From the Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cellular calcium (Ca2+)
and the Ca2+-binding protein calmodulin (CaM) regulate the
activities of Ca2+/CaM-dependent protein
kinases and protein phosphatase 2B (calcineurin). Functional
interactions between CaM kinases and mitogen-activated protein (MAP)
kinases were described. In this report, we describe cross-talk between
calcineurin and mitogen-activated protein kinase signaling. Calcineurin
was found to specifically down-regulate the transcriptional activity of
transcription factor Elk1, following stimulation of this activity by
the ERK, Jun N-terminal kinase, or p38 MAP kinase pathways. Expression
of constitutively activated calcineurin or activation of endogenous
calcineurin by Ca2+ ionophore decreased the phosphorylation
of Elk1 at sites that positively regulate its transcriptional activity.
Calcineurin specifically dephosphorylates Elk1 at phosphoserine 383, a
site whose phosphorylation by MAP kinases makes a critical contribution to the enhanced transcriptional activity of Elk1. The cross-talk between calcineurin and MAP kinases is of physiological significance as
low doses of Ca2+ ionophore which by themselves are
insufficient for c-fos induction can actually inhibit
induction of c-fos expression by activators of MAP kinases.
Thus through the effect of calcineurin on Elk1 phosphorylation,
Ca2+ can have a negative effect on expression of Elk1
target genes. This mechanism explains why different levels of
intracellular Ca2+ can result in very different effects on
gene expression.
The second messenger Ca2+ plays a critical
regulatory role in a variety of physiological processes in many
different cell types (1, 2). One of the most important mediators of
Ca2+ signaling is the Ca2+-binding protein,
calmodulin (CaM)1 (3). The
Ca2+-CaM complex binds to and modulates the activities of
multiple key signal-transducing enzymes, including
Ca2+/CaM-dependent protein kinases (CaM-K) (4,
5) and the Ca2+/CaM-dependent protein
serine/threonine phosphatase calcineurin (6, 7). The CaM kinases have
several substrates involved in transcriptional control, such as CREB
(cAMP-responsive element binding protein) (8-10) and SRF (serum
response element binding protein) (11). These transcription factors can
therefore mediate some of the effects of elevated intracellular
Ca2+ on gene transcription (4). Calcineurin consists of a
catalytic subunit, CnA, and a regulatory subunit, CnB (6). Besides a catalytic domain, which is conserved among members of the protein phosphatase family, which includes protein phosphatases 1 and 2a and
bacteriophage An increasing number of calcineurin-dependent cellular
processes were identified through the use of these specific inhibitors and by expression of the constitutively active mutant ( An additional target for calcineurin that is involved in T cell
activation is a yet-to-be identified component of the signaling pathway
that leads to activation of JNK (Jun N-terminal kinase) in T cells
(28). Stimulation of T cells with a phorbol ester and Ca2+
ionophore results in synergistic JNK activation, which is blocked by
CsA or FK506 (29). Recent studies have shown that the target for
calcineurin is a T cell-specific component of the JNK pathway that acts
in synergy with protein kinase C In addition to JNK, which belongs to the MAP kinase (MAPK) family,
mammalian cells express two other MAPK cascades that link a variety of
extracellular stimuli to changes in gene expression (30). The most well
characterized MAPK cascade leading to ERK activation is stimulated most
efficiently by mitogens and growth factors, which activate receptor
tyrosine kinases (31). Like all MAPKs, the ERKs are phosphorylated and
activated by specific MAPK kinases (MAPKKs), the MEKs, which themselves
are activated by MAPKK kinases (MAPKKKs), one of which is Raf-1 (30,
32). The MAPK pathways that lead to JNK and p38 MAPK activation are most potently triggered by physical and chemical stresses (UV irradiation, osmotic shock, and heat shock) and proinflammatory cytokines (33, 34). JNK and p38 are also activated by specific MAPKKs,
JNKK1 (MKK4) (35, 36), and JNKK2 (MKK7) (37-39) for JNK, and MKK3, and
MKK6 for p38 (40-42). These MAPKKs are in turn activated by several
MAPKKKs, including MEKK1 (43), MEKK2, and -3 (44), TAK1 (45), ASK1
(46), and MLK3 (47). The three components (MAPK, MAPKK, and MAPKKK) of
each cascade are engaged in sequential phosphorylation reactions in the
cytoplasm (30). Activated MAPKs are then redistributed in the cell,
with a portion entering the nucleus (48), where they phosphorylate
several transcription factors. The ERKs are known to phosphorylate Elk1 (49), ETS2 (50), c-Myc (31), and c-Myb (31). The JNKs phosphorylate
c-Jun (51), ATF2 (52), and Elk1 (48). The p38s phosphorylate ATF2 (52),
Elk1 (49), CHOP (53), and MEF2C (54). In many of these cases, the
transactivating potential of the affected transcription factor is
augmented upon its phosphorylation, leading to changes in the
expression profile of target genes (33, 51). One target gene for all
three MAPK cascades is the c-fos proto-oncogene (49). The
c-fos promoter contains a composite binding site recognized
by SRF and ternary complex factors (TCFs) (55). The activity of the TCF
protein Elk1 is stimulated by all three MAPK cascades through
phosphorylation of sites within its C-terminal activation domain
(49).
