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Molecular and Cellular Biology, November 2001, p. 7207-7217, Vol. 21, No. 21
Department of Molecular Biology, Cell Biology, and
Biochemistry, Brown University, Providence, Rhode Island
029121; Department of Medicine and
Whittier Diabetes Program, University of California
Received 8 January 2001/Returned for modification 14 March
2001/Accepted 2 August 2001
The Raf kinase inhibitor protein (RKIP) acts as a negative
regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade
initiated by Raf-1. RKIP inhibits the phosphorylation of
MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by
disrupting the interaction between these two kinases. We show here that
RKIP also antagonizes the signal transduction pathways that mediate the
activation of the transcription factor nuclear factor kappa B (NF- The transcriptional
activator nuclear factor kappa B (NF- We report here that the recently discovered protein kinase inhibitor
protein RKIP (Raf kinase inhibitor protein) acts to inhibit NF- Active NF- The kinase activity that mediates the phosphorylation of I NF- Although considerable progress has been made in the identification of
kinases that activate the IKK complex, little is known about negative
regulators that may impinge on these pathways (61). We
show here that ectopic expression of RKIP is sufficient to downregulate
NF- Cell culture and biological reagents.
293 cells, 293/IL-1R1,
BOSC 23, and COS-1 cells were grown in Dulbecco's modified Eagle's
medium with high glucose and glutamine and supplemented with 10% fetal
bovine serum and penicillin-streptomycin. 293/IL-1R1 (Z. Cao, Tularik)
is a stably transfected 293 cell line overproducing the IL-1 receptor
(9). Rat1 fibroblasts were maintained in the same medium
supplemented with 10% calf serum. Recombinant human TNF- Plasmids and protein expression.
HA-tagged RKIP, ASK1,
N-terminally truncated MEKK1, TAK1, and FLAG-tagged NIK, IKK Construction of a stable cell line expressing HA-RKIP.
BOSC
23 cells (55) were transfected with 20 µg of plasmid
pWZL-HA-RKIP (55). At 72 h posttransfection, the
virus-containing medium was filtered and used to infect Rat1
fibroblasts. Forty-eight hours after infection, the cultures were
trypsinized and diluted into medium containing 5 µg of blasticidin
(ICN Pharmaceuticals, Inc.)/ml. Individual blasticidin-resistant
colonies were ring cloned and expanded. The expression levels of
HA-RKIP in each clone were monitored by immunoblotting with 12CA5 antibody.
In vitro kinase assays.
IKK assays were performed as
described (43). 293 cells were transiently transfected
with the indicated expression plasmids using Lipofectamine (Life
Technologies) according to the manufacturer's specifications.
Thirty-six hours posttransfection, the cells were treated for 5 h
with 50 ng of TNF- Transfection and reporter gene assays.
COS-1 cells were
transfected by the DEAE dextran/chloroquine method using 5 µg of DNA
per 100-mm-diameter dish. 293, 293/IL-1R1, and Rat1 cells were
transfected at 70 to 80% confluence with Lipofectamine using 3 µg of
DNA and 2 µg of Lipofectamine per 35-mm-diameter plate. Cells
were harvested 48 h after transfection, and total cell extracts
were assayed for luciferase activity. Where indicated, cultures were
treated with TNF- Fractionation of Rat1 S100 cytosolic extract.
To prepare
S100 cytosolic extract, 8 × 107 Rat1 cells
were suspended in buffer A (10 mM HEPES [pH 7.9], 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM dithiothreitol)
supplemented with protease inhibitors. After 15 min on ice, the cells
were disrupted by five passes through a 26-gauge needle. The lysate was
centrifuged at 12,500 × g for 5 min at 4°C, and the
pelleted nuclei and mitochondria were discarded. The supernatant was
centrifuged again at 12,500 × g for 10 min at 4°C to
remove residual mitochondria and heavy membranes. Subsequently, the
supernatant was centrifuged at 100,000 × g for 60 min
at 4°C. Glycerol was added to the S100 supernatant to 10%, and the
KCl concentration was adjusted to 0.1 M. One milliliter of this extract (total protein, 2 mg) was applied to a 1-ml HiTrap DEAE Sepharose Fast
Flow column (Amersham Pharmacia Biotech) equilibrated with buffer A
with 100 mM KCl. The column was washed with 3 column volumes of buffer
A with 0.1 M KCl and eluted with 10 ml of 0.1 to 1 M linear KCl
gradient. Fractions of 0.5 ml were collected. Chromatography was
performed at 4°C using an automatic fast protein liquid
chromatography station (Amersham Pharmacia Biotech).
