(Received for publication, January 3, 1996)
From the
The Rel family of transcription factors are important mediators
of various cytokine stimuli such as interleukin (IL)-1, tumor necrosis
factor (TNF)-, and CD28 costimulation in T cell effector
responses. These stimuli induce Rel family DNA-binding activity to the
B enhancer and CD28 response elements of many cytokine gene
promoters leading to cytokine production. Consistent with the
importance of Rel family induction during immune responses, c-Rel
knockout mice exhibit profound defects in T cell functions including
IL-2 secretion and T cell proliferative responses to CD28 plus T cell
receptor costimulation. The novel protein kinases, c-Jun
NH
-terminal kinases (JNKs)/stress-activated protein
kinases, are also activated by TNF-
, IL-1, and CD28 costimulation.
Because of the common regulation of c-Rel and JNK1 by these agents in T
cells, we investigated the role of JNK1 in c-Rel activation. We found
that MAP kinase kinase kinase (MEKK) 1, a JNK1 activator, induced
transcription from the human immunodeficiency virus-1 long terminal
repeat and IL-2R
promoters in a
B-dependent manner.
Coexpression of I
B
, a c-Rel inhibitor, inhibited the
MEKK1-induced transcriptional activity. JNK1 synergized with MEKK1 in
activating transcription from a
B-driven heterologous promoter.
Furthermore, JNK1 associated with c-Rel in vivo in Jurkat T
cells by coimmunoprecipitation assays and bound directly to c-Rel in a
yeast two-hybrid assay. c-Rel also competed with c-Jun in in vitro kinase assays. However, JNK1 did not phosphorylate c-Rel,
NF-
B, and I
B
in vitro, indicating that c-Rel
may serve as a docking molecule to allow JNK1 phosphorylation of
certain Rel-associated proteins. Transactivation of the IL-2R
and
HIV-
B-driven promoters by c-Rel was augmented by coexpression of
MEKK1. These results demonstrate the first significant role for the
MEKK1 kinase cascade module in c-Rel-mediated transcription.
Characterization of signal transduction pathways mediating
cellular stress responses has expanded rapidly with the discovery of
multiple, parallel, mammalian mitogen-activated protein kinase (MAPK) ()modules homologous to those in yeast (reviewed in (1, 2, 3, 4) ). Recently, a growing
family of mammalian kinases, c-Jun NH
-terminal kinases
(JNKs)(5, 6) , and their rat homologs,
stress-activated protein kinases(7) , were identified based
upon their activation by environmental stimuli such as UV light and the
protein synthesis inhibitor anisomycin. Subsequently, kinase activation
by osmotic shock(6) , CD28 plus T cell receptor (TCR)
costimulation(8) , proinflammatory cytokines such as
TNF-
(9, 10) and IL-1(11) , and
DNA-damaging agents (12) has been demonstrated. Delineation of
the pathway linking receptors to JNK activation reveals similarity to
the Raf
MEK
MAPK pathway. MEKK1 activates JNKK/MKK4/SEK1,
a MEK homolog, which then activates
JNK1(13, 14, 15) . MEKK1 activation is
apparently specific for the JNK cascade at physiological levels as only
overexpression of MEKK1 induces MAPK
activity(14, 15, 16) .
While many of the
activating inputs of JNK have been defined, few substrate outputs have
been identified. JNK1 has been shown to bind, phosphorylate, and
activate the transcription factors c-Jun(5) , ATF2 (12, 17, 18) , and Elk-1(19) . Many
of the agents which activate JNK1 such as TNF-, IL-1, UV light,
and CD28 plus TCR costimulation also activate the Rel/NF-
B family
of transcription factors causing transcriptional activation of
promoters containing
B enhancers such as the HIV-1 LTR and the
IL-2R
promoters. Because of the common regulation of JNK1 and
c-Rel by a number of agents, we investigated the role of the MEKK1/JNK1
kinase module in
B enhancer activation. In this report, we
demonstrate the induction of the HIV-1 LTR and IL-2R
promoter by
the MEKK1 signaling cascade. Furthermore, JNK1 directly bound c-Rel,
and MEKK1 synergized with c-Rel to activate transcription from
B-driven promoters.