Interaction between Ca2+ signals and three MAPKs cascades
have been reported. In several cell types, elevated intracellular Ca2+ can stimulate MAPK activity (29, 56). Specifically,
CaM-Ks may activate MAPK cascades in fibroblasts (57). As mentioned above, in T cells, calcineurin can participate in JNK activation through synergy with protein kinase C Cell Culture--
COS7 cells and Jurkat cells were maintained in
Dulbecco's modified Eagle's medium and RPMI 1640, respectively,
supplemented with glutamate (1 mM), penicillin (100 units/ml), streptomycin (100 µM), and 10% fetal bovine
serum (Life Technologies, Inc.).
Plasmids--
Mammalian expression vectors (Raf-1, MEKK1, JNKK2,
MKK6, MEK1, JNK1, ERK2, p38, CnA( Transfection--
COS7 cells were seeded at 2 × 105 cells/well into 6-well plate (35-mm diameter) the day
before transfection. For reporter assay, cells were transfected with
Superfectamine (Qiagen) according to the manufacturer's instruction.
5×GAL4-LUC vector (1.5 µg), and mammalian expression vectors of
Raf-1, MEKK1, JNKK2, MKK6, MEK1, p38, GAL4-c-Jun, GAL4-Elk1, GAL4-ATF2
(5 ng of each, alone or in combination as indicated), and
CnA( Immunoblotting Analysis--
Anti-phospho-ERK,
anti-phospho-Elk1, anti-Elk1, and anti-ERK antibodies were purchased
from New England Biolabs. Transfected cells were washed with cold
phosphate-buffered saline, lysed in whole cell extract (WCE) buffer (25 mM Tris, pH 7.5, 0.25 M NaCl, 0.5% Nonidet
P-40, supplemented with protease and phosphatase inhibitors) (43), and
rotated for 30 min at 4 °C, followed by centrifugation at 13,000 rpm
for 10 min to clarify the lysate. The protein concentration of the
supernatant (WCE) was determined by Lowry Assay (Bio-Rad). Protein
samples (50 µg of each) were separated by SDS-PAGE (10%
polyacrylamide), transferred to nylon membrane (Immobilon-P,
Millipore), and blotted according to the manufacturer's instructions.
The HA-tagged proteins (CnA and CnB) were detected with anti-HA
polyclonal antibody (Santa Cruz Biotechnology, Inc.). The subsequent
procedures were performed with the enhanced chemiluminescence detection
kit (ECL, Amersham Pharmacia Biotech). In the case of Jurkat cells,
nuclear extract (NE) was prepared as described (22). NE samples (200 µg of protein each) were separated by SDS-PAGE and blotted with
anti-Elk1. NE samples (4 mg of each) were also incubated with a DNA
affinity resin prepared by coupling SRE (DNA-binding site for SRF
and TCFs) to cyanogen bromide-activated Sepharose (Amersham
Pharmacia Biotech). Bound proteins were eluted with SDS loading buffer,
separated by SDS-PAGE, and blotted with anti-phospho-Elk1.
Immunoprecipitation and in Vitro Phosphorylation--
After
24 h of transfection, transfected cells (HA-ERK2) were stimulated
with 150 nM 12-O-tetradecanoylphorbol-13-acetate
(Sigma) for 15 min, and transfected cells (HA-JNK1) were treated with UV light at 40 J/cm2 for 20 s as described previously
(36, 39). The WCE was incubated with HA monoclonal antibody (12CA5) and
20 µl of protein A-Sepharose (Amersham Pharmacia Biotech) and rotated
at 4 °C for 2 h. The immunocomplex was then washed extensively
with WCE buffer and kinase buffer (25 mM Tris, pH 7.5, 5 mM MgCl2, supplemented with phosphatase
inhibitors). GST fusion protein was purified as described before (39).
0.5 µg of GST fusion protein was incubated with the immunocomplex in
kinase buffer with addition of 4 µl of [ RNA Analysis--
Total RNA from Jurkat cells was prepared with
the RNeasy mini kit (Qiagen). Reverse transcriptase (RT)-polymerase
chain reaction (PCR) was conducted with the Superscript One-Step RT-PCR
system (Life Technology, Inc.) according to the manufacturer's
instructions. For c-fos, 20 ng of RNA was used. The
sequences of the two primers were 5'-CACCGACCTGCCTGCAAGATC-3' and
5'-CTGTGCAGAGGCTCCCAGTC-3'. For gapdh, 1 ng of RNA was used.
The sequences of the two primers were 5-ACCACAGTCCATGCCATCAC-3' and
5'-TCCACCACCCTGTTGCTGTA-3'. The PCR products were separated on 1.5%
agarose gel and stained with ethidium bromide. The optical density of
the bands was quantified by using Alphaimager (Alpha Innotech). A
standard curve was made by RT-PCR under the same conditions with a
serial dilution of RNA prepared from stimulated Jurkat cells.
