From the Center for Ligand and Transcription, Chonnam National University, Kwangju 500-757 and the ¶ School of Earth and Environmental Sciences, Seoul National University, Seoul 151-742, Korea
Received for publication, November 9, 2000, and in revised form, February 23, 2001
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ABSTRACT |
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Calmodulin-dependent protein
kinase IV (CaMKIV) is a key mediator of Ca2+-induced
gene expression. In this study, CaMKIV was found to directly associate
with and phosphorylate the nuclear factor- Calmodulin (CaM)1 is the
most ubiquitous and abundant Ca2+-binding protein in cells
that is an essential protein that serves as a receptor to sense changes
in calcium concentrations and, in this fashion, mediates the second
messenger role of this ion (reviewed in Ref. 1). Calcium binds to CaM
by means of a structural motif called an EF-hand, and a pair of
these structures is located in both globular ends of the protein. CaM
binds to and activates target enzymes. These
Ca2+/CaM-dependent protein kinases include
CaM-kinase kinase, CaMKI and CaMKIV, which are phosphorylated and
activated by CaM-kinase kinase, and CaMKII. In particular, CaMKIV is a
monomeric multifunctional enzyme that is expressed only in
subanatomical portions of the brain, T lymphocytes, and postmeiotic
male germ cells. CaMKIV is present in the nucleus of the cells in which
it is expressed and has been implicated in regulation of transcription
of a number of genes including those encoding interleukin 2, members of
the immediate early gene family such as c-fos, tumor
necrosis factor family members such as CD40L, FasL, and tumor necrosis
factor, the neurotrophin, BDNF, an Epstein-Barr virus gene
involved in the switch to the lytic cycle called BZLF1, and orphan
members of the steroid receptor superfamily such as ROR and COUP-TF
(1). However, the only direct substrates for CaMKIV involved in
transcription that have been defined to date are CREB and CREM
(2, 3) although the transcription coactivator CREB-binding protein
(CBP) has also been indirectly implicated as a possible substrate
(4).
Nuclear factor- Transcriptional coactivators either bridge transcription factors and
the components of the basal transcriptional apparatus and/or remodel
the chromatin structures (reviewed in Ref. 6). In particular, CBP and
its functional homologue p300, as well as steroid receptor
coactivator-1 and its family members, were shown to be essential for
the activation of transcription by a large number of regulated
transcription factors, including NF In this work, we found that CaMKIV specifically interacted with and
phosphorylated the NF Plasmids, Chemicals, Cells, and Antibodies--
The polymerase
chain reaction-amplified fragments for the full-length CaMKIV, CaMKIVc
(the CaMKIV residues 1-313), and CREB were subcloned into
EcoRI and XhoI (or SalI) restriction
sites of the LexA fusion vector pEG202PL, the B42 fusion vector
pJG4-5, the mammalian two hybrid vectors pCMX/Gal4 and
pCMX/VP16, and the mammalian expression/in vitro translation
vector pcDNA3. The polymerase chain reaction-amplified fragments
for CaMKIV, CaMKIVc, human p65, and p65C (the p65 residues 431-551)
were inserted into EcoRI and XhoI restriction
sites of the glutathione S-transferase (GST) fusion vector
pGEX4T-1. Expression vectors for various CaMKs and their C-terminal
deleted forms with constitutive activities were obtained from
Dr. Tony Means at Duke University. The mammalian two hybrid vectors
pCMX/VP16-SMRT-D, pCMX/VP16-CBP-A, pCMX/Gal4-p65, and
pCMX/VP16-p65, the mammalian expression vectors for p65, CBP, CBP-A,
SMRT-D, GFP/SMRT, and a constitutively active form of MEKK-1, and the
yeast expression vectors encoding B42 fusions to p50 and p65C were as
described (9, 16, 22-24). CV-1 and HeLa cells were obtained from ATCC
(Manassas, VA), and polyclonal antibodies against p65 and CaMKIV were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Calcium
signaling activator ionomycin and CaMK inhibitor KN-93 were purchased
from Calbiochem.
Cell Culture, Transfection, and Microscopy--
HeLa and CV-1
cells (5 × 104 cells/well) were grown in 24-well
plates with Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum for 24 h and transiently transfected using
SuperFect (Qiagen, Valencia, CA) according to the manufacturer's instructions. The cells were harvested 48 h later, the luciferase assays were done as described (25), and the results were normalized to
the LacZ expression. For treatment with ionomycin and KN-93, the cells, 24 h post-transfection, were replaced with fresh medium containing ionomycin or KN-93. The cells were lysed 24 h later. For the localization studies of SMRT, ~105 CV-1 cells
were seeded in a chambered coverslip cell culture system (Nalge-Nunc,
Rochester, NY) and were transfected with the pCMV-GFP-SMRT vector (24),
together with an appropriate expression vector for the activated form
of MEKK-1 (24) or various CaMKs, as indicated (or an equivalent empty
vector as a control) using the SuperFect procedure. One day after
transfection, the subcellular location of the GFP-SMRT fusion
polypeptide was visualized using a Zeiss AxiosKop 2 microscope.
