From the Functional Genomics, Division of Molecular
and Genetic Medicine, University of Sheffield, S10 2JF, United Kingdom
and the § Department of Pathology, School of Medicine,
University of Washington, Seattle, Washington 98195
Received for publication, July 28, 2000, and in revised form, October 3, 2000
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ABSTRACT |
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In the present study, we show that Ras activity
differentially controls interleukin (IL)-1 induced transcription factor
activation by selective regulation of responses mediated by receptor
complex components. Initial experiments revealed that stimulation with IL-1 caused a rapid, matrix-dependent activation of Ras.
The effect was transient, peaking at 5 min and returning to base levels
after 30 min. Activation correlated with pronounced changes in cell shape in EGFPH-Ras transfected cells. Transfection with the
dominant negative mutant, RasAsn-17, inhibited IL-1 induced
activation of the IL-8 promoter as well as of NF- Cellular responses to cytokines and growth factors are influenced
by the extracellular milieu (1). These regulatory effects are mediated
to a large extent by receptors of the integrin family and involve
modulation of signaling pathways that link receptor activation and
changes in cell shape, the cytoskeleton, and gene expression (2, 3).
Ras acts cooperatively and/or hierarchically with the Rho subfamily of
guanine nucleotide triphosphatases (GTPases) to regulate focal adhesion
and polymerized actin turnover (4). It is thought to be in their
arrangement of signaling proteins and receptors via the cytoskeleton
that the GTPases are able to channel a diverse range of stimuli, from
growth factor and cytokines to cell attachment, into common downstream
signaling cascades (5) such as the stress-activated protein
kinase (6, 7) and nuclear factor- The membrane proximal molecular events that take place following
interleukin-1 receptor (IL-1R) binding and leading to transcription factor activation involves initially the heterodimerization of the
IL-1R with accessory protein (IL-1RAcP) (10), followed by binding of
the adaptor protein MyD88 (11). MyD88 in turn recruits the Ser/Thr
kinases: IL-1R associated kinase (IRAK) 1, IRAK-2, and IRAK-3 or M
(11-13), which have recently been shown to be pre-associated with a
further adaptor protein, Tollip (14). Subsequently, the IL-1R
associated kinase 1s are freed and interact with the tumor necrosis
factor receptor-associated factor (TRAF) 6, followed by activation of
the stress-activated protein kinase and NF- The majority of IL-1 receptors are located at focal adhesions (16), and
matrix attachment results in alterations in IL-1 receptor function
(17). Subsequent IL-1-induced signal transduction and gene activation
are regulated by cell attachment and the cytoskeleton (18-20). Early
alterations following IL-1 receptor binding in adherent cells include a
rapid and transient phosphorylation of transmembrane linkage protein
Talin and changes in the cytoskeleton (21), suggesting an immediate
effect on the turnover of structural components at these sites. Aspects
of Ras, Rho, Rac, and Cdc42 involvement in downstream IL-1 signaling
pathways have recently been elucidated (22-25), suggesting they play a
part in mediating these structural changes localized around the
receptor complex. This may underpin the, as yet, unclear mechanism by
which the GTPases interact with members of the classical IL-1 signaling
cascade. So far, however, little is still known of how these effects
are initiated, and how they may modulate IL-1 signaling and influence
downstream cascades.