Phosphatase Assay--
The purified substrates were incubated
with calcineurin and calmodulin (Sigma) in phosphatase buffer (20 mM Tris, pH 7.5, 1 mM CaCl2, 50 mM NaCl, 0.02% Triton X-100, 1 mg/ml bovine serum albumin). The reactions were carried out at 30 °C for 20 min and terminated by addition of SDS loading buffer. Phospho-substrates and
free phosphate were separated by SDS-PAGE, and the results were
obtained by autoradiography and quantified by PhosphorImager analysis.
A Constitutively Active Calcineurin Mutant Blocks Elk1 but Not
c-Jun or ATF2 Activation--
To investigate specifically the effect
of calcineurin on different MAPK pathways, the constitutively active,
Ca2+-independent, CnA mutant ( Calcineurin Has No Effect on the Phosphorylation and Activity of
ERK--
Upon cell-surface receptor stimulation, such as treatment
with epidermal growth factor (EGF), or transient expression of
constitutively active Raf-1, the ERKs are activated through
phosphorylation at the conserved sites, threonine 202 and tyrosine 204, by the MEKs (60). We examined the effect of CnA(
ERK2 activity was also measured by an immunocomplex kinase assay.
Transiently expressed HA-ERK2 was immunoprecipitated, and the
immunocomplexes were incubated with GST-Elk1 and
[ Elk1 Is the Target for Calcineurin Activation--
Transcription
factor Elk1 is phosphorylated not only by the ERKs but also by the JNK
and p38 MAPKs (61). Through phosphorylation of sites in its C-terminal
activation domain, all of these kinases stimulate Elk1 transcriptional
activity (61). Therefore, to dissect at which level transient
expression of activated calcineurin inhibits the stimulation of Elk1
activity, the GAL4-Elk1 vector was cotransfected with an MEKK1
expression vector in the absence or presence of CnA(
Does calcineurin affect Elk1 abundance or phosphorylation? To answer
this question we conducted immunoblotting experiments with Elk1
antibodies that recognize both non-phosphorylated and phosphorylated
Elk1 or only phospho-Elk1 that is phosphorylated at serine 383. CnA( Cyclosporin A and FK506 Can Block the Effect of Calcineurin on Elk1
Phosphorylation--
As mentioned above, treatment with CsA enhanced
Elk1 phosphorylation. To examine further the effect of different
phosphatase inhibitors, COS7 cells transiently transfected with an Elk1
expression vector were pretreated with CsA, FK506, or okadaic acid
prior to EGF stimulation. As shown in Fig.
5, coexpression of CnA( Calcineurin Activation Inhibits c-fos Induction and Elk1
Phosphorylation--
Next we examined whether activation of
calcineurin has an adverse effect on one well characterized Elk1 target
gene, c-fos. It is well established that elevated
intracellular Ca2+ can induce c-fos
transcription through CaM kinase activation which phosphorylates CREB
and other related transcription factors that bind to a CRE in the
c-fos promoter (64). We therefore had to find a way to
stimulate calcineurin activity without activating CaM kinases. Since
calcineurin is more sensitive to Ca2+ than CaM kinases
(65), we first determined the minimal dose of ionomycin required for
c-fos induction. As shown in Fig.
6A, treatment of Jurkat cells
with more than 10 nM ionomycin can induce c-fos
transcription, but treatment with 1 nM or less had no
effect. We therefore examined whether treatment with 1 nM
ionomycin can reduce c-fos induction by inducers that do not
affect intracellular Ca2+. We chose anisomycin, a potent
activator of the JNK and p38 MAP kinase pathways (48), as the inducing
agent. As expected, anisomycin induced c-fos transcription
(Fig. 6B). This induction was inhibited by pretreatment with
1 nM ionomycin. If this inhibitory effect is mediated by
calcineurin, it should be prevented by cyclosporin A. Indeed, treatment
with cyclosporin A prevented the inhibition of c-fos
induction. Next we examined the effect of these treatments on the
phosphorylation status of endogenous Elk1. The endogenous Elk1 protein
was enriched by affinity purification on an SRE resin and its
phosphorylation state determined by immunoblotting with anti-phospho-Elk1. Indeed, the effect of the different treatment on
c-fos expression correlated very well with their effect on Elk1 phosphorylation at Ser-383. As the source of Elk1 in these experiments was nuclear extracts, it is very likely that the
calcineurin-mediated dephosphorylation occurred in the nucleus.
Calcineurin Directly Dephosphorylates Phospho-Elk1--
Since
calcineurin is a serine/threonine protein phosphatase and its
expression or activation has a marked effect on the phosphorylation of
Elk1 at Ser-383 in intact cells, we examined whether Elk1 is a direct
substrate for calcineurin. The substrate used in this experiment was a
GST-Elk1 fusion protein that was phosphorylated in vitro by
various MAPKs. As shown in Fig.
7A, calcineurin
dephosphorylated phospho-Elk1 but not phospho-c-Jun or phospho-ATF2.