RKIP is an inhibitor of the NF-
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.21.7207-7217.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Raf Kinase Inhibitor Protein Interacts with
NF-
B-Inducing Kinase and TAK1 and Inhibits NF-B
Activation
San Diego, La
Jolla, California 92093-06732; and
Beatson Institute for Cancer Research, CRC Beatson
Laboratories, Bearsden, Glasgow G61 1BD, United
Kingdom3
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
B)
in response to stimulation with tumor necrosis factor alpha (TNF-
)
or interleukin 1 beta. Modulation of RKIP expression levels affected
NF-B signaling independent of the MAPK pathway. Genetic epistasis
analysis involving the ectopic expression of kinases acting in the
NF-B pathway indicated that RKIP acts upstream of the kinase complex
that mediates the phosphorylation and inactivation of the inhibitor of
NF-B (IB). In vitro kinase assays showed that RKIP antagonizes
the activation of the IB kinase (IKK) activity elicited by TNF-.
RKIP physically interacted with four kinases of the NF-B activation
pathway, NF-B-inducing kinase, transforming growth factor
beta-activated kinase 1, IKK, and IKK
. This mode of action bears
striking similarities to the interactions of RKIP with Raf-1 and MEK1
in the MAPK pathway. Emerging data from diverse organisms suggest that
RKIP and RKIP-related proteins represent a new and evolutionarily
highly conserved family of protein kinase regulators. Since the MAPK
and NF-B pathways have physiologically distinct roles, the function
of RKIP may be, in part, to coordinate the regulation of these pathways.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
B) is required for
the upregulation of a large number of genes in response to
inflammation, viral and bacterial infection, and other stress stimuli.
Genes that respond to NF-B encode a variety of cytokines, cell
adhesion molecules, and acute-phase response proteins as well as
apoptotic suppressor and effector proteins. It is believed that this
reprogramming of gene expression is essential for cell survival during
situations of physiological crisis (61). The activation of
NF-B in response to stimulation by the proinflammatory cytokines
tumor necrosis factor alpha (TNF-) and interleukin 1 beta (IL-1)
has been extensively studied (17, 30); however, the
mechanisms that modulate and eventually limit these responses are still
poorly understood (61).
B
activation. RKIP was first identified as an interacting partner of
Raf-1 and shown to function as a negative regulator of the
mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by
Raf-1 (75, 76). The Raf-1-initiated pathway is comprised of three sequentially acting protein kinases: a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAPK. This basic
relationship has now been found to be conserved in several protein
kinase pathways. In the Raf-1 pathway the MAPK is ERK1/2 (extracellular
signal-regulated kinase 1 and 2), the MAPKK is MEK1 (MAP/ERK
kinase 1), and the MAPKKK is Raf-1 itself. Functional studies
using both gain-of-function and loss-of-function approaches demonstrated that RKIP disrupts the interaction between Raf-1 and MEK1
(75, 76). Depletion of endogenous RKIP upregulated Raf-1
kinase activity and MAPK signaling, whereas ectopic expression of RKIP
suppressed Raf-1 kinase activity and MAPK signaling as well as
v-Raf-mediated transformation. Biochemical studies showed that RKIP
efficiently dissociated preformed Raf/MEK complexes and behaved
kinetically as a competitive inhibitor of MEK phosphorylation. In vivo,
the association of endogenous RKIP with Raf-1 correlated inversely with
Raf-1 kinase activity during serum stimulation of quiescent cells.
B is a dimer that can be assembled from several members of
the Rel family of transcription factors, and some form of NF-B is
expressed in most cell types (61). In unstimulated cells,
NF-B is retained in the cytoplasm in an inactive form bound to a
family of inhibitory proteins known as IB (inhibitors of B).
Activation of NF-B requires the phosphorylation and degradation of
IB, which allows the NF-B dimer to translocate into the nucleus. Virtually all of the many stimuli that can activate NF-B cause the
phosphorylation of IB on two serine residues (Ser-32 and Ser-36 in
IB). This event targets IB for rapid polyubiquitination and
degradation by the 60S proteosome (2, 31).