The Yeast GST Pull Down Assays--
Equal amounts of GST alone or GST
fusion proteins, expressed in Escherichia coli and
purified, were bound to glutathione-Sepharose 4B beads and incubated in
the reaction buffer (100 mM NaCl, 25 mM Hepes
(pH 7.9), 20% glycerol, 0.2 mM EDTA, 1 mM
dithiothreitol, and 1.5% bovine serum albumin) with labeled proteins
expressed by in vitro translation by using the
TNT-coupled transcription-translation system, with conditions as
described by the manufacturer (Promega, Madison, WI). Specifically
bound proteins were eluted from beads with 40 mM reduced
glutathione in 50 mM Tris (pH 8.0) and analyzed by SDS-PAGE
and autoradiography as described (25).
Phosphorylation in Vivoand in Vitro--
For
in vivo phosphorylation, HeLa cells were cultured in 100-mm
dish at density of 1 × 105 cells/ml for 24 h,
transfected with 5 µg of the indicated expression vectors using
SuperFect, grown for 24 h, starved in serum-free Dulbecco's
modified Eagle's medium for 30 min, and treated with 100 µCi of
[32P]orthophosphate. 3 h later, the cells were
washed three times with phosphate-buffered saline, lysed in 0.2 ml of
lysis buffer (50 mM Tris (pH 7.5), 300 mM NaCl,
1 mM EDTA, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 20 mM NaF, 5 mM sodium orthovanadate, and 1×
phosphatase inhibitor), and centrifuged. The supernatant was
immunoprecipitated with 10 µl of p65-specific antibody for 2 h
and incubated with protein A-agarose for 16 h. The
immunoprecipitates were washed three times with lysis buffer and
analyzed by SDS-PAGE and autoradiography. Purified GST alone and
GST fusion protein to p65C were subjected to phosphorylation with
either cold ATP or [ The Direct Interaction of CaMKIV with NF Phosphorylation of p65 by CaMKIV--
The direct physical
interactions between the NF Recruitment of CBP and Release of SMRT by CaMKIV-phosphorylated
p65--
The fact that the CaMKIV-directed phosphorylation site was
mapped to the C-terminal transactivation domain of p65, which was previously shown to be the interaction interfaces of both transcription coactivator CBP and corepressor SMRT/N-CoR (7, 8, 16), led us to test
whether CaMKIV-directed phosphorylation of p65 affects its interactions
with these transcription cofactor molecules. Indeed, the mammalian two
hybrid-based assays demonstrated that coexpressed CaMKIVc strengthened
the interactions of Gal4/p65 and VP16/CBP-A (Fig.
3A). In contrast, the
interactions between Gal4/p65 and VP16/SMRT-D were significantly
impaired by coexpressed CaMKIVc. CBP-A (i.e. the
CBP residues 1-446) and SMRT-D (i.e. the SMRT
residues 1060-1495) are the previously defined p65 interaction interfaces (7, 8, 16). Consistently, these results were also
recapitulated in the in vitro GST pull down assays.
Radiolabeled CBP-A interacted with p65C, which was reacted with
CaM-activated CaMKIV but not CaMKIV alone and washed extensively prior
to being added to the GST pull down assays (Fig. 3B).
Consistent with the mammalian two hybrid tests, interactions of
unphosphorylated GST/p65C and radiolabeled SMRT-D were lost when p65C
was phosphorylated by CaM-activated CaMKIV (Fig. 3B). As
expected, GST alone did not interact with radiolabeled CBP-A or SMRT-D
under any condition. Overall, these results clearly demonstrate that
CaMKIV-directed phosphorylation of p65 results in efficient recruitment
of transcription coactivator CBP while repulsing corepressor SMRT.
CaMKIV-mediated Translocation of SMRT--
Recently, CaMK
signaling was shown to promote myogenesis by disrupting MEF2·HDAC
complexes and stimulating HDAC nuclear export (26, 27). Similarly,
phosphorylation of SMRT by MEKK-1 was shown to inhibit the ability of
SMRT to physically tether to its transcription factor partners and led
to a redistribution of the SMRT protein from a nuclear compartment to a
more perinuclear or cytoplasmic compartment (24). These results led us
to explore whether CaMKIV has a similar modulatory role with SMRT. As
shown in Fig. 4, cotransfection with
CaMKIVc expression vector resulted in more cells that express GFP/SMRT
in the cytoplasm. In the absence of CaMKc, ~73% of cells had the
previously described, speckled pattern of exclusive nuclear expression
with SMRT (6). In contrast, however, ~75% of cotransfected cells
exhibited cytoplasmic staining of SMRT in the presence of CaMKIVc. In
these cells, nuclear staining was still observed, but the number of
speckles was significantly decreased. Similar results were also
obtained with CaMKIc, whereas CaMKIIc was without any significant
effect. As reported (24), the active form of MEKK-1 also led to the
cytoplasmic translocation of SMRT. These results strongly suggest that
CaMKI and CaMKIV may stimulate SMRT nuclear export. Currently, we are
investigating whether CaMKIV, either directly or indirectly,
phosphorylates SMRT. However, close examinations of the SMRT sequences
revealed that they lack the known consensus phosphorylation sequences
for CaMKIV, (R/K)XX(S/T) (1).