Figure 1:
MEKK1 induces B enhancer-dependent
IL-2R
and HIV-LTR transcription. A, 5 µg of the
IL2R
-CAT or IL-2R
(
B)-CAT (22) reporter was
transfected into Jurkat cells with 5 µg of pEE-CMV (lanes 1 and 4), 5 µg of pCMV-
MEKK1 (lanes 2 and 5), or 5 µg of pCMV-
MEKK1 + 10 µg of
pRSV-I
B
(lanes 3 and 6) (21) by
electroporation. B, 3 µg of the HIV-CAT or
HIV(
B)-CAT (34) reporter was transfected into Jurkat
cells with 5 µg of pEE-CMV (lanes 1 and 5), 5
µg of pCMV-
MEKK1 (lanes 2 and 6), 5 µg
of pCMV-
MEKK1 + 10 µg of pRSV-I
B
(lanes 3 and 7), or 10 µg of pHIV-Tat (lanes 4 and 8) by electroporation. Control vectors normalized the amount
of transfected DNA.
Rel/NF-B family members are retained
in the cytoplasm by various inhibitory molecules including
I
B
. Rel/NF-
B induction involves the phosphorylation and
degradation of I
B
, resulting in the release of Rel/NF-
B
and subsequent translocation to the nucleus. To demonstrate that the
MEKK1-induced transcriptional responses involve the Rel family of
transcription factors,
MEKK1 and I
B
were co-transfected
into Jurkat cells with the IL-2R
-CAT and HIV-CAT reporter
constructs. While
MEKK1 alone induced transcription from these
reporters (Fig. 1), coexpression of I
B
inhibited
MEKK1-mediated induction (Fig. 1, A and B, lane 3). These results demonstrate that the
NF-
B family of transcription factors are necessary for
MEKK1-induced transcriptional effects from these
B-driven
promoters.
MEKK1, which has a growth factor-induced, Ras-dependent
kinase activity (29) and a kinase domain that binds directly to
Ras in vitro(30) , was first identifed as an upstream
kinase in the MAPK cascade(28) .However, recent work in several
laboratories has positioned MEKK1 as an upstream kinase in the JNK
cascade. Induction of stably transfected MEKK1 resulted in JNK and not
MAPK activity. Only overexpression of MEKK1 induced some MAPK
activity(14, 16, 31, 32) suggesting
that MEKK1 induction of the JNK cascade is more physiologically
relevant. To substantiate JNK1 involvement in the MEKK1-induced
NF-
B transcription which is shown in Fig. 1, suboptimal
amounts of
MEKK1 alone, JNK1 alone, or
MEKK1 plus JNK1 were
co-transfected with the 2xNF
B-CAT reporter construct, 6tkCAT. JNK1
increased
MEKK1-induced transcription of the reporter 4-fold when
compared to a suboptimal dose of
MEKK1 or JNK1 alone (Fig. 2). No synergy was observed with the control plasmid
BLCAT2.
Figure 2:
MEKK1-activated JNK1 induces transcription
through B sites. 1 µg of the 2xNF
B-CAT (6tkCAT) or BLCAT2 (24) reporter was transfected into Jurkat cells with 1 µg
of pEE-CMV (lane 1), 1 µg of pCMV-
MEKK1 (lane
2), 10 µg of pCMV-JNK1 (lane 3), or 1 µg of
pCMV-
MEKK1 + 10 µg of pCMV-JNK1 (lane 4).
Control vectors normalized the amount of transfected
DNA.
Since
JNK1 binds to c-Rel in a two-hybrid assay, we performed two-cycle
immunoprecipitations (20) of
[S]methionine-labeled Jurkat T cells (Fig. 3, A and B) to demonstrate JNK1-c-Rel
interactions in mammalian cells. This assay identifies specific members
of protein complexes in vivo. Primary immunoprecipitations
isolate a targeted protein from a complex which is then dissociated by
boiling. Secondary immunoprecipitations of this disrupted protein
complex identify members associated with the original target protein
from the first immunoprecipitation. Jurkat cells were stimulated with
phorbol 12-myristate 13-acetate and ionomycin during 16-20 h
labeling times. Whole cell extracts were subjected to two cycles of
immunoprecipitation using either anti-c-Rel or anti-JNK1 anitbodies in
the first cycle followed by an anti-c-Rel antibody in the second cycle.