We report here that IL-1-induced structural alterations in adherent
cells involve activation of the Ras GTPase as an early signaling event,
which regulates IL-1-induced NF- Cell Cultures and Transfection--
Human gingival (4) and skin
fibroblast strains (1) (transfer 5-15) and HeLa cells were used. Cells
were propagated in monolayer cultures in Dulbecco's modified Eagle's
medium containing 5 mM sodium pyruvate, 100 µg/ml
penicillin and streptomycin, and 10% heat inactivated fetal calf serum
(Life Technologies) in humidified atmosphere (95% air, 5%
CO2) at 37 °C. Cells were plated subconfluently and
grown for 1-3 days and transfected using the calcium phosphate co-precipitation method with glycerol shock (60 s, 20% glycerol in
phosphate-buffered saline) or using Superfect (Qiagen). Transfection efficiency, measured by determining EGFP-C1 expression in confocal experiments or renilla levels using the TK-RL or CMV-RL vectors in
luciferase assays, was 15-30% ± 3% for fibroblasts and 25-40% ± 5% for HeLa cells. Twenty-four or 48 h after transfection cells were stimulated directly, or detached using EDTA (5 mM) and
replated onto plates coated with fibronectin (10 µg/ml, Sigma) or on
bare tissue culture plastic (26) for 3-4 h prior to stimulation with IL-1 Constructs--
BamHI to EcoRI fragments
from pGEXH-Ras, pGEXRhoA, pGEXRac1,
and pGEXCdc42 and a HindIII to EcoRI
fragment from pBluescriptRELA (27) were subcloned in-frame to the
carboxyl terminus of EGFP downstream of the CMV promoter in the vector:
pEGFP-C1 (CLONTECH), to give fusion constructs:
pEGFPH-Ras, pEGFPRac1, pEGFPRhoA,
pEGFPCdc42, and pEGFPRelA. The
pEGFPN17Ras vector was made using the mutagenic primer:
5'-GGTCAGCGCGTTCTTGCCCAC-3' and "Muta-Gene" kit (Bio-Rad). The
expression vectors pCMVH-Ras and pCMVN17Ras were
constructed by PCR amplification of pEGFPH-Ras and
pEGFPN17Ras using the primers: 5'-GGGGCTAGCATGACCGAATACAAGCTTGTTGTT-3' (sense) and
5'-ACGATGAATTCTCAGGAGAGCACACACTT-3' (antisense); underlined
residues indicate engineered restriction sites and a transcriptional
start site on the sense strand. A 600-base pair
NheI-EcoRI fragment was subcloned into the
pEGFP-C1 vector (corresponding digest removes EGFP) downstream of the
CMV promoter. The expression plasmids pCMVMyD88 and
CMVV12Ras were gifts from Filipo Volpe, Glaxo, and Julian
Downward, International Cancer Research Fund, respectively, and
the pRK-TRAF6 and pRK- Ras Activation Assays--
The ratio of GTP to GDP bound to Ras
was determined essentially as described in Ref. 28 for both transfected
and nontransfected cells to assess the activity in exogenous and
endogenous Ras protein, respectively. Briefly, cells were plated onto
fibronectin-coated or bare tissue culture plastic on 10-cm plates as
described above at 2 × 106 cells/plate. Cells
transfected with EGFPH-Ras were replated in the same manner
24 h after transfection with 6.6 µg/dish of
EGFPH-Ras, as above. Cells were washed (2 times) and
incubated in phosphate-free Dulbecco's modified Eagle's medium (ICN)
with 10% dialyzed fetal bovine serum containing HEPES (20 mM), L-glutamine (5 mM) (1 h, 37 °C, pH 7.4) alone, and for a further 1 h with the same
medium containing 200 µCi/ml [32P]orthophosphate
(PerkinElmer Life Sciences). Following stimulation with 1 nM IL-1 or 40 ng/ml EGF (Promega) for varying times, cells were washed 2 times with phosphate-buffered saline and lysed in 1 ml of
RIPA buffer (50 mM HEPES buffer, pH 7.4, 100 mM
NaCl, 5 mM MgCl2, 1% Triton X-100, 1 mg/ml
bovine serum albumin, 10 mM benzamidine, 0.5%
deoxycholate, 0.05% SDS, 10 µg/ml leupeptin, 10 µg/ml aprotinin,
10 µg/ml soybean trypsin inhibitor). Lysates were precleared with 20 µl of protein A-Sepharose (10%, Sigma) coupled to 1 µl of rabbit
anti-rat Ig (DAKO) for 1 h at 4 °C. Precleared lysates were
incubated with rat anti-human Ras (1 µg) (259, Santa Cruz) and rabbit
anti-rat Ig coupled to protein A-Sepharose (60 µl, 2 h,
4 °C). Immunoprecipitations of EGFPRas from transfected cells were done using a rabbit polyclonal anti-GFP (1 µg)
(CLONTECH) and a goat anti-rabbit Ig coupled to
protein G-Sepharose (60 µl, 2 h, 4 °C). Immunoprecipitates
were transferred to IMMUNOCATCHER spin columns (Cytosignal) and washed
6 times with wash buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 5 mM MgCl2, 0.1% Triton
X-100, 0.005% SDS) and eluted with 2 mM EDTA, 2 mM dithiothretiol, 0.2% SDS, 0.5 mM GTP, 0.5 mM GDP for 20 min at 68 °C. Eluted nucleotides were
spotted onto polyethyleneimine-cellulose thin layer plates (Merck) and
developed in 1.2 M ammonium formate, 0.8 M HCl.
