From the Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain
Received for publication, December 15, 2000
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ABSTRACT |
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Transactivation by c-Rel (nuclear factor
Transcription factors belonging to the nuclear factor NF- Analysis of the transactivation domain of p65 by CD and NMR
spectroscopy revealed no defined structure (19). Two differentiated acidic regions (termed TA1 and TA2) were identified as essential for
its transcription promoting activity. Only TA2, however, was responsible for the activation by phorbol ester stimulation by a
mechanism that involved phosphorylation of Ser residues (8). Moreover,
the high Ser content of the transactivation domain of the avian
c-Rel-related oncogene v-rel has been demonstrated to be
essential for its transforming capabilities (20). Furthermore, the
mutation of the Ser residue 471 in the human c-Rel transactivation domain abrogated TNF In this work we have characterized the regulation of c-Rel
transactivation domain. This domain seems to belong to the family of
the phosphorylation-dependent Ser-rich acidic
transactivation domains. We have revealed the critical role of several
Ser residues for TNF Cells and Reagents--
Jurkat cells and COS-7 cells were grown
in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5%
heat-inactivated fetal bovine serum (FBS; Life Technologies, Inc.) and
containing 100 µg/ml streptomycin, 100 units/ml penicillin, 2 mM L-glutamine, plus nonessential amino acids,
at 37 °C in a 7% CO2-in-air atmosphere saturated with
water vapor incubator.
Recombinant human tumor necrosis factor
Sera from rabbits hyperimmunized with peptides derived from human c-Rel
(no. 265), kindly provided by Dr. Nancy Rice (NCI-FCRDC, Frederick, MD)
were used to detect the corresponding protein on Western blots, used at
a dilution of 1:10,000. Monoclonal anti-epitope HA antibody used for
immunoprecipitation studies was purchased from Roche Molecular
Biochemicals (Mannheim, Germany).
Plasmids--
The pNF3TK Luc reporter plasmid contains a trimer
of the NF-
Gal4 c-Rel-(309-588) wild type was made by cloning the corresponding
c-Rel PCR fragment into the XhoI-BglII site of
the Gal4 c-Jun-(1-166) plasmid, thus removing the c-Jun fragment. The
template for PCR reactions was pRc-hc-Rel, which consists of pRcCMV
with c-Rel cDNA inserted in the HindIII-XbaI
site. Gal4 DNA binding domain (DBD) fusions with different c-Rel
transactivation domain deletion mutants were made using the same
approach as Gal4 c-Rel-(309-588). The fragments fused to Gal4 DBD
were:Gal4-(309-318), Gal4-(309-372), Gal4-(309-421),
Gal4-(309-455), Gal4-(309-497), Gal4-(309-540), Gal4-(422-588),
Gal4-(456-588), Gal4-(498-588), Gal4-(541-588), Gal4-(498-540),
Gal4-(456-497), and Gal4-(422-455).
Substitutions Ser Cell Transfection--
Jurkat T cells or COS-7 cells were washed
once and resuspended at 106 cells/ml in Opti-MEM (Life
Technologies, Inc.). Cells were transfected with the LipofectAMINE Plus
reagent (Life Technologies, Inc.) preparing the LipofectAMINE
Plus-plasmid mixtures in accordance with the manufacturer's
instructions. The mixtures were incubated at 37 °C in a 7%
CO2 incubator for 3 h before washing with fresh Dulbecco's modified Eagle's medium + 5% FBS, and incubated for another 18 h. The cells were then washed once with, and
resuspended at the same concentration in, Dulbecco's modified Eagle's
medium + 5% FBS. Culture medium with or without stimuli, as indicated in text (10 ng/ml TNF Western Blots and Immunoprecipitation--
Whole cell extracts
(WCE) were made using TNT buffer as lysis buffer (20 mM
Tris-HCl, pH 7.6, 200 mM NaCl, 1% Triton X-100) supplemented with protease inhibitors (2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin, 0.1 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride) and phosphatase
inhibitors (5 mM NaF, 1 mM Na3VO).
For immunoprecipitation, WCE were incubated for 30 min at 4 °C with 1 µg of anti-HA antibody. Precipitates were collected on protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden), separated in
a 10% SDS-PAGE, and subsequently transferred to a polyvinylidene difluoride membrane. Membranes were analyzed by Western blot. For
Western blot, WCE were separated on a 10% SDS-PAGE and transferred to
a polyvinylidene difluoride membrane (Immobilon, Amersham Pharmacia Biotech). Rabbit anti-human c-Rel was used as first antibody, and goat
anti-rabbit IgG peroxidase as secondary antibody. The enhanced
chemiluminescent (ECL) developing kit (Amersham Pharmacia Biotech) was
used to identify the relevant band(s).
Solid Phase in Vitro Phosphorylation Assay--
c-Rel
transactivation domain constructs from position 422-588 or 422-540
(using as template pRc-hc-Rel wild type) were cloned into the
BamHI-EcoRI site of plasmid pGEX2T (Amersham
Pharmacia Biotech) in order to express recombinant GST-c-Rel fusion
protein. These recombinant proteins were purified from E. coli induced cultures according to the manufacturer's
instructions. 25 µl of GSH-agarose-GST-c-Rel were used as substrate
of an in vitro phosphorylation reaction in which whole cell
extracts from non-stimulated or stimulated Jurkat cells were assayed.