c-Rel is specifically isolated after two cycles of immunoprecipitation
with the anti-c-Rel antibody, Ab265 (Fig. 3A, lane
1). To test JNK1-c-Rel interactions, JNK1 was immunoprecipitated
with antibodies against different JNK1 epitopes (lanes
2-6, J1-J5). These JNK1-immunoprecipitated
complexes were tested for the presence of c-Rel by boiling and
reimmunoprecipitating with anti-c-Rel during the second cycle. c-Rel
was isolated from these JNK1 complexes after the second
immunoprecipitation (lanes 2-6), indicating that c-Rel
associates with JNK1 in vivo.
Figure 3:
JNK1
associates with c-Rel in Jurkat cells in vivo. 10 ng/ml
phorbol 12-myristate 13-acetate + 1 µM ionomycin
stimulated (16 h), S-Met-labeled Jurkat cell lysate was
subjected to two-cycle immunoprecipitations (20) with
anti-c-Rel and anti-JNK1 antibodies. A, JNK1 associates with
c-Rel. Immunoprecipitations (1st IP) were performed with
anti-c-Rel Ab265 (
Rel, lane 1), anti-JNK1 COOH
terminus (
J-1, lane 2), anti-JNK1 Ab102 (
J-2, lane 3), anti-JNK1 Ab101 (
J-3, lane 4), anti-JNK1 NH
terminus (
J-4, lane 5), anti-JNK1 recombinant protein (
J-5, lane 6), and normal rabbit sera (nrs, lane 7). Subsequently, the precipitates were
boiled in SDS and immunoprecipitated a second time (2nd IP)
with the anti-c-Rel Ab265 (
Rel). B,
immunoprecipitations (1st IP) were done with anti-c-Rel Ab265 (
R-1, lane 1), and anti-JNK1 (
J-4, lanes 2 and 3) followed by boiling and
re-immunoprecipitation (2nd IP) with anti-c-Rel Ab265 (
R-1, lanes 1 and 3), anti-c-Rel Ab1135 (
R-1, lane 2).
This interaction was also
seen with anti-c-Rel antibody, Ab1135 (Fig. 3B), which
was generated against a different c-Rel epitope. c-Rel is detected in
these lysates after immunoprecipitating first with an antibody against
the NH terminus of JNK1 followed by Ab1135 or Ab265 (lanes 2 and 3, respectively). The c-Rel complexed
with JNK1 in vivo corresponds to the c-Rel isolated by
two-cycle immunoprecipitations with Ab265 (lane 1). These
co-immunoprecipitation data, using multiple JNK1 and c-Rel antibodies,
show that JNK1 associates with c-Rel in Jurkat cells in vivo.
Furthermore, the use of five different anti-JNK1 antibodies raised
against distinct JNK1 epitopes eliminates the possibility of
cross-reactivity by anti-JNK1 antibodies with c-Rel. Similarly, the use
of two anti-c-Rel antibodies raised against different c-Rel epitopes
eliminates possible cross-reactivity of the anti-c-Rel antibodies with
JNK1. Finally, the interaction is specific, since a primary
immunoprecipitation with preimmune sera did not result in isolation of
c-Rel after a second cycle of anti-c-Rel immunoprecipitation (Fig. 3A, lane 7). The combination of the
immunoprecipitation and two-hybrid system data indicate that c-Rel and
JNK1 interact with each other in vivo.
JNK1 has been shown
to bind to and phosphorylate the transcription factors c-Jun, ATF2, and
Elk-1. The in vivo interaction of JNK1 with c-Rel suggests
that JNK1 may phosphorylate c-Rel or other NF-B family members. To
test this hypothesis, we performed immunocomplex kinase assays with an
anti-JNK1 antibody, Ab101, using recombinant NF-
B family members
as substrates. Ab101 specifically recognized a UV- and
anisomycin-stimulated 46-kDa protein kinase in an in-gel kinase assay
using GST-Jun as a substrate (Fig. 4A), indicating the
antibody was not immunoprecipitating multiple kinases. Furthermore, the
antibody recognized endogenous and recombinant JNK1 in a Western blot
(data not shown). In the immunocomplex kinase assays, however, JNK1 did
not phosphorylate any of the recombinant NF-
B family members
tested either alone or in combination with I
B
(Fig. 4B and data not shown).