The latter two substrates were prepared by phosphorylation of the
relevant GST fusion proteins with activated JNK. Calcineurin also did
not significantly dephosphorylate phospho-Ets2, even though Elk1 and Ets2 belong to the same transcription factor family and they both are
substrates for ERK (50, 56). When 2.5 µM calcineurin was incubated with 0.5 µg of phospho-Elk1, 60% of the substrate was dephosphorylated within 20 min (Fig. 7B). Similar results
were obtained when dephosphorylation of phospho-Elk1 was followed by immunoblotting with an antibody specific to phospho-Elk1 phosphorylated at Ser-383 (Fig. 7C). Immunoblotting with the general Elk1
antibody indicated that incubation with calcineurin reduced the
phospho-Elk1 signal by dephosphorylation of phospho-Ser-383 rather than
through nonspecific proteolysis.
Calcineurin is a ubiquitously expressed protein phosphatase that
is activated upon binding of Ca2+-CaM (6, 67). So far the
roles of calcineurin in signal transduction and gene regulation have
been investigated only in lymphoid cells, brain, heart, and other
limited cell types (28, 68, 69). The functions of calcineurin in other
cell types are poorly understood. The work described above links
calcineurin to a ubiquitous sequence-specific transcription factor,
Elk1, which serves as a target for all three MAPK pathways and plays an
important role in induction of immediate-early gene transcription.
We found that activation of calcineurin can inhibit the stimulation of
Elk1 transcriptional activity by all three MAPK cascades. MAPK
activation is known to be inhibited by specific MAPK phosphatases, such
as MKP-1, which directly dephosphorylate the activating phosphoacceptor sites within the T-loop regions of MAPKs (70). In the case reported here, however, the target for the phosphatase is a specific MAPK substrate rather the MAPK themselves. However, it is likely that MAPK
phosphatases and calcineurin, although structurally not related, may
act synergistically in vivo. Indeed, in fission yeast,
disruption of the calcineurin gene (ppb1+)
results in a dramatic chloride-sensitive growth arrest (71). A novel
gene pmp1+, which encodes a MAPK phosphatase,
suppresses this defect (71). Although the biochemical mechanism
accounting for this functional interaction is not known, in light of
our results it is possible that calcineurin (Ppb1) dephosphorylates a
MAPK substrate involved in regulation of chloride ion transport. In the
absence of calcineurin, the substrate accumulates in its phosphorylated
state, which prevents chloride export. One way to reverse this effect
is to inhibit the activity of the MAPK responsible for the
phosphorylation event.
Elk1 is the most abundant of the TCFs, first characterized as
transcription factors involved in induction of c-fos and
other members of its family by diverse extracellular stimuli (64). The
Fos proteins are basic leucine zipper (bZIP) transcription factors,
which dimerize with the Jun proteins to form stable AP-1 heterodimers
(72). The proteins are involved in the regulation of many target genes.
Transcription of the c-fos gene is activated by a variety of
stimuli such as serum, EGF and other growth factors, UV irradiation,
Ca2+, cAMP, phorbol esters, and various cytokines (49,
64).
The inactivation of Elk1 by calcineurin-mediated dephosphorylation adds
another layer to the complexity of c-fos promoter regulation
and Ca2+ signaling (summarized in Fig.
8). The c-fos promoter
contains two major cis elements, the CRE and the SRE (64).
The CRE (cAMP response element) is recognized by the CREB/ATF group of
bZIP proteins. The activity of CREB is stimulated by phosphorylation at
Ser-133, which can be catalyzed by a number of protein kinases including the cAMP-dependent protein kinase A (73), the
Ca2+/CaM-dependent CaM kinases (74), phorbol
ester-responsive protein kinase C (75), and growth factor-responsive
ribosomal S6 kinases (76). The activity of ATF2 is stimulated by JNK
and p38-mediated phosphorylation at threonine 71 (52), whereas the
mechanisms that control the activity of other ATFs are not yet known.
This probably is the reason why calcineurin cannot completely block the
induction of c-fos transcription by anisomycin (Fig.
6B), since ATF2 can also be phosphorylated by activated JNK
and p38 and is not dephosphorylated by calcineurin (Fig.
7A). The SRE is a composite binding site for the SRFs and
the TCFs (64). Several TCFs were identified, including Elk1 (49),
Sap1/2 (77), and Erp/Net (78). The TCFs belong to the Ets family,
characterized by a conserved Ets DNA binding domain (66). The TCFs
interact with the SRE only in conjunction with binding of SRF (79).
Although SRF may respond to signals that are transmitted by members of the Rho family of small G-proteins (80), or CaM kinases (11), most of
the signals sensed by the SRE are received by the TCFs (49, 64). The
ability of Elk1 and Sap1/2 to activate transcription is stimulated by
phosphorylation of conserved sites at their C-terminal activation
domain (58). Phosphorylation of these sites is mediated by various
MAPKs, including ERK, p38, and JNK (49).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phosphatase (7), CnA contains a C-terminal autoinhibitory domain as well as CaM and CnB binding domains (12). Ca2+/CaM binding activates CnA by relieving autoinhibition,
which, in the absence of Ca2+/calmodulin, blocks access of
substrates to the catalytic site (13). A truncation that removes the
autoinhibitory domain and a portion of the CaM binding region results
in a constitutively active form (
CaM-AI) that no longer requires
Ca2+ (14, 15). Calcineurin is a target for
immunosuppressive drugs, such as cyclosporin A (CsA) and FK506 (16).