WCE were made from 106 Jurkat cells in 25 µl, as
described above. The reaction mixture (kinase buffer) contained 20 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM Mapping of the c-Rel Transactivation Domain
Mouse c-Rel transactivation domain has been previously localized
in the C-terminal region of the protein, between positions 403 and 568 (23). In order to delineate the transcriptionally active region of
human c-Rel we constructed several fusion plasmids between the Gal4 DBD
and c-Rel, providing a system where transcriptional activity of this
protein could be assayed without interference of IB) was dependent on phosphorylation of several serines in the
transactivation domain, indicating that it is a
phosphorylation-dependent Ser-rich domain. By Ser
Ala
mutational and deletion analysis, we have identified two regions in
this domain: 1) a C-terminal region (amino acids 540-588), which is
required for basal activity; and 2) the 422-540 region, which responds
to external stimuli as tumor necrosis factor (TNF)
or phorbol
myristate acetate plus ionomycin. Ser from 454 to 473 were shown to be
required for TNF
-induced activation, whereas Ser between 492 and 519 were required for phorbol myristate acetate plus ionomycin activation.
Phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC)
were identified as downstream signaling molecules of TNF
-activation
of c-Rel transactivating activity. Interestingly, dominant negative
forms of PI3K inhibited PKC
activation and dominant negative PKC
inhibited PI3K-mediated activation of c-Rel transactivating activity,
indicating a cross-talk between both enzymes. We have identified the
critical role of different Ser for PKC
- and PI3K-mediated responses.
Interestingly, those c-Rel mutants not only did not respond to TNF
but also acted as dominant negative forms of nuclear factor
B activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-
B)1 family
regulate several of the most important genes induced during T cell
activation (for review, see Ref. 1). The NF-
B family of
transcription factors is composed of homo- and heterodimers of a family
of proteins, which include the Dorsal gene of
Drosophila and the mammalian genes
nf
b1, nf
b2,
c-rel, relA (p65), and relB (for
review, see Ref. 2). All members share a conserved 300-amino acid
region in their N terminus that includes the dimerization, nuclear
localization, and DNA binding regions. c-Rel, RelB, and RelA also have
C-terminal transactivation domains, which strongly activate
transcription from NF-
B sites. NF-
B is rapidly activated by the T
cell receptor complex, but, at later phases of T cell activation,
autocrine or paracrine secreted TNF
takes control of NF-
B
activation (3). Tumor necrosis factor (TNF)
is a pleiotropic
cytokine with biological effects ranging from promoting growth and
differentiation to induction of apoptosis. Those effects rely, at least
in part, in the activation of the transcription factor NF-
B (for
review, see Ref. 4). In T cells, the initial phase of NF-
B
activation after T cell receptor triggering mainly relies on p65
translocation, whereas the later phase is controlled by c-Rel. We have
previously shown that autocrine or paracrine TNF
secretion controls
the c-Rel levels in T cells (3). Thus, c-Rel activation emerges as a
key point for the later phase of T lymphocyte activation, a fact that
is supported by the functional unresponsiveness of T lymphocytes from
the c-Rel knock out mice (5, 6).
B activity is regulated, at least in part, by its subcellular
localization. Thus, functional NF-
B complexes are held in the
cytoplasm of resting T cells in an inactive state complexed with
members of the I
B family. In response to different activators, which
include T cell receptor and TNF
, I
B is phosphorylated by I
B
kinases (IKKs), and subsequently degraded, liberating the active
NF-
B complex, which translocates to the nucleus and activates transcription (for review, see Ref. 7). Recently, a second level of
regulation of NF-
B activity independent of I
B, which relies in
the activation of the transcriptional activity of p65, has been
described (8-10). Thus, the catalytic subunit of protein kinase A was
shown to be bound to inactive NF-
B complexes, and upon I
B
degradation this catalytic subunit phosphorylated p65, resulting in an
enhanced transcription promoting activity (11). Moreover, TNF
treatment of cells results in phosphorylation of Ser529 in
the transactivation domain of p65, resulting in the activation of the
transcriptional activity of the protein (10). The small GTP-binding
protein Ras enhanced p65/RelA transcriptional activity through a
pathway that required the stress-activated protein kinase p38 or a
related kinase (12), although it was not demonstrated whether this
kinase was directly involved in activating NF-
B or instead a
transcriptional co-activator. The activity of Ras as well as the
atypical protein kinase C
(PKC
) has been also shown to be
essential for the transcriptional activity of p65/RelA in endothelial
cells (13). This activation relies in the phosphorylation of the
N-terminal Rel homology domain and not on the C-terminal transactivation domain. PKC
was able to phosphorylate and activate IKK2 (14), thus demonstrating its direct implication in the NF-
B
activation process by participating in I
B degradation. A recently
identified 62-kDa protein (named p62) might function as a bridge
between PKC
and the TNF receptor-associated protein RIP (15). On the
other hand, PI3K activity seems to be required for interleukin-1- and
TNF
-induced NF-
B activity (16, 17). The PI3Ks are a family of
lipid kinases that catalyze the addition of a phosphate group to the
3'-OH position of the inositol ring of phosphoinositides. The
3-phosphoinositides are second messengers that exert specific
regulatory functions inside the cells (18). PI3K is composed of two
different subunits, a regulatory subunit (p85) and a catalytic subunit,
termed p110. Upon stimulation, p85 becomes associated to the cytosolic
portion of tyrosine-phosphorylated receptors via its SH2 domains, which
in turn promotes its association with the catalytic subunit p110 and
its subsequent activation. The activation of PI3K triggers a signaling
cascade that leads to the specific phosphorylation of p65/RelA subunit.