B has
been recently identified as part of a large (700 to 900 kDa)
multisubunit cytoplasmic IKK (IB kinase) complex (25). Three components of the IKK complex involved in the phosphorylation of
IB on the serines at positions 32 and 36 have been cloned and
designated as IKK, IKK, and IKK
(also known as NEMO or IKKAP1)
(14, 47, 48, 58, 62, 72, 74). The and subunits
display kinase activity for IB, whereas no recognizable kinase
domain is found in IKK. It has been proposed that two molecules of
IKK associate with a catalytic IKK/IKK heterodimer, and that
the C terminus of IKK links this core particle to upstream signaling
molecules (25, 47, 78). The importance of these molecules
in NF-B signaling has been confirmed by genetic analysis utilizing
knockout mice (23, 39, 41, 45, 63, 68, 69).
B can be activated by several kinase signaling cascades and is
subject to multiple levels of regulation. Some of the kinases in
NF-B-activating pathways are related on the basis of sequence conservation to kinases in MAPK pathways. The kinase domains of IKK
and IKK contain an activation loop with the motif SXXXS, common to
all MAPKKs, whose phosphorylation on both serine residues activates the
kinase. Furthermore, overexpression as well as interference studies
with dominant-negative mutants have suggested that some kinases in the
MAPKKK family, including NIK (NF-B-inducing kinase), MEKK1
(MEK kinase 1), TAK1 (transforming growth factor beta-activated kinase 1), mixed-lineage kinase 3, and Cot/TPL2 are involved in the
phosphorylation and activation of IKKs in response to specific stimuli
(20, 37, 42, 46, 50). Protein kinases other than MAPKKKs,
such as protein kinase C and B, have also been reported to act
upstream of IKK (27, 35, 54, 60), and a homologue of IKK
designated NAK (NF-B-activating kinase) has been implicated as an
upstream activator of IKK (57, 70).
B activity, whereas ablation of endogenous RKIP activity
upregulates the NF-B pathway. Specifically, RKIP can negatively
modulate the activating phosphorylations of IKK and IKK by
upstream kinases. In mechanistic terms, RKIP physically associates with
NIK and TAK1 and modulates the response of the NF-B pathway to
TNF-- and IL-1-mediated signaling. This mode of action bears
striking similarities to the interactions of RKIP with Raf-1 and MEK in
the MAPK pathway.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
and
IL-1 were purchased from Life Technologies and Calbiochem,
respectively. Anti-FLAG M2 antibody and affinity resin were purchased
from Sigma. Rabbit anti-NIK polyclonal antibody was from Santa Cruz
Biotechnology. Monoclonal anti-actin and anti-hemagglutinin (HA)
12CA5 antibodies were purchased from Amersham and Boehringer Mannheim,
respectively. Rabbit anti-TAK1 antibody was a gift from K. Matsumoto
(73). MEK inhibitor U0126 was purchased from Promega.
,
IKK, and NAK expression plasmids have been previously described
(10, 24, 46, 50, 58, 70, 72, 76). To construct FLAG-RKIP,
the RKIP cDNA was PCR amplified and cloned in frame into pCMV2-FLAG
(Sigma). Glutathione S-transferase (GST)-IB was
expressed in Escherichia coli and purified as
described (26). To construct an HA-RKIP retrovirus
expression vector, HA-RKIP (75) was cloned between the
EcoRI and SalI sites of the retrovirus vector
pWZL-Blast (J. Morgenstern, Millenium). The reporter plasmids E-Sel
lacZ, CRE-lacZ, NF-B-Luc, AP-1-Luc, Gal4-Luc,
and Hsp-Luc and effector plasmids CMV-Gal4 (amino acids 1 to
94), CMV-Gal4(Sp1), CMV-Gal4(Sap1), and CMV-RKIP have been previously reported (34, 49, 76, 77).
/ml. After TNF- treatment, cells were washed
with cold phosphate-buffered saline and lysed in 50 mM HEPES (pH 7.6),
250 mM NaCl, 10% glycerol, 1 mM EDTA, and 0.1% Nonidet P-40
supplemented with protease and phosphatase inhibitors. Cell lysates
were cleared by centrifugation and incubated for 4 h at 4°C with
anti-FLAG M2 antibody conjugated to agarose beads. In vitro kinase
assays were performed on the immune complexes using purified
recombinant GST-IB protein as a substrate in 20 µl of kinase
buffer containing 20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 0.5 mM dithiothreitol, 100 µM ATP, and 5 µCi of [-32P]ATP at room temperature for
30 min.