Position of GTP and GDP was identified by using nonlabeled GTP/GDP and viewing under UV lamp. Plates were exposed for 1 week on film and the
GTP/GDP ratios determined by autoradiograph scanning and the NIH image
"gel plotter" macro.
Confocal Fluorescence Microscopy--
Fibroblasts
(106/10-cm dish) were transfected using 6.6 µg of total
DNA/dish and replated onto fibronectin-coated or bare tissue culture
plastic 8-well slides (Nunclon). Cells were visualized using a
Molecular Dynamics confocal laser scanning microscope with a 37 °C
stage incubator coupled to a Nikon Diaphot microscope and a Silicon
Graphics work station. Localization of the fusion proteins following
transfection with constructs containing the GTPase cDNAs was done
by confocal microscopy of single cells over time in the presence or
absence of IL-1 Luciferase Reporter Assays--
Fibroblasts (1.5 × 104/well) were plated in 24-well plates and transfected
with 2 µg of total DNA/well, including 1 µg of luciferase reporter
plasmid, containing the IL-8 promoter or NF- IL-1 Induces an Attachment-dependent Activation of Ras--
The
effect of IL-1 on activation of the Ras GTPase was determined by
immunoprecipitation and thin layer chromatography, comparing fibroblasts plated on fibronectin-coated plates or on bare tissue culture plastic. Analysis of the ratio of GTP to GDP bound Ras showed
that stimulation with 1 nM IL-1 induced a matrix dependent increase in endogenous Ras activity. Thus, in the presence of fibronectin attachment IL-1 induced a 4-5-fold increase in activity within 5 min (Fig. 1, A and
B). The response was transient and the activity returned to
base levels by 30 min. The level of activity was comparable, but less
pronounced than that induced by stimulation with EGF, a potent
activator of Ras (31). EGF also caused a more sustained activity, which
was maintained throughout the experiment. In contrast, cells on bare
tissue culture plastic showed no change in the levels of GTP-bound Ras
during 60 min stimulation with IL-1 but demonstrated the same base
levels prior to stimulation as cells plated for 4 h on the
fibronectin matrix (Fig. 1B).
Ras Mediates IL-1-induced Shape Changes in Attached
Cells--
Confocal microscopy and serial observations of single cells
demonstrated that IL-1 stimulation resulted in pronounced and successive changes in shape of cells transfected with
EGFPH-Ras, correlating with the IL-1 induced Ras activity
(Fig. 2A). The reduction in
cross-sectional area coincided with an apparent concentration of the
fusion protein at the plasma membrane, which in unstimulated cells, was
the same as that of the endogenous protein (4). In comparison,
transfection with EGFPRac1, EGFPCdc42, or
EGFPRhoA had no effect on cell shape or localization of the
protein (data not shown). The Ras-induced changes were nontransient and
constituted retraction of the peripheral flattened areas followed by
progressive rounding, initially occurring at 5-15 min, and resulting
in reduction in cross-sectional area of about 50% after 60 min (Fig.
2B). Less pronounced effects were observed following Ras
transfection alone without matrix attachment or IL-1 stimulation, and
total inhibition of the IL-1 induced changes was noted following
transfection with a dominant negative mutant of EGFPH-Ras
(EGFPN17Ras) (Fig. 2B).
Ras Mediates Activation of IL-1 Responsive Genes--
The
involvement of Ras in IL-1-induced gene activation was demonstrated by
co-transfection experiments. These experiments showed a pronounced
enhancement in IL-1 induced activation of the IL-8 promoter in the
presence of excess Ras (Fig.
3A) demonstrating additive
effects over a range of IL-1 concentrations. Transfection of cells with
RasAsn-17 prior to stimulation with IL-1 abolished
activation of the IL-8 promoter, and caused a marked decrease in basal
levels of transcription in fibroblasts (Fig. 3A) showing a
direct effect of Ras in IL-1 mediated gene regulation.