This phosphorylation enhances p65-mediated transcription without
affecting I
B degradation, nuclear translocation of NF-
B, or the
ability of NF-
B to bind to DNA (16).
-induced NF-
B activity in a Jurkat T cell clone (21). Taken together, all these works point to a key functional role of the regulation of the transactivation domain of c-Rel family
proteins for NF-
B function.
-dependent activation.
Interestingly, mutations of those Ser residues not only abrogated c-Rel
transactivating activity, but also acted as dominant negative forms of
NF-
B activation, further stressing the importance of this regulation
in the activity of NF-
B. Additionally, we have identified PI3K and
PKC
as enzymes participating in the signaling route that leads to
c-Rel activation by TNF
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF
) was
purchased from Genzyme (Cambridge, MA). Phorbol myristate acetate
(PMA), calcium ionophore A23187 and HA1004, and
L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone (TPCK)
were purchased from Sigma. Cyclosporin A, cypermethrin, and LY294002
were purchased from Biomol (Plymouth Meeting, PA). The inhibitors
wortmannin and D609 were obtained from Calbiochem (San Diego, CA). The
inhibitor SB203580 was a kind gift of Dr. J. M. Redondo (Centro de
Biologia Molecular Severo Ochoa, Madrid, Spain). The lipid
phosphatidylinositol 3,4,5-trisphosphate (PIP3) was
obtained from Alexis Biochemicals (San Diego, CA).
B-binding motif of the H-2k gene upstream of the TK
minimal promoter and the luciferase reporter gene (22). The reporter
Gal4 Luc contains five tandem repeats of the Gal4 element upstream from the luciferase reporter gene (kindly provided by Dr. J. M. Redondo). Expression plasmids encoding either wild-type or
dominant-negative mutant of PKC
were kindly provided by Dr. J. Moscat (Centro de Biologia Molecular Severo Ochoa, Madrid, Spain).
p85 expresses deletion mutant of p85 subunit of human PI3K enzyme
incapable of binding to catalytic subunit p110, rendering a
dominant-negative form of the enzyme (kindly provided by Dr. J. Downward, Imperial Cancer Research Fund, London, United Kingdom).
Ala were made with the QuickChange site-directed
mutagenesis kit (Stratagene), using as template for mutation the
Gal4-c-Rel deletion mutant
7-(422-588). Mutation was confirmed by
sequencing in each case. Substitutions were as follows: A1 = Ser426; A2 = Ser443,
Ser444, Ser447; A3 = Ser454;
A4 = Ser460; A5 = Ser463; A6 = Ser470, Ser471, Ser473; A7 = Ser484; A8 = Ser491, Ser494;
A9 = Ser508, Ser509, Ser510,
Ser511, Ser513; A10 = Ser518;
A11 = Ser525, Ser527; A12 = Ser533, Ser536; A13 = Ser541;
A14 = Ser546, Ser549, Ser551;
A15 = Ser563, Ser566; A16 = Ser577, Ser579. Similar substitutions were made
using pRc-hc-Rel as template.
or PMA (10 ng/ml) + calcium ionophore (1 µM)) was added to duplicate wells containing 0.5 ml of
these cell suspensions, which were then incubated under the same
conditions for 6 h. The cells were lysed with Passive Cell Culture
Lysis Reagent (Promega, Madison, WI) and microcentrifuged at full speed for 5 min at 4 °C, and 20 µl of each supernatant was used to
determine firefly luciferase activity in a Monolight 2010 luminometer
(Analytical Luminescence Laboratory). The results were expressed as
-fold increase in luminescence relative to the value obtained with the non-stimulated control after normalization with respect to protein concentration, determined by the bicinchoninic acid spectrophotometric method (Pierce). For normalization of transfection efficiency, cells
were co-transfected with the reporter plasmid pTK Renilla (Promega) and luciferase activity recorded using the Dual Luciferase assay (Promega). Results are always expressed as values normalized to
Renilla activity.
-glycerophosphate, 20 µM ATP, and 1 µCi of [32P]ATP (specific activity, 3,000 Ci/mol).
After 20 min at 30 °C, the reaction was terminated by washing with
TNT buffer. Phosphorylated protein was boiled in 25 µl of Laemmli
sample buffer and resolved in 10% SDS-PAGE, followed by
autoradiography. For PKC
phosphorylation assay, anti-HA precipitates
from WCE from transfected COS-7 cells were incubated in the kinase
buffer described before, containing 1 µg of soluble recombinant GST
c-Rel-(422-588).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B association
and/or degradation. The fragment of c-Rel from position 309 to 588 was
fused to Gal4 DBD, and several deletion mutants of this region were
generated (Fig. 1). These constructs were
transfected into Jurkat T cells along with a 5xGal4 Luc reporter plasmid and luciferase activity was recorded. As a negative control, a
construction which covered only the c-Rel fragment from position 309 to
318 fused to Gal4 DBD was used.
