .
The
finding that CN associates with phosphorylated and dephosphorylated
NFATp favors a model of a direct interaction of CN with NFATp for the
following reasons. (i) It has been demonstrated that dephosphorylated
NFATp is able to translocate to the nucleus(19, 20) .
In case NFATp is released from a cytosolic anchoring protein after
activation it is unlikely that CN associates with NFATp via a cytosolic
anchoring protein, since NFATp would already be released. (ii) The
known NFB anchoring proteins, I
B
and
I
B
degrade 10 min and 4 h after stimulation,
respectively(32) . Nuclear translocation of NFATp takes place
5-10 min after stimulation(19, 20) , indicating
that if NFATp is retained in the cytosol by an I
B-like anchoring
protein it must be released from this protein and from CN within
5-10 min. Since we could demonstrate that CN/CaM beads
precipitate NFATp from lysates of T cells stimulated for up to 6 h (Fig. 4) it is unlikely that NFATp would still associate with CN
via an I
B-like protein.
Figure 4: CN associates with NFATp up to 6 h after stimulation. CN/CaM-Sepharose beads were incubated with 500-µl lysates of T cells stimulated with 2 µM ionomycin for the indicated amount of time. After a 60-min incubation, calmodulin-Sepharose beads were washed, and precipitated proteins were resolved by SDS-PAGE and immunoblotted with NFATp antiserum. The arrow indicates the dephosphorylated form of NFATp.
Therefore, our observation of a physical interaction of CN and NFATp, together with the fact that phosphorylated NFATp can serve as a substrate for CN in vitro(21) , supports a model in which CN is directly involved in the dephosphorylation and subsequent nuclear translocation of NFATp.