Stimulation of the NF
Several reports have demonstrated that inhibition of CaMK activity is
associated with apoptosis and proliferation. Inhibition of CaMK
activity with specific inhibitors induces apoptosis in NIH 3T3 cells
(28) and sensitizes etoposide-resistant cells to apoptotic challenge
(29). Thymic T cells from transgenic mice expressing a catalytically
inactive form of CaMKIV showed defects in survival and proliferation
(30). Similarly, CaMK inhibitor KN-62 was shown to reduce DNA synthesis
in small cell lung carcinoma (31). It is interesting to note that
NF
In conclusion, we have shown that CaMKIV specifically interacts with
and phosphorylates the NFB (NF
B) component p65
both in vitro and in vivo. The phosphorylation
of p65 by CaMKIV resulted in recruitment of transcription coactivator cAMP-response element-binding protein-binding protein and
concomitant release of corepressor silencing mediator for retinoid and
thyroid hormone receptors, as demonstrated by the glutathione
S-transferase pull down and mammalian two hybrid assays. In
addition, cotransfection of CaMKIV resulted in cytosolic translocation
of the silencing mediator for retinoid and thyroid hormone receptors.
Consistent with these results, cotransfected CaMKIV dramatically
stimulated the NF
B transactivation in mammalian cells. From these
results, NF
B is suggested to be a novel downstream effector molecule
of CaMKIV.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B (NF
B), composed of homo- and heterodimeric
complexes of members of the Rel (NF
B) family of polypeptides, is
important for the inducible expression of a wide variety of cellular
and viral genes (reviewed in Ref. 5). In vertebrates, this family
comprises p50, p65 (RelA), c-rel, p52, and RelB. These proteins share a 300-amino acid region, known as the Rel homology domain, which binds to DNA and mediates homo- and heterodimerization. This domain is also the target of the I
B proteins, which include I
B
, I
B
, I
B
, Bcl-3, p105, and p100. In the majority of
cells, NF
B exists in an inactive form in the cytoplasm, bound to the inhibitory I
B proteins (5). In response to various inducers, a
multisubunit protein kinase, the I
B kinase, is rapidly activated and
phosphorylates two critical serine residues in the N-terminal regulatory domain of the I
Bs. Phosphorylated I
Bs are recognized by a specific E3 ubiquitin ligase complex and undergo
polyubiquitination, which targets them for rapid degradation by the 26 S proteasome. NF
B dimers, which are spared from degradation,
translocate to the nucleus to activate gene transcription (5).
B (7-9). Interestingly, steroid
receptor coactivator-1 and its homologue ACTR, along with CBP
and p300, were recently shown to contain histone acetyltransferase
activities and associate with yet another histone acetyltransferase
protein P/CAF (6). In contrast, nuclear receptor corepressor
(N-CoR) and its homologue-silencing mediator for retinoid and thyroid
hormone receptors (SMRT) harbor transferable repression domains that
can associate with various histone deacetylases (HDAC). In humans,
three highly homologous class I (HDAC1, HDAC2, and HDAC3) and four
class II (HDAC4, HDAC5, HDAC6, and HDAC7) HDAC enzymes have been
identified to date. The class I deacetylases HDAC1 and HDAC2 are
components of multisubunit complexes mSin3A and the NuRD complex
(10, 11). It is interesting to note that N-CoR/SMRT serves as an
adapter molecule between the core mSin3 complex and sequence-specific
transcriptional repressors without stably associating with the mSin3
complex. More recently, however, SMRT/N-CoR was found to be a direct
component of a newly isolated HDAC3 complex (12, 13). N-CoR and SMRT
have also been reported to partner with HDAC4, HDAC5, and HDAC7 (14,
15). These results are consistent with the notion that acetylation of
histones destabilizes nucleosomes and relieves transcriptional repression by allowing transcription factors to access to recognition elements, whereas deacetylation of the histones stabilizes the repressed state (6). Interestingly, N-CoR/SMRT is also known to mediate
transcriptional repression from a wide variety of other non-receptor-mediated pathways (16-21). These include AP-1,
NF
B, SRF, MyoD, the bHLH-LZ proteins Mad and Mxi that mediate
repression of Myc activities and tumor suppression, E2F-repressive
retinoblastoma protein, and the oncoproteins PLZF-RAR and LAZ3/BCL6,
which are involved in acute promyelocytic leukemia and non-Hodgkin's
lymphomas, respectively.