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Fig. 1.
Deletion mapping of the C terminus of
c-Rel. Left panel, structure of c-Rel
transactivation domain deletion mutants. RHD, Rel homology
domain; NLS, nuclear localization signal. Right
panel, effect of deletion mutants on the transcriptional
activity of c-Rel fusions to Gal4 DBD. Jurkat T cells were transfected
with each of the mutants described and with a reporter plasmid
containing five tandem repeats of Gal4 site upstream from the
luciferase gene. Results are expressed as percentage of activity
compared with the wild-type construct. Transfection efficiency was
normalized using the Dual Luciferase assay (Promega). Additionally,
similar amounts of the different construction were expressed in
transfected cells as detected by EMSA assays (data not shown). The
results shown are the mean ± S.D. of three independent
experiments.
Constructions spanning the c-Rel region from 309 to 421 had no basal transcriptional activity, as the control construction Gal4-(309-318) (Fig. 1, right). A minimal transcriptional activation was observed when Gal4-(309-455) was transfected. By contrast, Gal4-(422-588) induced an activity of the Gal4 reporter that was 236% of the wild-type fusion, indicating that this region possessed all the transcriptionally active sequences of c-Rel and even behaved as a better autonomous transactivation domain that the whole c-Rel C-terminal region (from position 309 to 588). Progressive deletions toward the C terminus were introduced in this region (Gal4-(456-588), Gal4-(498-588), and Gal4-(541-588)), which increasingly reduced the transcriptional activity. However, the smallest construction Gal4-(541-588) still evidenced a significant transcription promoting activity. That opened the possibility of the co-existence of several subdomains within region 422-588. To test this, smaller fragments covering this region were fused to Gal4 DBD (Gal4-(498-540), Gal4-(456-497), and Gal4-(422-455)). As shown in Fig. 1, none of them were transcriptionally active, indicating that region 422-588 behaved as a single transcription activation domain.
Identification of the Regions in c-Rel Transactivation Domain
Activated by TNF and PMA + Ionophore
We used the Gal4 DBD fusion plasmids described in the preceding
section to map the region responsible for the transcriptional activation of c-Rel by TNF and compared it with the activation produced by PMA + ionophore. Table I
lists the TNF
and PMA + ionophore inducibility of the different
constructions. The region responsive to activation mapped between
positions 422 and 540. Although region 540-588 showed a strong basal
transcription activity (Fig. 1), it did not respond to stimulation,
suggesting that this C-terminal region was necessary for the basal
transcriptional activity of c-Rel but was not involved in TNF
or PMA + ionophore stimulation.
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Analysis of c-Rel Region 422-588 by Ser Ala Substitutions
The above results indicate the region 422-588 includes the
transcription promoting region of c-Rel, whereas the region 422-540 was responsible for integrating signals derived from activation by
TNF and PMA + ionophore. This region contains 33 Ser residues (20%), 20 acidic residues (12%), and 8 Pro residues (5%), suggesting that it could be an acidic transcription activation domain, despite not
having any significant homology to conventional acidic and Pro-rich
transactivation domains (24). The existence of 20% Ser residues could
confer it with the properties of a transcription activation domain
regulated by phosphorylation. Those Ser residues are strongly conserved
between the human and murine proteins, suggesting the relevance of
those residues for function. In order to study their functional
relevance, we introduced Ser
Ala mutations into the
Gal4-c-Rel-(422-588) (Fig.
2A) fusion. We subsequently assayed for the basal and the TNF
- or PMA/ionophore-induced
transcriptional activity of the substitution mutants transfected into
Jurkat T cells. However, we did not observe significant differences in the basal activity in any of the Ser
Ala substitutions (Fig. 2B), indicating that none of them is absolutely necessary
for the basal transcriptional activity of c-Rel. Interestingly, when we
assayed for activation by TNF
or PMA + ionophore, we observed that
mutants A3, A4, A5, and A6 failed to respond to TNF
stimulation by
increasing its transactivating activity, whereas mutants A8, A9, and
A10 showed a reduced response to either PMA + ionophore or TNF
.
Mutants A13-A16, included within the constitutively active region
541-588, showed a reduced response, as well. This results identified
one region essential for the activation of c-Rel transcriptional capabilities by TNF
, which includes the Ser residues 454, 460, 463, 470, 471, and 473. Substitution of those residues abrogated the
activation of c-Rel by TNF
. A second region including Ser residues
491, 494, 508, 509, 510, 511, 513, and 518 was identified to be
involved in c-Rel activation, although it was not essential (Table
II).
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|
Identification of the Signaling Route Involved in TNF Activation
of c-Rel Transactivating Activity
Implication of Phosphatidylinositol 3-Kinase (PI3K)--
We used
several commercial inhibitors of putative signaling enzymes to define
the route leading to c-Rel activation by TNF. We first transfected
Jurkat T cells with a NF-
B reporter plasmid and tested the effect of
different inhibitors in TNF
-stimulated NF-
B activity. As a
control, the proteasome inhibitor TPCK was used as a generic inhibitor
of NF-
B activity since it interferes with the degradation of I
B.