(50 ng/ml) or IL-1 (10 ng/ml) for 6 h
immediately before harvest. A Renilla luciferase gene (Promega) driven by the constitutive thymidine kinase promoter was
included in all transfection experiments as an internal control to
correct for transfection efficiency. Dual luciferase assays were
performed using a kit obtained from Promega. Microinjection experiments
with affinity-purified RKIP antiserum and NF-B and TRE-lacZ reporter plasmids were performed as described
(75, 76).
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
B signaling pathway.
In
experiments to investigate the role of RKIP in MAPK pathway signaling
(75, 76), NF-B reporters were used as controls and
discrete effects on activity were noticed. We therefore
performed a series of directed experiments to examine the role of RKIP
on NF-B signaling. First, we inhibited endogenous RKIP activity by
antibody microinjection and monitored the effects on NF-B activity
by measuring the expression of a coinjected NF-B lacZ reporter, E-sel lacZ (Fig.
1A). Microinjection of affinity-purified anti-RKIP antibodies strongly activated the NF-B lacZ
reporter in Rat1 fibroblasts to approximately the same extent as that
elicited by the microinjection of a plasmid encoding the p65 subunit of NF-B (Fig. 1A). This effect was specific because the injection of a
control immunoglobulin G (IgG) was ineffective and the anti-RKIP IgG
did not affect the expression of a cyclic AMP-dependent reporter gene,
CRE × 5 lacZ (Fig. 1A, bars 1 and 7). Ectopic
expression experiments corroborated these results by showing that the
transfection of an RKIP expression construct diminished the basal
activities of NF-B and AP-1 reporters to approximately the same
extent (Fig. 1B). The repression was specific since the effect was not
observed with a reporter lacking NF-B binding sites (Fig. 1B, bars 5 and 6).
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FIG. 1.
Ablation of RKIP activates and overexpression of RKIP
represses basal NF- B activity. (A) Ablation of RKIP activity by
antibody injection activates an NF-B-dependent reporter (E-sel) but
not a cyclic AMP-stimulated reporter (CRE × 5). E-sel is an
E-selectin promoter DNA fragment containing one copy of the consensus
NF-B binding site (71). Quiescent Rat1 cells were
microinjected with the indicated reporter plasmids and antibodies and
either left unstimulated or treated with 20 µg of forskolin per ml,
an activator of adenyl cyclase. (B) Overexpression of RKIP represses
NF-B- and AP-1-dependent reporters. RKIP was placed under the
control of the cytomegalovirus (CMV) promoter (CMV-RKIP). RKIP
or an empty vector control (CMV) was cotransfected with the indicated
reporter plasmids into exponentially growing NIH 3T3 cells, and 48 h later extracts were assayed for luciferase activity. The activities
of reporters in combination with the empty CMV vector were set to
100%. In all panels the means and standard deviations of at least two
independent experiments are shown.
B signaling (15,
40, 52), we considered the possibility that the observed repression by RKIP could be the result of cross talk from the Raf/MEK/ERK pathway. To address this, we examined the effect of pharmacological MEK inhibitors, such as U0126, on the repression of an
NF-B luciferase reporter. These experiments showed that significant
inhibition of NF-B signaling in unstimulated, actively growing cells
can occur at concentrations of U0126 that abolish MEK activity (Fig.
2A and B, lanes 4 to 6). The effect of
U0126 was specific since it had no effect on Sp1 transactivation
activity (Fig. 2B, lanes 1 to 3). Cross talk between the MAPK and
NF-B pathways does occur, but its magnitude is insufficient to
explain the influence of RKIP on NF-B activity. For example, we
observed that approximately 20% of the NF-B basal activity found in
exponentially cycling 293 cells could be inhibited by MEK inhibitors,
and it is thus likely elicited by cross talk from the Raf/MEK/ERK
pathway (Fig. 2A, lanes 1 and 2).
|
Interaction of RKIP with IKKs and upstream kinase activators of
NF-B.
RKIP was first identified as an inhibitor of Raf-1, a
kinase in the MAPKKK family. Three kinases belonging to the MAPKKK
family, TAK1, MEKK1, and NIK, have recently been implicated as upstream activators of the NF-B pathway (37, 38, 46, 50). Based on their functional relatedness to Raf-1, we considered these kinases
as possible targets of RKIP, and therefore, we tested directly for
evidence of physical interactions. COS-1 cells were transfected with
FLAG-tagged RKIP and various HA-tagged MAPKKKs, cell lysates were
immunoprecipitated with a FLAG tag-specific antibody, and
coimmunoprecipitation of the MAPKKKs was visualized by immunoblotting
with an HA tag-specific antibody. Clearly detectable amounts of TAK1
were coimmunoprecipitated with RKIP (Fig.