B component p65, and the CaMKIV-mediated phosphorylation of p65 resulted in enhanced recruitment of CBP with
concomitant dissociation of SMRT. In addition, cotransfection of CaMKIV
resulted in cytosolic translocation of SMRT. Accordingly, CaMKIV
dramatically stimulated the NF
B transactivation. From these results,
we concluded that NF
B is a novel downstream effector molecule of CaMKIV.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-Galactosidase Assay--
The cotransformation and
-galactosidase assay in yeast were done as described (25). For each
experiment, at least three independently derived colonies expressing
chimeric proteins were tested.
-32P]ATP for labeling. CaMKIV was
expressed by in vitro translation by using the TNT-coupled
transcription-translation system and purified by immunoprecipitation
with CaMKIV-specific antibody. Alternatively, HeLa cells cotransfected
with indicated expression vectors were lysed, immunoprecipitated with
p65 antibody, washed three times with phosphate-buffered saline, and
utilized as a source for putative p65-associated kinase(s) in
vivo. Kinase reactions were carried out for 30 min at 30 °C
with 25 mM Hepes (pH 7.6), 10 mM
MgCl2, 5 µM [
-32P]ATP, 1 mM dithiothreitol, and 5% glycerol. To activate the
full-length CaMKIV, 1 mM CaCl2 and 10 µg/ml
of CaM were added.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B--
It is
interesting to note that there are significant overlaps in function
between NF
B and CaMKIV, such as their involvement in anti-apoptosis
and pro-proliferation (1, 5). Furthermore, CaMKIV is known to reside in
the nucleus in vivo and directly phosphorylate the
transcription factors CREB and CREM (2, 3). Thus, we tested whether
CaMKIV is also functionally linked to NF
B. As shown in Fig.
1A, the constitutively active
mutant form of CaMKIV consisting of the CaMKIV residues 1-313
(i.e. CaMKIVc) was found to interact with the
NF
B components p50 and p65 in yeast. In the case of p65, p65C
consisting of the C-terminal transactivation domain of p65
(i.e. the p65 residues 431-551) was utilized,
because the full-length p65 was not readily expressed. As expected, the positive control CREB was also found to directly interact with CaMKIVc
in yeast. Similar results were also obtained with the full-length
CaMKIV (data not shown). Consistent with these results, coexpression of
a fusion protein consisting of the transactivation domain VP16 and
CaMKIVc stimulated the Gal4/p65-directed transactivation in HeLa cells,
suggesting that p65 directly interacts with CaMKIV in vivo
(Fig. 1B). In this experiment, it is notable that 20 µM of the CaMK inhibitor KN-93 was used, because
VP16/CaMKIVc alone was an effective stimulator of the Gal4/p65
transactivation in the absence of KN-93 (data not shown). Coexpression
of VP16/p65 also stimulated the Gal4/CaMKIV-mediated level of
transactivation directed by the Gal4-Luc reporter construct (data not
shown). Finally, labeled p65 protein expressed by using the TNT-coupled in vitro transcription-translation system specifically
interacted with GST fusions to the full-length CaMKIV and CaMKIVc but
not with GST alone (Fig. 1C). Interestingly, the p50-CaMKIV
interaction was much weaker in the mammalian two hybrid tests and not
readily observed in the GST pull down assays (data not shown),
suggesting that an intermediary protein(s) may mediate the observed
in vivo interactions between CaMKIV and p50 in yeast.
Nevertheless, these results strongly suggest that specific interactions
may occur between CaMKIV and NF
B in vivo.
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Fig. 1.
Direct interactions of CaMKIV and
NF B. A, the indicated B42 and
LexA plasmids were transformed into a yeast strain containing an
appropriate LacZ reporter gene as described (25).
Open and closed boxes indicate the presence of
LexA alone and LexA fusion to CaMKIVc, respectively. The p65 residues
431-551 were included in p65C. The data are representative of at least
two similar experiments, and the error bars are as
indicated. B, HeLa cells were transfected with
LacZ expression vector and VP16 fusion to CaMKIVc, along
with an expression vector for Gal4/p65 and a reporter gene, Gal4-Luc,
as indicated. Normalized luciferase expressions from triplicate samples
were calculated relative to the LacZ expressions, and the
results were expressed as -fold activation (n-fold) over the
value obtained with Gal4-Luc alone. To suppress the intrinsic
transcriptional stimulatory effect of VP16/CaMKIVc on Gal4/p65, 20 µM of the CaMK inhibitor KN-93 was added. Similar results
were also obtained with CV-1 cells. The data are representative of
three similar experiments, and the error bars are as
indicated. C, the full-length p65 was labeled with
[35S]methionine by in vitro translation and
incubated with glutathione beads containing GST alone, GST/CaMKIV, and
GST/CaMKIVc, as indicated. Beads were washed, and specifically bound
material was eluted with reduced glutathione and resolved by SDS-PAGE.