RasAsn-17 similarly inhibited induction of the IL-8
promoter in HeLa cells (Fig. 3B), resulting in a reduction
in transcription of around 80%. Similar experiments using an NF- Ras Regulated IL-1-induced Gene Transcription Is Mediated through
TRAF6--
Further analyses focused on the upstream regulatory
mechanism involved in mediating the Ras-induced response and the role of the adapter proteins TRAF6 and MyD88 in structural regulation of the
IL-1-induced pathways. For these experiments, cells were transfected
with the IL-8 promoter reporter and signaling protein constructs.
Consistent with previous results from this laboratory, transfection
with both TRAF6 and MyD88 typically resulted in activation of the IL-8
promoter corresponding to 70-120-fold and 20-30-fold induction,
respectively (30) (Fig. 4A).
Co-transfection with RasAsn-17 demonstrated a
dose-dependent inhibition of the TRAF6-mediated response of
up to about 60%. In contrast, transfection with RasAsn-17
had no effect on the MyD88 induced activity (Fig. 4A).
Furthermore, we tested potency of the RasAsn-17 inhibitory
effect in the presence of IL-1 stimulation and adaptor protein
overexpression. Similar to the observations in unstimulated cultures,
co-transfection with the TRAF6 containing construct resulted in
enhancement of the IL-1 induced activity. Thus, addition of IL-1 in the
presence of TRAF6 showed massively elevated levels of activation,
increasing induction from 70-120- to 300-370-fold (Fig.
4B), suggesting a synergistic effect with the soluble
agonist, while transfection with MyD88 had no significant effect on the IL-1 response. Furthermore, the presence of RasAsn-17
caused a pronounced reduction in the levels of TRAF6 + IL-1 induced activity corresponding to around 50%. In contrast, and similarly to
the nonstimulated levels of activity, activation induced by IL-1 in
MyD88-transfected cells was unaffected by co-transfection with
RasAsn-17. Levels of TRAF6 + IL-1 activation of IL-8
promoter activity, even in the presence of RasAsn-17, were
still significantly higher than those induced by 1 nM IL-1 or TRAF6 alone, agreeing with the notion that Ras regulation of IL-1
responses is mediated through particular facets of the signaling complex.
NF- Ras Regulation of the NF- In the present study, we demonstrate that IL-1 transiently
activates Ras in a matrix-dependent fashion, correlating
with translocation of EGFPH-Ras fusion protein and structural effects
on IL-1 responses in transfected cells. We also show that the
regulation of IL-1-mediated NF- The IL-1-induced Ras activation occurs during the initial steps of
signal transduction involving heterodimerization of the receptor and
the accessory protein and in matrix-attached cells, recruitment of a
heparan sulfate to the receptor complex. Association of the adaptor
protein MyD88 results in activation of the receptor IRAKs and
association of TRAF6 (see Fig. 7). The
observed Ras activity correlate with the similarly rapid,
matrix-dependent IL-1 induced serine phosphorylation of the
transmembrane linkage protein, Talin preceding the alterations in cell
structure and the changes in the cytoskeleton (20, 21). Such
alterations are known to be directly associated with events related to
GTPase activation (33). These data agree with recent findings of
essential roles for other members of the GTPase family in IL-1
signaling (22-25), and together with the known interdependence between
members of the Ras and Rho subfamilies suggest that their roles in
IL-1-mediated responses to be interconnected.
B and AP-1
synthetic promoters in transient transfection assays. Furthermore,
overexpression of the IL-1 signaling proteins TRAF6 or MyD88 gave
characteristic activation of IL-8, which was accentuated in the
presence of IL-1. Co-transfection with RasAsn-17 gave a
dose-dependent inhibition of TRAF6-induced responses in the
presence and absence of IL-1, but had no effect on MyD88 mediated activity. Similarly, induction of NF-
B was abolished by
RasAsn-17 only in TRAF6-transfected cells. In contrast,
inhibiting Ras activity limited AP-1-mediated responses through both
receptor complex proteins. Constitutively active RasVal-12
increased the TRAF6 induced activity of the NF-
B pathway similar to
the effect induced by IL-1, while the RasVal-12 induced
activity was not inhibited by co-transfection with a dominant negative
TRAF6. Our data show that activation of the Ras GTPase is an early,
matrix-dependent response in IL-1 signaling which
participates in structural regulation of IL-1-induced genes. In
addition, they show that the Ras induced effect selectively regulates
TRAF6-mediated activation of the NF-
B pathway, suggesting that Ras
GTPase represents a convergence point in structural and cytokine
responses, with distinct effects on a subset of downstream signaling events.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-
B)1 pathways (8,
9).