These drugs inhibit calcineurin activity after forming complexes with
cytoplasmic immunophilins, cyclophilins, and FK506-binding proteins,
respectively (16). These immunophilin-immunosuppressant complexes bind
calcineurin and inhibit its function by sterically hindering the access
of substrates to the catalytic site (13, 17).
CaM-AI) (6).
The most well characterized functions of calcineurin are during T cell
activation (18). Upon binding of antigen to T cell receptor,
intracellular Ca2+ is elevated through the action of
protein tyrosine kinases and phospholipase C (19-21). Calcineurin is
then activated through binding of Ca2+/CaM and
dephosphorylates the cytosolic forms of the NF-ATc transcription factors (22). The dephosphorylated NF-ATc proteins translocate to the
nucleus, where either alone or in cooperation with AP-1 family members
bind specific cis elements in the promoter/enhancer regions
of cytokine genes, such as IL-2 (23, 24). This pathway makes
an important contribution to induction of IL-2 gene
transcription, a hallmark of T cell activation (18, 20). Genes
expressed in other cell types were identified to be affected by CsA and FK506, but NF-AT family members are not known to be involved in their
control (25-27). Therefore, there must be additional transcription factors whose activity is modulated by calcineurin.
(28). However, in other cell types
calcineurin activation or treatment with CsA has no effect on JNK
activity (28).
(28). In this report, we
investigated cross-talk between calcineurin and MAPK pathways in cell
lines of either fibroblast or lymphoid origin. We found that
calcineurin specifically dephosphorylates one substrate that is common
to all three MAPKs, the transcription factor Elk1, thus negatively
regulating its transcriptional activity. This mechanism explains the
regulatory versatility of intracellular Ca2+, which can
exert very different effects on gene expression depending on its level.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CaM-AI), CnB, GAL4-c-Jun,
GAL4-Elk1, and GAL4-ATF2) and vectors for GST fusion proteins
(GST-c-Jun, GST-Elk1, and GST-ATF2) were previously described (28, 39, 41, 43, 58, 59). GST-ETS2 vector encoding the human ETS2-(1-226) was
constructed by PCR and subcloned into pGEX-4T-2 (Amersham Pharmacia
Biotech) (50).
CaM-AI) expression vector (200 ng, as indicated) were
transfected into the cells. In each transfection, 50 ng of
pActin-
-gal was cotransfected to monitor the transfection
efficiency. The total amount of DNA was kept at 2 µg with empty
pSR
vector. The cells were incubated with DNA for 2 h and then
incubated with regular media (10% fetal bovine serum) for 1 h.
The cells were then incubated with media without serum. 24 h
later, the cells were harvested and lysed. The luciferase and
-galactosidase activities were measured as described before (36).
For immunoblotting analysis, COS7 cells were transfected with
LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instruction. pCMV-Elk1 and pSR
-ERK2 (50 ng of each),
pSR
-CnA(
CaM-AI) and pSR
-CnB (475 ng of each, unless otherwise
indicated), were transfected into the cells. Total amount of DNA was
kept to 1 µg. 24 h after transfection, cells were serum-starved for 3 h before treatment with EGF, ionomycin, and cyclosporin A
(purchased from Calbiochem).
-32P]ATP in
a 20-µl reaction volume. Kinase reaction was carried out at 30 °C
for 30 min. Then, 500 µl of kinase buffer supplemented with 1 M NaCl were added, and the mixture was rotated at 4 °C for 30 min. After spinning down, the supernatant was incubated with 30 µl of glutathione-Sepharose (Amersham Pharmacia Biotech), and the
mixture was rotated at 4 °C for 30 min. After extensively washing
with kinase buffer plus 1 M NaCl, the GST fusion proteins were eluted with 20 mM reduced glutathione (Sigma) in
kinase buffer. The eluted proteins were then concentrated with
Centrifugal Filter Device (Millipore).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CaM-AI) was used.
Cotransfection with an expression vector encoding a constitutively
active form of MEKK1 enhances the ability of a fusion protein between
the GAL4 DNA binding domain and the c-Jun activation domain
(GAL4-c-Jun) to activate a reporter plasmid whose promoter contains
five GAL4 DNA-binding sites (Fig.
1A). In previous experiments,
the enhancement of GAL4-c-Jun transcriptional activity by cotransfected
MEKK1 was shown to depend on phosphorylation of the c-Jun activation domain at serines 63 and 73 (36, 43). Coexpression of CnA(
CaM-AI) with MEKK1 had no effect on stimulation of GAL4-c-Jun activity (Fig.
1A). To examine the effect of CnA(
CaM-AI) on the ability of p38 to stimulate transcription, we used an activated MKK6 expression vector (40) to stimulate the activity of a GAL4-ATF2 fusion protein,
which contains the ATF2 activation domain. The potentiation of
GAL4-ATF2 activity by MKK6 was not significantly reduced by coexpression of CnA(
CaM-AI) (Fig. 1B). To examine the
effect on ERK-mediated signaling, a chimeric GAL-Elk1 activator, which contains the Elk1 activation domain (58), was cotransfected with the
5×GAL4-LUC reporter with or without an expression vector for activated
Raf-1 (43). As expected, coexpression of Raf-1 greatly enhanced the
ability of GAL4-Elk1 to stimulate expression of the reporter (Fig.