Approximately 20% of the total reaction mixture was loaded as
input.
B component p65 and CaMKIV in
vitro (Fig. 1C) suggested that p65 may serve as a
direct substrate for the kinase activity of CaMKIV. Thus, we tested
whether p65 can be phosphorylated by CaMKIV in vivo. HeLa
cells transfected with either the parental vector pcDNA3 or
pcDNA3-CaMKIVc were subjected to in vivo phosphorylation
by incubating the cells in the presence of 100 µCi of
[32P]orthophosphate. The cells were lysed and
immunoprecipitated by either preserum or antibody against p65. As shown
in Fig. 2A, fractionation of
the lysed extracts in SDS-PAGE and autoradiography showed that the p65
immunoprecipitates but not the preserum-directed immunoprecipitates
contained a labeled p65 band. The labeling intensity of the identical
band was significantly increased in CaMKIVc-transfected cell extracts.
The total amounts of p65 were identical between these samples (data not
shown), excluding the possibility that CaMKIVc increased the expression
level of p65. These results imply that p65 is either a direct or
indirect phosphorylation target of CaMKIV in vivo. To
distinguish between these possibilities, purified GST alone or GST
fusion proteins to p50, p65, and p65C were subjected to
[
-32P]ATP-phosphorylation with CaMKIV, either in the
absence or presence of CaMK-activating CaM. For these reactions, we
utilized in vitro-translated CaMKIV, which was purified
further by immunoprecipitating with specific antibody against CaMKIV to
exclude other kinase activities included in the in vitro
translation reactions. As expected, GST/p65C but not GST alone was
specifically labeled with 32P only in the presence of CaM
(Fig. 2B). Interestingly, phosphorylation of p50 was not
observed in these experiments, consistent with the lack of its direct
interactions with CaMKIV in vitro, whereas the overexposure
of the film revealed some weak labeling with the full-length p65 (data
not shown). These results suggest that efficient phosphorylation of the
full-length p65 in vivo may require other additional
proteins or signaling events that are not present in the in
vitro reactions. Nevertheless, these results clearly demonstrate
that the C-terminal transactivation domain of p65 is a direct
phosphorylation substrate of CaMKIV. Finally, we tested whether p65 is
associated with CaMK in vivo. To this end, p65 antibody-directed immunoprecipitates from lysates of HeLa cells transfected with either p65 or p65 plus CaMKIV were utilized as a
source for kinase to phosphorylate GST alone, GST/p65, and GST/p65C as
substrates. In these experiments, GST/p65C but not GST alone was
labeled with [
-32P]ATP only in the presence of
CaMK-activating CaM when using cells transfected with p65 alone (Fig.
2C, compare lanes 1 and 2). Similarly, HeLa cells cotransfected with both p65 and CaMKIV exhibited a detectable, basal level of phosphorylation with GST/p65C (Fig. 2C, lane 3), which was further strengthened with
addition of CaM (Fig. 2C, lane 4). In particular,
phosphorylation of GST fusion to the full-length p65 was readily
detectable under this condition (Fig. 2C, lanes 5 and 6). Thus, the putative proteins or signaling events
required for efficient phosphorylation of the full-length p65 in
vivo may exist in this p65 antibody-directed immunoprecipitate. Notably, CaMKIV is known to be expressed only in certain cell types
whereas CaMKI and CaMKII are ubiquitous in expression. Thus, the CaMK
found to be associated with p65 in vivo in the absence of
cotransfected CaMKIV (Fig. 2C, lane 2) is likely
to be either CaMKI or CaMKII. Overall, these results strongly
demonstrate that the endogenous CaMK in HeLa cells or cotransfected
CaMKIV specifically associates with and phosphorylates p65 in
vivo.
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Fig. 2.
Phosphorylation of p65 by CaMKIV.
A, HeLa cells were transfected with either vector alone
(i.e. pcDNA3) or pcDNA3-CaMKIVc and
subjected to in vivo phosphorylation by incubating the cells
in the presence of 100 µCi of [32P]orthophosphate. The
lysed cells were immunoprecipitated by either preserum or antibody
against p65 and analyzed by SDS-PAGE and autoradiography. The total
amounts of p65 were identical between these samples as judged by
Western analysis (data not shown). IP and T
indicate immunoprecipitation and cotransfection, respectively.