3A). The coimmunoprecipitation of TAK1
with RKIP is specific because HA-TAK1 was not found in the anti-FLAG
immunoprecipitates when FLAG-RKIP was not included in the transfection
(Fig. 3A). No HA tag cross-reactive material was observed with either
HA-MEKK1 or HA-ASK1, a MAPKKK implicated in TNF--mediated activation
of the JUN kinase pathway (11, 51). All the
proteins being tested except for HA-MEKK1 were expressed at
approximately equal levels in COS-1 cells (Fig. 3A). Using a similar
approach, we also tested for interaction of RKIP with NIK and observed
coimmunoprecipitation of FLAG-tagged NIK and HA-tagged RKIP. Although
the binding affinity of RKIP for NIK is apparently lower than that for
Raf-1, the binding is highly specific because FLAG-tagged protein
phosphatase 2A (catalytic subunit) did not coimmunoprecipitate with
HA-tagged RKIP in the same experiment (Fig. 3B).
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and IKK contain a canonical MAPKK
activation loop and that MEK, a MAPKK, binds RKIP both in vivo and in
vitro prompted us to extend the RKIP interaction studies to IKKs. Using
the methods described above, the interaction of IKK, IKK, and
RKIP was investigated by coimmunoprecipitation of FLAG-tagged IKKs and
HA-RKIP expressed in COS-1 cells. Detectable amounts of RKIP were
coimmunoprecipitated with both IKK and IKK (Fig. 3C). Although
the binding is weak, the interactions that we observed between RKIP,
IKK, and IKK appear to be specific because we did not detect any
association between RKIP and NAK, another IKK-related kinase (data not shown).
RKIP inhibits NIK- and TAK1-mediated activation of NF-B.
To
examine the functional consequences of the above described physical
associations between RKIP, NIK, TAK1, and IKK/, we made use of
the fact that ectopic expression of these activator kinases can
upregulate an NF-B-responsive reporter. 293 cells were therefore
transfected with different combinations of kinase and RKIP expression
vectors in the presence of an NF-B-luciferase reporter. For
experiments examining the effects of RKIP on TAK1-mediated activation
of an NF-B reporter, TAB1, an essential TAK1 coactivator, was also
included in the transfection in addition to TAK1 (50). All
four upstream kinase activators of IKK, namely NIK, NAK, TAK1, and
MEKK1, stimulated the NF-B reporter (Fig.
4). As previously reported, different
kinases stimulated NF-B activity to different extents, with NIK
being the most potent (50-fold), followed by NAK (20-fold), and TAK1
and MEKK1 (4- to 5-fold) (37, 38, 46, 49, 50, 70). RKIP
strongly antagonized the activation of NF-B elicited by NIK (up to
fivefold) and more weakly antagonized the activation elicited by TAK1
(up to twofold). Notably, RKIP did not antagonize the activation
elicited by either NAK or MEKK1 (Fig. 4A and B). The downregulation of
the NIK- and TAK1-mediated activation of the NF-B reporter by RKIP
was not a result of reduced expression levels of NIK and TAK1, because
we did not observe a decrease in endogenous NIK and TAK1 protein levels
when RKIP was overexpressed (data not shown). Taken together, the
results of these in vivo activation experiments are in complete
agreement with the coimmunoprecipitation results.
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B activation cascade, we examined its effect on
transactivation elicited by the transfection of the p65 subunit of
NF-B. RKIP had no effect on p65-mediated activation of an NF-B
reporter (Fig. 4C). The failure to block at this downstream point
indicates that RKIP does not interfere directly with the activity of
NF-B but acts upstream. Interestingly, RKIP also did not block the
activation of NF-B elicited by the overexpression of IKK and
IKK (Fig. 4C).
RKIP inhibits IL-1- and TNF--mediated activation of NF-B
and IKK activity.