1C). This stimulation was considerably reduced upon
coexpression of CnA(
CaM-AI).
View larger version (9K):
[in a new window]
Fig. 1.
Constitutively active calcineurin blocks Elk1
but not c-Jun or ATF2 activation. COS7 cells were transfected with
5×Gal4-LUC reporter construct (1.5 µg of DNA/plate) together with
expression vectors for truncated MEKK1, activated Raf-1 (BXB),
activated MKK6, p38, GAL-c-Jun, GAL4-ATF2 and GAL4-Elk1 (5 ng each), as
indicated, in the absence or presence of a CnA( CaM-AI) expression
vector (200 ng). The total amount of DNA was kept at 2 µg.
pActin-
gal reporter (50 ng) was included as an internal control.
After 24 h, luciferase and
-galactosidase activities were
measured. Results are expressed in fold activation of luciferase
expression relative to reporter alone (set as 1) after normalization
for transfection efficiency determined by
-galactosidase expression.
Shown are the average (± S.D.) of three separate experiments.
CaM-AI) on ERK
activation by cotransfecting it with an expression vector for
epitope-tagged ERK2. Immunoblotting the transiently expressed ERK2 with
an antibody specific for activated ERK (60) revealed that, as expected, ERK2 phosphorylation was induced by EGF treatment (Fig.
2A). This phosphorylation was
not inhibited upon coexpression of CnA(
CaM-AI).
View larger version (32K):
[in a new window]
Fig. 2.
Calcineurin has no effect on ERK activation
or activity. COS7 cells were transfected with pSR -HA-ERK2 (50 ng/plate) with or without pSR
-CnA(
CaM-AI) and pSR
-CnB (475 ng
each). Total amounts of DNA were kept constant at 1 µg. After 20 h, the cells were serum-starved for 3 h prior to EGF treatment (50 ng/ml for 15 min). The cells were then harvested and lysed.
A, protein samples (50 µg) were separated by SDS-PAGE and
analyzed by immunoblotting with an antibody specific for ERK
phosphorylated at Tyr-204. The same blots were stripped and reprobed
with an ERK2 specific antibody. B, protein samples (100 µg) were immunoprecipitated with anti-HA, and after extensive
washing, the activity of HA-ERKs was assayed with purified GST-Elk1 as
a substrate. The phosphorylated proteins were separated by SDS-PAGE,
and the extent of GST-Elk1 phosphorylation was quantified with a
PhosphorImager. C, expression of CnA was determined by
immunoblotting protein samples (15 µg) using an HA antibody.
-32P]ATP in kinase buffer. EGF treatment stimulated
ERK2 activity by about 5-fold (Fig. 2B). Coexpression with
CnA(
CaM-AI) had no effect on the activation of ERK2 and its ability
to phosphorylate GST-Elk1 in vitro.
CaM-AI). Whereas
coexpression of MEKK1 enhanced GAL4-Elk1 transcriptional activity by
20-fold, coexpression with CnA(
CaM-AI) resulted in a 3-fold decrease
in this stimulation (Fig. 3A).
Because expression of moderate levels of MEKK1 can activate either JNK
or p38 without exerting an effect on ERK activity (43, 62), we used
more specific and direct JNK and p38 activators, namely JNKK2 (39) and
MKK6 (40), respectively. We also used an expression vector for
activated MEK1 to activate specifically the ERKs (59). As expected,
coexpression with activated forms of all three MAPKKs resulted in
enhanced GAL4-Elk1 transcriptional activity (Fig. 3, B-D).
Regardless of the MAPKK used, coexpression of CnA(
CaM-AI) resulted
in a severely diminished effect on GAL4-Elk1 activity.
View larger version (15K):
[in a new window]
Fig. 3.
Elk1 is the target for calcineurin
activation. COS7 cells were transfected and analyzed as described
in Fig. 1, but with different expression vectors. Shown is the average
(± S.D.) fold stimulation of luciferase activity determined by three
separate transfections after normalization for -galactosidase
expression. CN, calcineurin.
CaM-AI) had no effect on the level of Elk1 expression, but it
dramatically decreased the extent of Elk1 phosphorylation induced by
EGF (Fig. 4A). Decreased Elk1
phosphorylation was also observed after treatment of EGF-stimulated
cells with the Ca2+ ionophore ionomycin, whereas treatment
with the specific calcineurin inhibitor cyclosporin A (CsA) enhanced
Elk1 phosphorylation. A titration experiment in which increasing
amounts of CnA(
CaM-AI) were coexpressed with Elk1 revealed a
dose-dependent decrease in the extent of EGF-induced Elk1
phosphorylation (Fig. 4B).
View larger version (42K):
[in a new window]
Fig. 4.