Of the different inhibitors used, only the PI3K inhibitor wortmannin
significantly inhibited TNF
stimulation of NF-
B activity (Fig.
3A). To corroborate the effect of wortmannin, another inhibitor of PI3K, LY294002, structurally unrelated to wortmannin, produced similar inhibition of NF-
B activity (Fig. 3B). Neither LY294002 was wortmannin affected
cell viability (data not shown). However, NF-
B activity results from the combined effect of I
B degradation and c-Rel and p65 activation. Thus, in order to study exclusively the implication of PI3K on c-Rel
transactivating activity, we transfected Jurkat cells with the Gal4
c-Rel-(309-588) construct and tested the effect of the PI3K
inhibitors. Both PI3K inhibitors prevented the transcription activity
of c-Rel stimulated by TNF
(Fig. 3C).
|
PKC Involvement in the Activation of the c-Rel Transactivation
Domain--
PKC
has been recently found to be involved in the
process of NF-
B activation by TNF
stimulation (25), so we studied
the effect of this kinase on c-Rel activation. Jurkat T cells were co-transfected with the NF-
B reporter along with a plasmid that expressed either a wild-type or a dominant-negative mutant form of
PKC
. Co-transfection of wild-type PKC
induced a strong increase in NF-
B-driven reporter activity, compared with cells co-transfected with an empty plasmid (Fig. 4).
Furthermore, co-transfection of the PKC
dominant-negative mutant
inhibited the activation of NF-
B transcriptional activity by TNF
(Fig. 4A). Parallel experiments were carried out assaying
the activity of the Gal4 reporter driven by Gal4 c-Rel-(309-588)
construct. Co-transfection of PKC
induced a strong activity of the
Gal4 reporter, in a similar way as it did with the NF-
B reporter,
and TNF
stimulation induced a still higher activation of the
reporter (Fig. 4B). Interestingly, co-transfection of a
PKC
dominant-negative mutant completely inhibited TNF
activation. Taken together, those results suggested the involvement of PKC
in
the activation of the c-Rel transactivation domain by TNF
and
subsequently in NF-
B activation.
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Cross-talk between PI3K and PKC--
The above results
indicated that PI3K and PKC
were involved in the activation of the
c-Rel transactivation domain by TNF
. On the other hand, the products
of PI3K activity, PIP2 and PIP3, have
been described to activate PKC
(26). Thus, these enzymes could be
participating in the same signaling pathway leading to c-Rel activation
by TNF
. To investigate this, Jurkat cells were transfected with a
dominant-negative mutant of PI3K (termed
p85), resulting in the
inhibition of both the NF-
B- and Gal4 c-Rel-driven activity induced
by co-transfection of active PKC
(Fig. 4). Additionally, the PI3K
inhibitors wortmannin and LY294002 inhibited the activation of
NF-
B-dependent reporter activity (Fig.
5A), as well as activation of
Gal4 c-Rel-driven activity (Fig. 5B) induced by transfection of wild-type PKC
. On the other hand, PIP3, the product
of PI3K activity, was able to induce the activity of Gal4 reporter
driven by Gal4 c-Rel when added exogenously. Addition of
PIP3 to cultures of Jurkat cells co-transfected with
wild-type PKC
, along with the Gal4 reporter and Gal4 c-Rel, resulted
in a reporter activity similar to that observed after TNF
stimulation (Fig. 5C), suggesting that PIP3 is
able to activate PKC
activity. However, PIP3 could not
revert the inhibition produced by co-transfection of the
dominant-negative mutant of PKC
(Fig. 5C). These data
indicate that both PI3K and PKC
are necessary for the activation of
c-Rel transactivation domain but do not allow establishment of the
relative position of each other in the signaling pathway.
|
Mapping of the Target Sites of PKC and PI3K on the c-Rel
Transactivation Domain
Results shown above indicate that TNF-dependent
activation of c-Rel transactivation domain relies on the Ser residues
defined by the Ser
Ala substitution mutants A3, A4, A5, A6, A8, A9, and A10. In order to identify the exact residues that were affected by
TNF
-induced PKC
activity, these mutants were co-transfected into
Jurkat cells with the wild-type form of PKC
. As shown in Fig.
6A, co-transfection of PKC
along with wild-type Gal4 c-Rel-(422-588) induced an average of
2.5-fold induction of reporter activity. Co-transfection of Ser
Ala
substitution mutants A3, A5, A6, A9, and A10 with PKC
produced a
similar activation of the reporter. However, mutants A4
(Ser460) and A8 (Ser491, Ser494)
were not activated by PKC
co-transfection. These results indicate that those sites were necessary for PKC
activation of c-Rel
transactivation domain. Strikingly, both sites are palindromes of the
sequence SNCS, not found in other part of the c-Rel transactivation
domain.