In light of previous findings that NIK and TAK1
are the physiological activators of IKK in response to the
proinflammatory cytokines IL-1 and TNF- (46, 50,
67), we also examined the effects of RKIP on TNF-- and
IL-1-mediated stimulation of NF-B activity. In 293 cells RKIP
consistently reduced TNF--mediated stimulation of NF-B activity
up to fivefold (Fig. 5, left panel). Both
the magnitude of TNF- stimulation of NF-B and the magnitude of
the RKIP inhibitory effect were strikingly parallel to the results we
obtained with NIK overexpression (compare Fig. 5, left panel, with Fig.
4A). Consistent with the results that TAK1 is a target of RKIP, we also
observed that RKIP elicited a clear inhibition of IL-1-mediated
activation of an NF-B reporter (Fig. 5, right panel). Importantly,
the magnitude of this effect was comparable to that elicited by RKIP on
ectopically expressed TAK1 (compare Fig. 5, right panel, with Fig. 4B).
|
or IKK with or without RKIP and subsequently stimulated the
cells with TNF-. IKK and IKK were immunoprecipitated with an
anti-FLAG antibody, and the immunoprecipitates were assayed in vitro in
the presence of [-32P]ATP for
autophosphorylation as well as phosphorylation of an exogenously added
GST-IB substrate (Fig. 6). As
reported, IKK activities toward IB were stimulated by TNF-
treatment (Fig. 6A). Interestingly, the inclusion of RKIP in the
transfection reduced the in vitro IKK activities by four- to fivefold
in both assays (Fig. 6A). In agreement with previous reports showing
that the extent of IKK autophosphorylation correlated directly with kinase activities, we observed a downregulation of IKK and IKK autophosphorylation in the presence of RKIP (Fig 6B). These results show that RKIP can antagonize the in vivo activation of IKK activity elicited by treatment of cells with TNF-. To examine whether RKIP
directly inhibits IKK activities, FLAG-tagged IKKs were expressed in
COS-1 cells, the cells were stimulated with TNF-, and FLAG-IKKs were
purified with anti-FLAG antibody. The effects of purified bacterial
recombinant RKIP on IKK activity were assayed using in vitro kinase
assays with GST-IB as a substrate. As seen in Fig. 6C, the
addition of increasing concentrations of RKIP (0 to 2.3 µM) resulted
in a dose-dependent reduction of the phosphorylation of IB. The
inhibition by RKIP was specific because addition of equivalent amounts
of bovine serum albumin had no effect on the IKK activities.
|
, we made use of the fact that
IB phosphorylated by activated IKKs is rapidly targeted for
degradation. 293 cells were transfected with FLAG-tagged IB
either alone or together with HA-tagged RKIP. Cells were treated with
TNF- to activate the endogenous IKK activities, and the effects of
RKIP overexpression were examined by monitoring the expression levels
of FLAG-IB by immunoblotting with anti-FLAG antibody. As
expected, the expression levels of IB were reduced after TNF-
treatment (Fig. 6D). Consistent with the result that RKIP inhibited IKK
activities in vitro, we observed that RKIP elicited a clear inhibition
of IKK-mediated degradation of IB in the transfected cells (Fig.
6D).
Endogenous NIK and TAK1 coimmunoprecipitate with RKIP.
To
investigate whether RKIP can associate with NIK and TAK1 under
physiological levels of protein expression, we immunoprecipitated RKIP
from extracts of Rat1 fibroblast cells with anti-RKIP antibodies. The
amounts of TAK1 and NIK proteins in the anti-RKIP immunoprecipitates were monitored with TAK1- or NIK-specific antibodies. As shown in Fig.
7A, detectable amounts of endogenous TAK1
were present in the anti-RKIP immunoprecipitate but not in the control
immunoprecipitate. In light of previous findings (50) that
IL-1 treatment stimulates the activity of TAK1, we decided to
examine whether IL-1 could affect the association of TAK1 with RKIP.
293 cells were transfected with HA-RKIP, serum starved, and stimulated
with IL-1 for 5 or 15 min. The association of endogenous TAK1 with
HA-RKIP was examined by coimmunoprecipitation followed by
immunoblotting with TAK1 antibodies. Although detectable amounts of
TAK1 were associated with RKIP in cycling cells (Fig. 7A), no TAK1 was
detected in the anti-HA immunoprecipitates from serum-deprived 293 cell
lysates (Fig. 7B). Interestingly, TAK1 was found to be associated with HA-RKIP 5 min after IL-1 stimulation, and the association was drastically reduced after 15 min (Fig. 7B). Note that the association and dissociation of RKIP with TAK1 correlate well with the previously reported activation kinetics of TAK1 (67). It remains to
be seen whether the RKIP could directly inhibit the kinase activity of
TAK1.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The first demonstrated function of RKIP was that of a negative
regulator of the Raf/MEK/ERK signaling pathway. Since RKIP is a much
more abundant protein than Raf-1, we considered the possibility that it
may have additional intracellular targets (75, 76). In
this study we found evidence that RKIP influences signaling by the
NF-B pathway. As was the case in the Raf/MEK/ERK pathway, RKIP
activity exerts a negative effect on the NF-B pathway.