Calcineurin expression or activation reduces
Elk1 phosphorylation. A, COS7 cells were transfected
with pCMV-Elk1 (50 ng/plate) with or without pSR -CnA(
CaM-AI) and
pSR
-CnB (475 ng DNA each). The total amount of DNA was kept constant
at 1 µg. After 24 h, the cells were treated with 0.2 µM ionomycin and 1 µM cyclosporin A
(CsA) for 10 min prior to EGF treatment for 15 min. Cells
were harvested, lysed, and analyzed by immunoblotting as described in
Fig. 2, except that anti-phospho-Elk1 and anti-Elk1 antibodies were
used. Results were quantified by using a PhosphorImager. B,
COS7 cells were transfected with pCMV-Elk1 (150 ng/plate) and
increasing amounts of pSR
-CnA(
CaM-AI) and pSR
-CnB (1:1 ratio).
After 15 min of EGF treatment, cells were harvested and lysed. Protein
samples (50 µg) were separated by SDS-PAGE and immunoblotted with
phospho-Elk1 and total Elk1-specific antibodies.
CaM-AI) decreased the extent of Elk1 phosphorylation at serine 383. Pretreatment with increasing amounts of CsA or FK506 enhanced the
extent of Elk1 phosphorylation and blocked the effect of
CnA(
CaM-AI). Treatment with okadaic acid at a concentration that
inhibits either protein phosphatase 2A (50 nM) or both
protein phosphatase 1 and 2A (500 nM) (63) had no effect on
the extent of Elk1 phosphorylation. None of the treatments had any
effect on Elk1 or calcineurin (either CnA or CnB subunits)
expression.
View larger version (41K):
[in a new window]
Fig. 5.
Effect of calcineurin and phosphatase
inhibitors on Elk1 phosphorylation COS7 cells were transfected
with pCMV-Elk1 (150 ng/plate) with or without pSR -CnA(
CaM-AI) and
pSR
-CnB (150 ng of DNA each). The total amount of DNA was kept
constant at 1 µg. After 24 h the cells were incubated with the
indicated concentration of cyclosporin A (CsA), FK506, and
okadaic acid (OA) for 10 min prior to EGF treatment. The
cells were lysed, and phospho-Elk1 and total Elk1 levels were
determined by immunoblotting as described in Fig. 4. CnA(
CaM-AI) and
CnB expression was monitored using HA antibody. CN,
calcineurin.
View larger version (43K):
[in a new window]
Fig. 6.
Calcineurin inhibits c-fos
induction and stimulation of Elk1 phosphorylation by
anisomycin. A, Jurkat cells were seeded at 2 × 106/ml the day before treatment. Cells were mixed with
equal amount of medium containing twice the indicated concentration of
ionomycin (IONO). Cells were harvested after 30 min, and RNA
was prepared. The relative levels of c-fos and
gapdh mRNA were determined by quantitative RT-PCR. The
PCR products were separated on 1.5% agarose gel, stained with ethidium
bromide, and quantified by using Alphaimager. B, Jurkat
cells were mixed with equal amount medium containing 100 ng/ml
anisomycin (An) for 30 min. Some cells were pretreated with
1 nM ionomycin and/or 500 nM cyclosporin A
(CsA) for 5 min prior to anisomycin addition as indicated.
The relative levels of c-fos and gapdh mRNA
were determined by RT-PCR. C, Jurkat cells were treated as
in B. After 20 min, nuclear extracts (NE) were
prepared and purified on an SRE-DNA-affinity Sepharose resin and the
bound proteins eluted in SDS loading buffer. The elute were separated
by SDS-PAGE and immunoblotted with anti-phospho-Elk1 antibody. Smaller
samples of each extract were also separated by SDS-PAGE and
immunoblotted with anti-Elk1, to determine the total level of Elk1 in
each extract. gapdh, glyceraldehyde-3-phosphate
dehydrogenase.
View larger version (30K):
[in a new window]
Fig. 7.
Calcineurin directly dephosphorylates
phospho-Elk1. A, GST-Elk1, GST-Ets2, GST-c-Jun, and
GST-ATF2 fusion proteins were expressed in E. coli and
purified on GSH-Sepharose. Each of the GST fusion proteins (0.5 µg
each) was in vitro phosphorylated by immunoprecipitated
HA-ERK2 (for GST-Elk1 and GST-Ets2) and HA-JNK1 (for GST-c-Jun and
GST-ATF2) and repurified on GSH-Sepharose. The 32P-labeled
substrates were incubated with the indicated concentrations of
calcineurin and calmodulin purified from bovine brain in phosphatase
buffer containing Ca2+ for 20 min at 30 °C. The proteins
were separated by SDS-PAGE and visualized by autoradiography.
B, 32P-labeled GST-Elk1 was incubated with the
indicated concentrations of calcineurin, and the level of GST-Elk1
phosphorylation was determined as described above. The average (± S.D.) values of relative GST-Elk1 phosphorylation determined in three
separate experiments were plotted against the concentration of
calcineurin (initial level of phosphorylation was set at 100%).
C, phospho-GST-Elk1 was incubated with 2.5 µM
calcineurin as described above. After separation by SDS-PAGE,
phospho-Elk1, and total Elk1 levels were determined by immunoblotting
as described in Fig. 4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 8.