B, GST alone and GST fusion protein to p65C were expressed
in E. coli, purified, and subjected to
[ -32P]ATP phosphorylation with in
vitro-translated and purified CaMKIV, either in the absence or
presence of CaM. The labeled bands were analyzed by SDS-PAGE and
autoradiography. C, lysates of HeLa cells cotransfected with
p65 alone or p65 plus CaMKIV-expression vectors were immunoprecipitated
with antibody against p65, washed extensively, and utilized as a
putative kinase to phosphorylate purified GST alone, GST/p65, or
GST/p65C as substrates. T indicates cotransfection.
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Fig. 3.
Recruitment of CBP and release of SMRT
by CaMKIV-phosphorylated p65. A, CV-1 cells were
transfected with LacZ expression vector and VP16 fusion to
CBP-A and SMRT-D, along with expression vectors for Gal4/p65 and
CaMKIVc and a reporter gene, Gal4-Luc, as indicated. Normalized
luciferase expressions from triplicate samples were calculated relative
to the LacZ expressions, and the results were expressed as
-fold activation (n-fold) over the value obtained with
Gal4-Luc alone. The data are representative of three similar
experiments, and the error bars are as indicated. CBP-A
(i.e. the CBP residues 1-446) and SMRT-D
(i.e. the SMRT residues 1060-1495) are the
previously defined p65 interaction interfaces (7, 8, 16). Similar
results were also obtained with HeLa cells. B, CBP-A and
SMRT-D were labeled with [35S]methionine by in
vitro translation and incubated with glutathione beads containing
GST alone and GST/p65C. Prior to the GST pull down reactions, both GST
proteins were subjected to CaMKIV-mediated kinase reactions, in either
the absence or presence of CaM, and washed extensively. CaMKIV was
expressed by in vitro translation by using the TNT-coupled
transcription-translation system and purified by immunoprecipitation
with CaMKIV-specific antibody. Beads were washed, and specifically
bound material was eluted with reduced glutathione and resolved by
SDS-PAGE. Approximately 20% of the total reaction mixture was loaded
as input.
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Fig. 4.
CAMKIV-mediated translocation of SMRT.
CV-1 cells were transfected with an expression vector encoding GFP
fused to SMRT (24), along with expression vectors encoding various
kinases, as indicated. Under a fluorescence microscope, cells were
counted against the location of GFP/SMRT expression
(i.e. cytoplasmic versus nuclear).
Each data point represents the average percentage of two independent
experiments of at least 200 cells per experiment. C,
cytoplasmic; N, nuclear.
B Transactivation by CaMKIV--
The above
results suggest that CaMKIV should serve as an activator of the NF
B
transactivation. This prediction was confirmed in experiments in which
Gal4/p65-directed transactivation of the Gal4-Luc reporter construct
(9, 16) was significantly enhanced by coexpressed CaMKIVc in a
dose-dependent manner in CV-1 cells (Fig.
5A). Similarly, coexpressed
CaMKIVc stimulated the NF
B transactivation with the
B-Luc
reporter construct (9, 16) (Fig. 5B). In addition,
CBP-dependent stimulation of the p65 transactivation (7, 8)
was synergistically enhanced by coexpressed CaMKIVc (Fig.
5B). Similar results were also obtained in HeLa cells (data not shown). The C-terminally deleted, constitutive active form of CaMKI
but not CaMKII showed dramatic stimulatory effects on the NF
B
transactivation (Fig. 5C). Thus, CaMKI is likely to
represent the CaMK proposed to be associated with p65 in
vivo (Fig. 2C). Treatment of HeLa cells with ionomycin,
the CaMK activator, either in the absence or presence of coexpressed
CaMKIV, also led to stimulation of the p65-directed transactivation of
the
B-Luc reporter construct in HeLa cells, whereas the
CaMKIVc-stimulated level of the p65 transactivation was impaired by
KN-93, the CaMK inhibitor, in a dose-dependent manner (Fig.
5D). As already noted, the ionomycin effect on the p65
transactivation in HeLa cells non-transfected with CaMKIV is probably
because of the endogenous CaMKI. Accordingly, the inhibition was
relatively minor with 3 µM KN-93, the concentration that
is known to specifically inhibit CaMKII, whereas 30 µM
KN-93, at which both CaMKI and CaMKIV are inhibited, completely blocked
the p65 transactivation (Fig. 5D). From these results, we
concluded that CaMKI and CaMKIV are capable of stimulating the NF
B
transactivation, likely through direct phosphorylation of p65 and other
proteins that appears to result in efficient recruitment of
transcription coactivator CBP while disrupting the p65-SMRT
interactions and stimulating nuclear export of corepressor SMRT.
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Fig. 5.