Two distinct models could explain RKIP-mediated repression of NF-B.
First, RKIP could inhibit NF-B activity as a result of its
previously demonstrated effects on the Raf/MEK/ERK pathway. Second,
RKIP could interfere directly with the pathways that lead to NF-B
activation. Several lines of evidence presented in this communication
strongly suggest a direct mode of action. First, we consistently
observed specific physical associations between RKIP and some, but not
all, of the upstream activating kinases of the NF-B pathway. These
associations were detected both in ectopic expression systems as well
as in cellular extracts under physiological levels of protein
expression. Second, RKIP could inhibit NF-B activity elicited by the
activation of some, but not all, of the upstream NF-B-activating
kinases. Most importantly, the results of physical interaction and in
vivo activation studies were in complete agreement. RKIP interacted
with and blocked NIK and TAK1, but it neither interacted with nor
blocked MEKK1 or NAK1. Importantly, the association between TAK1 and
RKIP was physiologically regulated and induced upon treatment of the
cells with IL-1, a known activator of TAK1 activity. Third, RKIP
could efficiently antagonize NF-B activity in the presence of
pharmacological inhibitors of MEK activity.
Our genetic epistasis analysis involving the overexpression of NIK,
IKK, IKK, TAK1, NAK1, MEKK1, and p65 NF-B showed that RKIP
exerted its inhibitory effects only on NIK- and TAK1-mediated activation of NF-B. The fact that RKIP did not interfere with MEKK1-mediated activation of NF-B indicates that RKIP does not have
a general effect on NF-B signaling through promiscuous interactions involving all MAPKKKs. The simplest interpretation of the genetic epistasis data is that RKIP acts upstream of IKK. The possibility that
RKIP acts in a pathway that is parallel to those of upstream activators
of IB and that bypasses IKK is unlikely because RKIP can block
activation of IKK activity by TNF-.
Our results indicate that RKIP physically interacts with TAK1 and NIK
but not with MEKK1. We also detected the physical association of RKIP
with IKK and IKK, both of which belong to the family of MAPKKs
(43). Since the association of RKIP with TAK1, NIK, IKK, and IKK has not yet been achieved in vitro using purified components, it is not known whether these interactions are direct. However, the interaction of RKIP with Raf-1 (a MAPKKK) and MEK1 (a
MAPKK) has been well studied and is known to be direct
(75). Thus, although the existence of a bridging partner
between RKIP and any of the NF-B pathway kinases cannot be ruled
out, it would not be predicted on the basis of studies with the
Raf/MEK/ERK pathway. Since TAK1, NIK, IKK, and IKK have all been
reported to be part of the 700-kDa TNF--induced IKK complex
(48), it is possible that RKIP is also part of this complex.
NF-B activation pathways involved in IL-1 and TNF- signaling
have been partially delineated and found to involve both shared and
unique components (33, 50, 58, 67). Both pathways converge
on NIK, which activates IKK through direct phosphorylation of IKK.
TAK1 was implicated in IL-1 signaling immediately upstream of NIK.
Consistent with this view, we consistently observed a repressive effect
of RKIP on TNF-- as well as IL-1-mediated activation of NF-B.
The inhibition may be partly due to the inhibition of IKK activation by
TAK1/NIK, since we observed a decrease in IKK and IKK
autophosphorylation in extracts of TNF--treated, RKIP-overexpressing
293 cells. The inhibition may also be a result of a direct interference
with IKK activity by RKIP, since we observed that RKIP inhibited the
kinase activities of purified TNF--activated IKK and IKK in
vitro. A direct inhibition of IKK activity is also consistent with the
result that RKIP coimmunoprecipitated with IKK and IKK.