Summary of major signaling pathways that
regulate the c-fos promoter. The c-fos
promoter contains two major cis elements, the CRE and the
SRE. The CRE is recognized by the CREB/ATF. Ser-133 of CREB can be
phosphorylated by protein kinase A (PKA), CaM-Ks, and
protein kinase C (PKC). Thr-71 of ATF2 can be phosphorylated
by JNKs and p38s. The SRE is recognized by SRF and TCFs. SRF is the
substrate for ribosomal S6 kinases, CaM-K and MAPKs. Elk1, the most
well characterized TCF, is a substrate for all three MAPKs, and
phospho-Elk1 can be dephosphorylated by calcineurin. The
phosphorylation of these transcription factors stimulates
c-fos promoter activity, whereas dephosphorylation of Elk1
by calcineurin may play a role in turning off c-fos
transcription. CN, calcineurin.
The most ubiquitous and prevalent TCF is Elk1. It was shown that c-fos promoter activity correlates temporally with the phosphorylation state of Elk1 (81). Thus not only Elk1 phosphorylation is an important mechanism for regulation of c-fos transcription but also its dephosphorylation is likely to be instrumental in shutting off c-fos transcription. Originally it was suggested that Elk1 might be inactivated by a constitutive phosphatase (81). Although such phosphatase may also be involved in Elk1 inactivation, the present study identified calcineurin as a physiologically relevant Elk1 phosphatase. While this manuscript was being prepared for publication, a similar conclusion was derived by Sugimoto et al. (82), although these authors documented the involvement of calcineurin only in the context of ERK-mediated Elk1 activation. Here we report that this dephosphorylation is specific to Elk1 (calcineurin does not affect c-Jun, Ets2 or ATF2) and that stimulation of Elk1 transcriptional activity by all three MAPK pathways is negatively regulated by calcineurin. Most importantly, activation of calcineurin can reduce the induction of c-fos transcription by inducers, such as anisomycin, which operate via MAPK pathways.
How and why Ca2+ signaling can both turn on and off c-fos transcription? One can posit several mechanisms that explain such a paradoxical effect. According to the first mechanism, the Ca2+-esponsive kinases and phosphatases have different kinetics of activation. If kinases such as CaM-Ks are activated first, they can stimulate c-fos transcription through direct phosphorylation of CREB or CREM or indirectly through stimulation of ERK, which then phosphorylates Elk1. Once calcineurin activity picks up, Elk1 is dephosphorylated and part of the Ca2+ generated stimulatory effect is attenuated. This may contribute to the transient nature of c-fos induction. A second possibility is that Ca2+-dependent protein kinases and phosphatases have different sensitivity to Ca2+ and different affinity for CaM, and their activity is independently controlled. For example, the Kd for CaM binding is 0.1 nM and 20-200 nM for calcineurin and CaM kinase II, respectively (65). Thus, a small increase in intracellular Ca2+ will have a negative effect on c-fos transcription through activation of calcineurin, whereas a large increase in Ca2+ will stimulate c-fos transcription through activation of CaM-K II and ERK. Indeed, as shown in Fig. 6, treatment of Jurkat cells with 1 nM ionomycin is not sufficient for induction of c-fos transcription presumably because it does not increase intracellular Ca2+ to a level competent for CaM kinase activation. However, that level of Ca2+ is sufficient for inhibition of c-fos induction by inducers that work via MAPK signaling pathways, probably because it is sufficient for activation of calcineurin, as evidenced by the dephosphorylation of Elk1. Such a mechanism certainly increases the regulatory potential of Ca2+ transients.
In order to exert its regulatory effect on Elk1 bound at the
c-fos or other similar promoters, calcineurin must act in
the nucleus. Calcineurin is widely considered to be a cytoplasmic protein (6). It has no nuclear translocation signal (22). However, it
has been detected in the nucleus of activated T cells (22). It was
suggested that after calcineurin dephosphorylates NF-AT1, it
accompanies it to the nucleus by virtue of its tight association with
this transcription factor (22). Thus by affecting the subcellular
location of calcineurin, the NF-AT proteins may modulate the ability of
calcineurin to inactivate transcription factors such as Elk1.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank R. Janknecht, T. Hunter, J. Han, R. Treisman, N. G. Ahn, M. Ostrowski, Z. Wu, Y. Xia, and W. Li for providing the necessary constructs. We thank C. Hauser for critically reading the manuscript and for helpful discussion.
![]() |
FOOTNOTES |
---|
* This research was supported in part by grants from the National Institutes of Health (to M. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a postdoctoral fellowship from the Leukemia Research Foundation.
§ To whom correspondence should be addressed: Laboratory of Gene Regulation and Signal Transduction, Dept. of Pharmacology, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0636. Tel.: 619-534-1361; Fax: 619-534-8158; E-mail: karinoffice{at}ucsd.edu.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CaM, calmodulin; CaM-K, Ca2+/CaM-dependent protein kinases; MAP, mitogen-activated protein; MAPK, MAP kinase; CsA, cyclosporin A; CREB, cAMP-responsive element binding protein; SRF, serum response factor; GST, glutathione S-transferase; WCE, whole cell extract; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; NE, nuclear extract; TCF, ternary complex factors; RT-PCR, reverse transcriptase-polymerase chain reaction; JNK, Jun N-terminal kinase; CRE, cAMP response element; MEK, MAPK kinases; ATF, activating transcription factor; EGF, epidermal growth factor; SRE, serum response element.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|