Stimulation of the
NF B transactivation by CaMKIV. CV-1 or
HeLa cells were transfected with LacZ expression vector and
various reporter genes and expression vectors, as indicated. CaMKIc
(the CaMKI residues 1-295) and CaMKIIc (the CaMKII residues 1-290)
are the C-terminal deletion mutant forms with constitutive kinase
activities (1), like CaMKIVc (C). ion indicates
CaMK activator ionomycin (0.12 µM), and KN
denotes CaMK inhibitor KN-93 (3 µM and 30 µM, respectively) (D). 25 ng of each CaMK was
cotransfected in C and D. Normalized luciferase
expressions from triplicate samples were calculated relative to the
LacZ expressions. and the results were expressed as -fold
activation (n-fold) over the value obtained with a reporter
alone. The data are representative of three similar experiments, and
the error bars are as indicated.
B has also been linked to both anti-apoptosis and
pro-proliferative activities (reviewed in Refs. 32 and 33). Thus,
NF
B may mediate these previously characterized anti-apoptotic and
pro-proliferative effects of CaMKIV, although other activities of
CaMKIV previously known and/or yet to be characterized may also turn
out to operate through NF
B. Similar to the work described in this
report, p65 was recently shown to be phosphorylated by the
I
B-associated PKAc subunit through a cyclic AMP-independent
mechanism, which promoted a novel bivalent interaction of p65 with the
coactivator CBP/p300 (34, 35). Thus, p65 appears to be directly
phosphorylated by at least two distinct kinases. Finally, the
C-terminal transactivation domain of p65 is noted to have a numerous
number of phosphorylation sites, including consensus CaMK sites
((R/K)XX(S/T)) (1), and an effort to precisely map the
regulatory phosphorylation sites of p65 by CaMKIV is currently under
progress.2
B component p65, which results in augmented
transcriptional activity of p65 by facilitated dissociation of SMRT
with concomitant, enhanced recruitment of CBP. CaMKIV also stimulated
the nuclear export of SMRT. Overall, these results suggest that the
NF
B component p65 may serve as a novel downstream phosphorylation
target of CaMKIV and act as one of its effector molecules in
vivo.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Tony Means for various CaMK plasmids.
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FOOTNOTES |
---|
* This work was exclusively supported by a grant from the National Creative Research Initiatives Program of the Korean Ministry of Science and Technology, Republic of Korea.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.
§ Present address: Salk Inst., San Diego, CA 92185.
To whom correspondence should be addressed. Tel.:
82-62-530-0910; Fax: 82-62-530-0772; E-mail:
jlee@chonnam.chonnam.ac.kr.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M010211200
2 M. K. Jang, J. Cheong, and J. W. Lee, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
CaM, calmodulin;
CaMK, Ca2+/CaM-dependent protein kinase;
CREB, cAMP-response element-binding protein;
CBP, CREB-binding protein;
NFB, nuclear factor-
B;
N-CoR, nuclear receptor corepressor;
SMRT, silencing mediator for retinoid and thyroid hormone receptors;
HDAC, histone deacetylases;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel electrophoresis;
GFP, green fluorescent
protein;
MEKK, mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase kinase;
Luc, luciferase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Means, A. R.
(2000)
Mol. Endocrinol.
14,
4-13 |
2. | Schulman, H., and Braun, A. (1999) in Calcium as a Cellular Regulator (Carafoli, E. , and Klee, C., eds) , pp. 311-343, Oxford University Press, New York |
3. | Means, A. R., Ribar, T. J., Kane, C. D., Hook, S. S., and Anderson, K. A. (1997) Recent Prog. Horm. Res. 52, 389-407[Medline] [Order article via Infotrieve] |
4. |
Chawla, S.,
Hardingham, G. E.,
Quinn, D. R.,
and Bading, H.
(1998)
Science
281,
1505-1509 |
5. | Karin, M., and Delhase, M. (2000) Semin. Immunol. 12, 85-98[CrossRef][Medline] [Order article via Infotrieve] |
6. | Westin, S., Rosenfeld, M. G., and Glass, C. K. (2000) Adv. Pharmacol. 47, 89-112[Medline] [Order article via Infotrieve] |
7. |
Gerritsen, M. E.,
Williams, A. J.,
Neish, A. S.,
Moore, S.,
Shi, Y.,
and Collins, T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2927-2932 |
8. |
Perkins, N. D.,
Felzien, L. K.,
Betts, J. C.,
Leung, K.,
Beach, D. H.,
and Nabel, G. J.
(1997)
Science
275,
523-527 |
9. |
Na, S.-Y.,
Lee, S.-K.,
Han, S. J.,
Choi, H. S.,
Im, S. Y.,
and Lee, J. W.
(1998)
J. Biol. Chem.
273,
10831-10834 |
10. | Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J., Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., Seto, E., Eisenman, R. N., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1997) Nature 387, 43-48[CrossRef][Medline] [Order article via Infotrieve] |
11. | Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S. L., and Evans, R. M. (1997) Cell 89, 373-380[Medline] [Order article via Infotrieve] |
12. |
Wen, Y. D.,
Perissi, V.,
Staszewski, L. M.,
Yang, W. M.,
Krones, A.,
Glass, C. K.,
Rosenfeld, M. G.,
and Seto, E.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7202-7207 |
13. |
Guenther, M. G.,
Lane, W. S.,
Fischle, W.,
Verdin, E.,
Lazar, M. A.,
and Shiekhattar, R.