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A similar approach was used to map the sites relevant in PI3K
activation of c-Rel. Exogenous PIP3 was used to mimic PI3K
activation. Wild-type Gal4 c-Rel-(422-588) was successfully activated
by PIP3 treatment, as well as the Ser Ala substitution
mutants A4, A5, A9, and A10 (Fig. 6B). Substitution mutant
A8 (Ser491, Ser494), which was not activated by
PKC
, was not activated either by PIP3 treatment, while
substitution mutants A3 (Ser454) and A6
(Ser470, Ser471, Ser473) displayed
only a reduced activation. These results suggest that the positions
defined by substitution mutant A8 may be the point of coincidence of
PKC
and PI3K activation on c-Rel transactivation domain. Noteworthy,
both substitution mutants A3 and A6 show a Ser residue close to an Asp
residue, pointing out to a possible unique kinase dependent on PI3K activity.
c-Rel Transactivation Domain Mutants Act as Dominant Negative Forms
in NF-B Activation by TNF
In order to test the functional significance of several of those
mutations in c-Rel functioning as well as in NF-B activation in T
cells, we transfected expression plasmid of c-Rel mutants into Jurkat
cells together with a NF-
B-luc reporter gene. Basal reporter
activity was not altered by overexpression of any c-Rel protein (data
not shown). As shown in Fig. 7,
transfection of wild-type c-Rel slightly, although significantly,
increased TNF
-induced stimulation. However, mutants A3, A4, A5, and
A8 could not support TNF
-induced activation of NF-
B activity in
contrast to wild-type c-Rel. More interestingly, those results indicate
that overexpression of these mutant proteins acted as dominant negative
forms for NF-
B activation (that includes both endogenous p65 and
c-Rel), further pointing out to the importance of these pathways in
NF-
B activity.
|
Phosphorylation of c-Rel Region 422-588
The above results suggested that Ser residues in region 422-588
may be activated by TNF through phosphorylation. Previous studies
have shown that activity of c-Rel and other NF-
B proteins are indeed
regulated by phosphorylation (27). In order to study the
stimulation-dependent phosphorylation of this region, we
made a recombinant GST fusion protein comprising region 422-588 of c-Rel transactivation domain. Solid phase phosphorylation assays using
this recombinant protein revealed that extracts from
non-stimulated cells already had strong c-Rel basal phosphorylation
activity. Nonetheless, addition of extracts made from Jurkat cells
stimulated either with TNF
or PMA + ionophore gave rise to a
significant increase (about 2-fold) in the level of phosphorylation
state of the recombinant protein (Fig.
8A). When a fusion construct lacking the region 541-588 was used, a great reduction in
phosphorylation by unstimulated extracts was observed (Fig.
8B). Interestingly, this construct, comprising region
422-540, was more heavily phosphorylated by extracts from
TNF
-stimulated Jurkat cells (about 5-fold increase). Those results
suggest that region 541-588 retains most of the basal phosphorylation
of c-Rel, but the phosphorylation dependent on TNF
activation
resides in the region 422-540, thus corroborating the results obtained
for transcriptional stimulation of the deletion mutants. In addition,
PI3K inhibitors prevented the increase of the in vitro
phosphorylation of c-Rel transactivation domain by cell extracts from
TNF
-stimulated cells (Fig. 8C). Although both TNF
and
PMA + ionophore stimulation induced the specific phosphorylation of the
c-Rel transactivation domain, we could not detect any kinase activity
in TNF
- or PMA/ionophore-treated cell extracts on this domain using
"in-gel" kinase phosphorylation assays (data not shown).
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DISCUSSION |
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The intermediate and late phase of T cell activation is controlled
by the autocrine or paracrine effect of the cytokine TNF, which in
turn modulates the transcription factor NF-
B through the sustained
activation of c-Rel (3). On the other hand, it is well established that
IKK activation by TNF
stimulation, and subsequent I
B degradation,
is a pre-requisite for NF-
B activation (28-30). However, a second
level of regulation of NF-
B activity that is independent of IKK
degradation has been recently described. This second level involves the
signal-dependent phosphorylation and activation of the
transactivation domain of p65 (8, 10, 16, 31), although the exact
mechanism has not yet been defined.
The transactivation domain of c-Rel has been previously defined as the region downstream the Rel homology domain to the C terminus of the protein (positions 309-588), as in this region reside all the transcription promoting capabilities of c-Rel (23, 32). The deletion analysis described in this work has indicated that positions 309-421 in the C terminus are dispensable for c-Rel-mediated transcription, further delimiting the location of this transactivation domain between positions 422 and 588 of the C terminus. However, a role of the region 309-421 in the regulation of c-Rel stability through degradation by the proteasome has been proposed (33). Furthermore, as the region 422-588 fused to the Gal4 DBD had a stronger transcriptional activity than the complete C-terminal region (309), this may indicate the existence of repressor sequences within the 309-421 region. Alternatively, region 422-588 could acquire a configuration in the Gal4 fusion that is more competent for transcriptional activation. Sequential deletions from position 422 to 588 (C terminus) were associated with parallel decrease in transcriptional activity. The region from position 541 to 588 still retained significant transcriptional activity, whereas smaller fragments (422-455, 456-497, and 498-540) showed no transcriptional activity, indicating that region 422-588 is a functional transactivation domain non divisible in smaller units. This is not the case of the Rel family member p65, where two different transcriptional activating regions were found in the transactivation domain, named TA1 and TA2 (8, 19).