Figure 4:
JNK1 does not phosphorylate Rel family
members in vitro.A, an inducible, 46-kDa protein
kinase is recognized by anti-JNK1 Ab101 in Jurkat cells. In-gel kinase
assay (6) using Ab101 immunoprecipitates from untreated Jurkat
cells (lane 1) or Jurkat cells treated with 100 J/m UV irradiation (lane 2) or 50 µg/ml anisomycin (lane 3) using G-Jun(1-331) as a substrate. B,
immunocomplex kinase assays using anti-JNK1 Ab101 reveal no
phosphorylation of Rel family members in vitro. 40 µg
Jurkat whole cell extract were immunoprecipitated with 4 µl of
anti-JNK1 Ab101. The JNK1 precipitate was then used in kinase assays
with 3 µg of GST-c-Jun(1-331) (lane 1),
GST-c-Jun(1-79) (data not shown), GST-c-Rel (lane 2),
GST-p50 (lane 3), or GST-p65 (lane
4).
Since JNK1 bound c-Rel in vivo but did not phosphorylate Rel family members in vitro, we investigated c-Rel's ability to compete with c-Jun and interfere with JNK1 activity. JNK1 was immunoprecipitated from anisomycin-stimulated extracts, washed, and preincubated with either buffer, GST, or GST-c-Rel at room temperature (Fig. 5). GST-c-Jun(1-79) was then added and a kinase reaction was performed. When GST was added alone (lane 2), no inhibition of c-Jun phosphorylation was observed when compared with buffer preincubation (lane 1). However, as an equal or greater number of moles of GST-c-Rel compared to c-Jun were added, inhibition of c-Jun phosphorylation was observed (lanes 4-6). Preincubation with half the number of GST-c-Rel moles had no detectable effect (lane 3). This competition most likely results from c-Rel binding to JNK1 and not through binding to the c-Jun substrate. The c-Jun used (amino acids 1-79) lacks the carboxyl-terminal leucine zipper region shown to interact with Rel family proteins(33) . This in vitro competition of kinase activity further supports the binding between JNK1 and c-Rel observed in the two-hybrid assay and two-cycle immunoprecipitation ( Table 1and Fig. 3).
Figure 5:
c-Rel blocks JNK1 phosphorylation of c-Jun in vitro. JNK1 was immunoprecipitated from anisomycin lysates,
washed, and preincubated with buffer (lane 1), GST (lane
2), or GST-c-Rel(2-587) (lanes 3-6) for 35
min at room temperature. GST-c-Jun(1-79) was added and kinase
reactions performed. n equals the number of moles of
GST-c-Rel added relative to GST-c-Jun.
Figure 6:
MEKK1 protein kinase synergizes with c-Rel
in activating the IL-2R and HIV
B-driven promoters. A, 5 µg of the IL2R
-CAT or IL2R
(
B)-CAT
reporter was transfected into Jurkat cells with 15 µg of pEE-CMV (lanes 1 and 5), 10 µg of pCMV-c-Rel (lanes 2 and 6)(22) , 5 µg of pCMV-
MEKK1 (lanes 3 and 7), or 5 µg of pCMV-
MEKK1
+ 10 µg of pCMV-c-Rel (lanes 4 and 8) by
electroporation. B, 1 µg of the 2xNF
B-CAT (6tkCAT) or
BLCAT2 reporter was transfected into Jurkat cells with 15 µg of
pEE-CMV (lanes 1 and 5), 10 µg of pCMV-c-Rel (lanes 2 and 6), 5 µg of pCMV-
MEKK1 (lanes 3 and 7), or 5 µg of pCMV-
MEKK1
+ 10 µg of pCMV-c-Rel (lanes 4 and 8) by
electroporation. C, 5 µg of the HIV-CAT or
HIV(
B)-CAT reporter was transfected into 293T cells with 10
µg of pCMV-c-Rel, 5 µg of pCMV-
MEKK1, or 5 µg of
pCMV-
MEKK1 + 10 µg of pCMV-c-Rel by calcium phosphate
precipitation. The DNA amount in each transfection was equalized with
pEE-CMV.