(2000)
Genes Dev.
14,
1048-1057 |
14. |
Kao, H. Y.,
Downes, M.,
Ordentlich, P.,
and Evans, R. M.
(2000)
Genes Dev.
14,
55-66 |
15. |
Huang, E. Y.,
Zhang, J.,
Miska, E. A.,
Guenther, M. G.,
Kouzarides, T.,
and Lazar, M. A.
(2000)
Genes Dev.
14,
45-54 |
16. |
Lee, S.-K.,
Kim, J. H.,
Lee, Y. C.,
Cheong, J.,
and Lee, J. W.
(2000)
J. Biol. Chem.
275,
12470-12474 |
17. |
Bailey, P.,
Downes, M.,
Lau, P.,
Harris, J.,
Chen, S. L.,
Hamamori, Y.,
Sartorelli, V.,
and Muscat, G. E.
(1999)
Mol. Endocrinol.
13,
1155-1168 |
18. | Laherty, C. D., Yang, W. M., Sun, J. M., Davie, J. R., Seto, E., and Eisenman, R. N. (1997) Cell 89, 349-356[Medline] [Order article via Infotrieve] |
19. |
Lai, A.,
Lee, J. M.,
Yang, W. M.,
DeCaprio, J. A.,
Kaelin, W. G., Jr.,
Seto, E.,
and Branton, P. E.
(1999)
Mol. Cell. Biol.
19,
6632-6641 |
20. | Lin, R. J., Nagy, L., Inoue, S., Shao, W., Miller, W. H., Jr., and Evans, R. M. (1998) Nature 391, 811-814[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Dhordain, P.,
Albagli, O.,
Lin, R. J.,
Ansieau, S.,
Quief, S.,
Leutz, A.,
Kerckaert, J. P.,
Evans, R. M.,
and Leprince, D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10762-10767 |
22. |
Lee, S.-K.,
Anzick, S. L.,
Choi, J. E.,
Bubendorf, L.,
Guan, X. Y.,
Jung, Y. K.,
Kallioniemi, O. P.,
Kononen, J.,
Trent, J. M.,
Azorsa, D.,
Jhun, B. H.,
Cheong, J.,
Lee, Y. C.,
Meltzer, P. S.,
and Lee, J. W.
(1999)
J. Biol. Chem.
274,
34283-34293 |
23. |
Lee, S.-K.,
Na, S.-Y.,
Jung, S. Y.,
Choi, J. E.,
Jhun, B. H.,
Cheong, J.,
Meltzer, P. S.,
Lee, Y. C.,
and Lee, J. W.
(2000)
Mol. Endocrinol.
14,
915-925 |
24. |
Hong, S. H.,
and Privalsky, M. L.
(2000)
Mol. Cell. Biol.
20,
6612-6625 |
25. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1995) Current Protocols in Molecular Biology , Greene Associates, NY |
26. | McKinsey, T. A., Zhang, C. L., Lu, J., and Olson, E. N. (2000) Nature 408, 106-111[CrossRef][Medline] [Order article via Infotrieve] |
27. |
McKinsey, T. A.,
Zhang, C. L.,
and Olson, E. N.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
14400-14405 |
28. | Tombes, R. M., Grant, S., Westin, E. H., and Krystal, G. (1995) Cell Growth Differ. 6, 1063-1070[Abstract] |
29. | Kawamura, K., Grabowski, D., Krivacic, K., Hidaka, H., and Ganapathi, R. (1996) Biochem. Pharmacol. 52, 1903-1909[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Anderson, K. A.,
Ribar, T. J.,
Illario, M.,
and Means, A. R.
(1997)
Mol. Endocrinol.
11,
725-737 |
31. | Williams, C. L., Phelps, S. H., and Porter, R. A. (1996) Biochem. Pharmacol. 51, 707-715[CrossRef][Medline] [Order article via Infotrieve] |
32. | Natoli, G., Costanzo, A., Guido, F., Moretti, F., and Levrero, M. (1998) Biochem. Pharmacol. 56, 915-920[CrossRef][Medline] [Order article via Infotrieve] |
33. | Rayet, B., and Gelinas, C. (1999) Oncogene 18, 6938-6947[CrossRef][Medline] [Order article via Infotrieve] |
34. | Zhong, H., SuYang, H., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1997) Cell 89, 413-424[Medline] [Order article via Infotrieve] |
35. | Zhong, H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell. 1, 661-671[Medline] [Order article via Infotrieve] |