Stimulation of the cells transfected with the Gal4 c-Rel fusion
constructs revealed that only region 422-540 was activated by TNF,
as well as PMA + ionophore, stimulation. Region 541-588 was not
further activated by these stimuli, even though it showed a strong
basal transcriptional activity. Thus, those analyses indicate that this
region is necessary for the function of c-Rel as a transcription factor
but dispensable for inducible activation of this factor. In
vitro solid phase phosphorylation assays demonstrated that this
region (541) was strongly phosphorylated by cell extracts obtained
from non-stimulated cells, thus supporting its role in basal
transcriptional activity of c-Rel. In contrast, region 422-540 was
weakly phosphorylated by cell extracts from unstimulated cells, whereas
extracts from TNF
- or PMA/ionophore-stimulated Jurkat cells strongly
phosphorylated it. This indicate that it is within this region where
stimulus-induced activation occurs. These data suggested that the
activation of this region requires extracellular signal-dependent phosphorylation.
This region (422) is negatively charged (15 acidic residues, 3 basic) and is rich in Pro and Ser. These characteristics resemble to
acidic transactivation domains (24). Although this region is not
conserved among different Rel family members, the acidic and Ser
residues are well conserved between human and mouse c-Rel, suggesting a
critical role for c-Rel functioning. This region does not show homology
to other described acidic transactivation factors. However, its high
Ser content is typical of other transcription factors like CREB,
TCF/Elk-1, or STAT that are regulated by phosphorylation (34). Thus,
phosphorylation may provide the additional negative charges necessary
to constitute an acidic transactivation domain. The effect observed in
Ser Ala mutants of the transactivation domain of c-Rel corroborates
this hypothesis. Although none of the mutants had any effect on the
basal transcriptional activity of c-Rel, the substitution of the Ser
residues at position 455 (mutant A3), 461 (mutant A4), 464 (mutant A5),
or 470, 471, and 473 (mutant A6) completely prevented the activation of
this domain by TNF
. Any of these positions is absolutely required
and must be activated, as the substitution of any of them was enough to abrogate the activation of this domain by TNF
. Additionally, the
substitution of the Ser residues at position 492 and 494 (mutant A8),
509-512 and 514 (mutant A9), or 519 (mutant A10) had a less pronounced
inhibitory effect on both TNF
- and PMA/ionophore-induced activation
of c-Rel transactivation domain, indicating that they may participate
in c-Rel activation although they are probably not essential.
Transcription factors like NF-B activate gene transcription through
the interaction with basal transcriptional machinery, or with
co-factors that modulate that machinery (35). In this regard, both
c-Rel and v-Rel have been found to interact with the basal
transcription factors TBP and TFIID directly through their
transactivation domains (36). However, other reports indicate that only
the first 50 amino acids were necessary for TBP interaction (37). p65
could interact with TBP and TFIID through its transactivation domain
(8, 38). Furthermore, other proteins that are able to interact with
NF-
B have been described: the HMG(I)Y nuclear protein (39), the SP1
factor (40), or a 40-kDa protein, which acts as a target for the
quinone derivative E3330 in the inhibition of NF-
B activity (41).
The mutations described in this work may be very useful for the study
of the interactions of c-Rel with the transcriptional machinery and to
define protein-protein interactions that regulates the process of
transcription activation.
We were unable to identify a kinase activity, capable of
phosphorylating c-Rel transactivation domain, by in-gel phosphorylation assays. However, only a fraction of cellular kinases can remain active
after the renaturalization process required in those assays. Furthermore, no kinase that requires a co-factor or association with
other proteins to form an active complex will remain active in those
assays. On the other hand, the use of commercially available inhibitors
revealed the dependence of NF-B activation by TNF
on the activity
of PI3K. PI3K activity was previously described as necessary for
NF-
B activity in several cell types but not in lymphocytes (42).
Furthermore, PI3K-dependent phosphorylation and activation
of p65 has been described as a requisite for interleukin-1 activation
of p65 transactivation domain, without affecting I
B degradation or
DNA binding activity of p65 complexes (16). In a similar manner,
TNF
-dependent activation of NF-
B-induced
transcription in HepG2 cells has been shown to depend on PI3K activity,
which does not affect NF-
B binding to DNA or I
B degradation
induced by the cytokine (17). Our results indicate that a similar
mechanism is taking place for c-Rel activation by TNF
. Thus, PI3K
inhibitors wortmannin and LY294002 inhibited not only NF-
B dependent
activity, but also c-Rel activation and phosphorylation of its
transactivation domain, supporting a critical role of PI3K activity for
TNF
activation of c-Rel. However, whether PI3K can associate with
TNF receptors or any of its associated factors is not known yet.
Furthermore, the PI3K metabolite, PIP3, was capable of
activating Gal4 c-Rel when transfected into Jurkat cells but not
NF-
B reporter activity (data not shown). Thus, TNF
might activate
several signaling routes, leading to the direct modulation of
transcriptional abilities of c-Rel, as well as to activation of IKKs
for I
B degradation and subsequent nuclear translocation of active
NF-
B heterodimers. PI3K may therefore be implicated in the
activation of c-Rel transactivation domain but not in the activation of
IKK by TNF
. Nevertheless, one of the well known targets of PI3K
activity, the protein kinase Akt, has been recently demonstrated to
interact with IKK upon TNF
stimulation (43, 44). However, our
results with PIP3 stimulation, as well as other recent
reports looking directly at NF-
B binding activity (16, 17), suggest
that PI3K may not be essential for IKK activation and subsequent
nuclear translocation of NF-
B. Although those discrepancies cannot
be explained yet, PI3K might differentially affect NF-
B activation
depending on the cell type.