Concordant with the in vitro kinase assay results, RKIP also
antagonized the TNF--induced degradation of IB, presumably by
interfering with the activity of endogenous IKK. Unexpectedly, RKIP did
not interfere with the activation of the NF-B reporter by ectopic
expression of either IKK or IKK. The discrepancy may be due to
the possibility that overexpression of IKKs may result in the
phosphorylation and activation of other IBs or other unknown IKK
substrates. It is thus possible that RKIP does not inhibit the
phosphorylation of these other IKK substrates. Taken together, we
proposed that RKIP acts as a brake on TNF- and IL-1 signaling by
antagonizing the activation of IKKs by NIK and TAK1 as well as by
directly down-modulating the activity of the IKK complexes.
Prior to these studies, the IBs and the A20 protein were the only
known negative regulators of NF-B signaling (44, 66). A20 is a cytoplasmic zinc finger protein that is induced by TNF- in
a variety of cells (53). Knockout studies in mice
indicated that A20 was required for the downregulation of IKK activity
after TNF- stimulation but not IL-1 stimulation
(36). Both A20 and the IKK complex were recruited to the
TNF receptor after TNF- stimulation. It was proposed that A20
antagonizes IKK activity by interacting with the IKK subunit of the
complex (21, 79).
The data presented in this communication indicate that RKIP antagonizes
NF-B activity by a mechanism that is distinct from both IB and
A20. Unlike IBs, which inhibit NF-B by direct interaction with
the Rel proteins, our epistasis analysis shows that RKIP acts upstream
of p65. Whereas A20 was reported to act primarily on TNF- signaling
(36), RKIP can inhibit both TNF-- and IL-1-mediated NF-B activation. Furthermore, epistasis analysis based on the overexpression of TRADD, TRAF2, RIP, and NIK indicated that A20 acts upstream of NIK (21), whereas our data implicate NIK
as a direct target of RKIP. It remains to be determined whether RKIP is
recruited to the cytokine receptors upon their stimulation. It is also
not known whether RKIP can interact with IKK.
Proteins that are highly homologous to RKIP (85 to 95% identity) have
been found in all mammalian species examined to date (18,
56). A family of RKIP-related proteins with 29 to 55% sequence
identity to the mammalian proteins have also been identified in
Drosophila, Caenorhabditis elegans,
Saccharomyces cerevisiae, several parasites, including
Onchocerca volvulus and Toxocara canis, and the
flowering plants Antirrhinum and Arabidopsis
(5, 6, 16, 18, 56, 59, 64). Despite limited sequence conservation between mammalian RKIP and some of these orthologues, recent studies of the crystal structures of two mammalian RKIPs and
Antirrhinum CEN revealed that they displayed an
almost identical novel -fold topology (3, 4, 65). The
close conservation in structure between mammalian RKIPs and plant RKIP
orthologues raises the possibility that they may have similar
functions. Indeed, genetic studies indicate that RKIP orthologues in
plants function in signal transduction pathways that control plant
architecture and development (1, 5, 6, 29, 32).
Emerging data from diverse organisms suggest that RKIP and RKIP-related
proteins represent a new and evolutionarily highly conserved family of
protein kinase regulators (4). The fact that RKIP impinges
on both the Raf/MEK/ERK MAPK pathway and the NF-B pathways raises
the intriguing possibility that RKIP and related proteins may play
roles in a number of different protein kinase signaling pathways. Since
MAPK and NF-B pathways have physiologically distinct roles, the
function of RKIP may be, in part, to coordinate the regulation of these pathways.
| |
ACKNOWLEDGMENTS |
|---|
We thank Z. Cao, S. Frisch, D. Goeddel, A. H. Ichijo, A. Lin, K. Matsumoto, M. Nakanishi, M. Rothe, and D. Wallach for plasmid constructs, antibodies, and cell lines.
This work was supported by NIH grants to K.C.Y. (R01 GM64767) and J.M.S. (R01 GM55435 and GM 41690) and an AICR grant to W.K. This work was also supported in part by the COBRE award P20RR15578 from the NIH.
| |
FOOTNOTES |
|---|
* Corresponding author. Present address: Medical College of Ohio, Department of Biochemistry & Molecular Biology, 3035 Arlington Ave., Toledo, OH 43614-5804. Phone: (419) 383-6658. Fax: (419) 383-6228. E-mail: kyeung{at}mco.edu.
Present address: Rhode Island Hospital, Providence, RI 02903.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, November 2001, p. 7207-7217, Vol. 21, No. 21
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.21.7207-7217.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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