To demonstrate that MEKK1 was activating JNK1
specifically in this transfection system, immunocomplex kinase assays
were performed. JNK1, p38, and ERK2 were immunoprecipitated from empty
vector and MEKK1-transfected 293T cells (Fig. 7). JNK1 was
activated 50-fold in the
MEKK1-transfected cell lysates (lane
2) compared to control transfected lysates (lane 1),
while ERK2 and p38 activity remained relatively unchanged (compare lanes 4 and 6 with lanes 3 and 5,
respectively). These data show that MEKK1 activates JNK1 and not the
other MAPKs, ERK2 and p38, in transient transfection assays.
Furthermore, the transfection and kinase results suggest a synergistic
role for MEKK1-activated JNK1 and c-Rel in
MEKK1-mediated
activation of the
B elements.
Figure 7:
Preferential JNK1 activation by
transfected MEKK1. 5 µg of pEE-CMV or 5 µg of pCMV-MEKK1
was transfected into 293T cells, and JNK1, p38, and ERK2 activation was
measured by immunocomplex kinase assays. 2.5 µg of
GST-c-Jun(1-79) was used as a JNK1 substrate and 5 µg of
myelin basic protein was used as a p38 or ERK2
substrate.
c-Rel and JNK1 are critical molecules involved in CD28
signaling and T-cell activation. Both are activated by CD28
costimulation leading to c-Rel-mediated IL-2 transcriptional activation
and IL-2 production. This study provides evidence that the MEKK1
signaling module and c-Rel interact in T cells. The MEKK1 module drives
transcription from the HIV-LTR and IL-2R reporters in a
B-dependent manner and synergizes with c-Rel in activating
transcription from these reporters. JNK1 involvement in mediating
MEKK1-activated NF-
B transcription is suggested by the fact that
JNK1, not ERK2 nor p38, is activated in MEKK1-transfected cell lysate (Fig. 7). Cotransfection of a dominant negative JNK1 construct
with MEKK1 would solidify JNK1's role in this MEKK1-activated
NF-
B pathway, but attempts thus far have failed possibly due to an
inefficient inhibitory effect of the JNK1 mutant (
)and/or
high endogenous JNK1 activity in Jurkat cells (data not shown).
Nevertheless, this study provides the first demonstration of
MEKK1-activated NF-
B transcription.
Additional evidence for
interaction between the JNK1 and NF-B activation pathways is
demonstrated through binding studies. JNK1 bound directly to c-Rel in vivo in a yeast two-hybrid assay (Table 1) and
associated with c-Rel in vivo in Jurkat cells as shown by
two-cycle immunoprecipitations (Fig. 3). Furthermore, GST-c-Rel
blocked JNK1 phosphorylation of a truncated GST-c-Jun substrate in
vitro in immunocomplex kinase assays (Fig. 5). The results
from these three assays indicate that c-Rel binds to JNK1 in vivo and that c-Rel binds to the activated form of the JNK1 protein
kinase in vitro. As JNK1 binds and phosphorylates other
transcription factors like c-Jun and ATF2, NF-
B phosphorylation
may be the result of the JNK1-c-Rel interaction. However, no
phosphorylation of Rel/NF-
B family members was observed in
vitro ( Fig. 4and data not shown). While the lack of
phosphorylation in vitro does not rule out Rel family members
as substrates, other c-Rel-associated proteins may be the in vivo targets for JNK1. For example, c-Rel may serve as a docking or
stabilizing molecule for the JNK1 protein kinase and cause enhanced
phosphorylation of or access to c-Rel-associated JNK1 substrates. As
many common agents activate the MEKK1 module and c-Rel, this
interaction between JNK1 and c-Rel would potentiate the signaling
effects of these proteins as suggested by the observed synergy between
c-Rel and MEKK1 (Fig. 6). Our finding of the physical
association of c-Rel and JNK1 in T cells, coupled with their
involvement in common T cell signaling cascade, will lead to the
discovery of other, novel JNK1 substrates that are important in T cell
signal transduction. This lays the foundation to investigate the
cross-talk between JNK1 and c-Rel in signaling cascades.