On the other hand, PKC has been implicated in NF-
B activation,
through mechanisms that involve IKK activation (14, 45, 46) or directly
through the phosphorylation and activation of p65 (13). Thus, PKC
may play a dual role in the activation of NF-
B, activating IKK and
also participating in the activation the transactivation domain of
members of the Rel family. Our results clearly support this hypothesis,
indicating that PKC
, besides participating in IKK activation, is
also involved in the activation of c-Rel transactivation domain. Thus,
co-transfection experiments into Jurkat cells of PKC
wild-type with
Gal4 c-Rel showed a strong potentiation of c-Rel transcriptional
promoting capabilities. Furthermore, a dominant-negative mutant of
PKC
abrogated TNF
-induced c-Rel activation. Surprisingly, PKC
activation of c-Rel transactivation domain was inhibited in the
presence of wortmannin and LY294002, both inhibitors of PI3K activity,
as well as the co-transfection of a dominant-negative mutant of PI3K,
suggesting that PI3K might act downstream of PKC
. In addition, the
activation of c-Rel transactivation domain by PIP3, a
product of PI3K activity, was inhibited by a dominant-negative mutant
of PKC
. However, PKC
has been shown to be activated by the
protein kinase PDK-1, an effector of PI3K activity (47), suggesting
that PKC
would be downstream of PI3K in the route of c-Rel
activation. A possible explanation for this apparent different
positioning in the signaling pathway of PI3K and PKC
is that both
pathways are parallel and required for activity.
The fact that PIP3 and active PKC seem to act on
different Ser residues of the c-Rel transactivation domain would be in
agreement with the above hypothesis. The Ser
Ala substitution
analysis showed that positions defined by the mutants A3, A4, A5, and
A6 are essential and in lesser extent by the mutants A8, A9, and A10
for c-Rel activation by TNF
. Thus, the failure to activate one of
them resulted in the inability of c-Rel to be activated by TNF
.
Mapping of the sites activated by PKC
and PIP3 revealed that the only Ser residues substituted in mutant A8 are the point of
convergence for PKC
and PI3K activation. However, PKC
also required the Ser residue defined by mutant A4, whereas PIP3
required Ser residues defined by mutants A3 and A6. Thus, the
inhibition of either PKC
or PI3K would result in an inhibition of
c-Rel transactivation domain. The sites defined by mutants A4 and A8 showed a striking palindromic similarity (SNCS for A4 and SCNS for A8).
Mutant A5, however, which substituted the second Ser residue downstream
mutant A4, was not essential for PKC
activation. Thus the Ser
residue close to Asn (Ser460 and Ser494) may be
the actual target of PKC
activation. Furthermore, A8, which defined
the point of convergence between PI3K and PKC
, mutants A3
(Ser454) and A6 (Ser471, Ser473,
and Ser474), failed to be activated by PIP3.
These mutants involve Ser residues that are in close proximity of an
Asp residue, suggesting that the same kinase might be activating both
sites. Furthermore, a natural mutant of Ser471 to Asn
produced a form of c-Rel that could not be activated by TNF
stimulation (21), suggesting that PI3K may be an essential part of the
signaling mechanisms activated by TNF
, resulting in the activation
of c-Rel transactivation domain. Furthermore, the different responses
of several Ser mutants to the different stimuli clearly discard
nonspecific effects of the pathways or activators used.
More interestingly, those mutants not only are defective in
transactivating activity, but they prevent
NF-B-dependent reporter activity. Those results indicate
that they act as dominant negative forms of NF-
B activation, either
by recruiting the kinases required for phosphorylation of
transactivation domains of c-Rel and/or p65 or by binding to NF-
B
sites on DNA and then prevent active NF-
B complexes (either p65 or
endogenous c-Rel) for binding. The fact that the spontaneous
Ser471 mutant failed to activate at all NF-
B (21) tends
to support the first hypothesis.
In summary, our results have revealed an important level of
TNF-induced NF-
B activation mediated through PI3K and PKC
, which are absolutely required for c-Rel transactivating activity (Fig.
9).
|
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ACKNOWLEDGEMENTS |
---|
We thank María Chorro and Lucía Horrillo for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from Dirección General de Investigación Científica y Técnica, Fondo de Investigaciones Sanitarias, Comunidad Autónoma de Madrid, and Fundación Ramón Areces.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.
To whom correspondence should be addressed. Tel.: 34-913978413;
Fax: 34-913974799; E-mail: mfresno@cbm.uam.es.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M011313200
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ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear
factor
B;
DBD, DNA binding domain, FBS, fetal bovine serum;
GST, glutathione S-transferase;
IKK, I
B kinase;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
PI3K, phosphatidylinositol 3-kinase;
PMA, phorbol myristate acetate;
TNF, tumor necrosis factor;
TPCK, L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone;
WCE, whole cell extract;
PKC, protein kinase C;
HA, hemagglutinin;
PIP3, phosphatidylinositol 3,4,5-trisphosphate.
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