B pathways (15).
B transactivation of inflammatory
genes. In addition, we show that Ras regulation of NF-
B affects
TRAF6-mediated activation, but not that induced through MyD88,
indicating that IL-1 induced pathways leading to NF-
B activation
diverge upstream of TRAF6. Furthermore, the data suggest that Ras
represents a convergence of IL-1 and matrix-mediated signaling events
induced downstream of the adaptor proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(a gift from the Immunex Corp., and Steve Poole, National Institute for Biological Standards and Control).
TRAF6-(289-522) were donated by David
Goeddel, Tularik Inc. The IL-8 promoter was a PCR product of the
primers: 5'-GAAGATCTAACTTTCGTCATACTCCG-3' (sense) and
5'-CCGGTACCCTTCACACAGAGCTCGAG-3' (antisense). A 220-base pair BglII-HindIII fragment, containing the
promoter and transcriptional start site of IL-8 was subcloned into the
pGL3 vector (Promega). The NF-
B and AP-1 synthetic
promoter-luciferase constructs were purchased from Stratagene. The
CMV-RL renilla luciferase internal control vector was constructed by
subcloning a NheI to XbaI fragment containing the
luciferase gene into pcDNA3.1(
) (Invitrogen) downstream of the
CMV promoter.
stimulation. Excitation (488 nm line of the Kr/Ar
laser at 10 mW, split equally between 488, 568, and 647 emission lines)
was attenuated to 10-30% with a neutral density filter. Emitted green
fluorescence was collected with a × 60 Plan Apo oil immersion
objective (NA 1.4) passed through a 530-nm band pass (±15 nm) filter,
amplified with a photomultiplier at 650-700 V, and digitized to 8 bits. ×60 magnification digital images obtained of 512 × 512 voxels of dimensions: 0.21(X) × 0.21(Y) × 0.54(Z) µm gave
an output directly proportional to the concentration of GFP in the
voxel scanned (not shown). The digital images were transferred to a
Power Macintosh. For quantitation of cell size, horizontal sections
were scanned at the base of transfected cells, and cross-sectional area
was measured using NIH image. For the quantitation of RelA (p65)
translocation, cells were transfected with EGFPRelA and
CMVH-Ras and analyzed as in Ref. 29. Briefly, nuclear and
cytoplasmic protein levels were quantitated by scanning horizontal
sections through the nucleus of transfected cells. Using NIH image,
relative fluorescence was calculated by measuring the mean intensity of
representative areas of nucleus and cytoplasm and dividing by the
attenuation setting.
B/AP-1 binding elements
alone, and 1 µg of CMV expression vectors including 0.5 µg of
CMVH-Ras or CMVN17Ras and 0.4 µg of CMV-RL
internal control. Remaining DNA was made up using pcDNA3.1 (empty
control vector). HeLa cells (6 × 104/well) were
plated in 24-well plates and transfected with 1.125 µg of total
DNA/well including 0.5 µg of luciferase reporter plasmid, 0.375 µg
of expression vectors or pcDNA3.1 (empty control vector) and 0.3 µg of TK-RL internal control. Amounts of transfected IL-1 signaling
proteins (1.25 µg of TRAF6 or MyD88) was based on previous data
showing maximal stimulation at these levels (30). Forty-eight (fibroblasts) or 24 h (HeLa cells) after transfection, cultures were stimulated with 10
13,
10
11, or 10
9 M
IL-1
, 6 h before lysis in Passive Lysis Buffer (Promega), transferred to microtiter plates (Dynex) and assayed for luminescence intensity using Standard Luciferase Assay and Stop "n" Glo
Substrates (Promega) and an ML3000 plate reader (Dynatech). Control for
matrix requirement, using cultures plated on bare plastic, showed no induction of activity by IL-1. Induction of the IL-8, NF-
B, and AP-1
luciferase reporter constructs ranged between 15-50-, 2-6.4-, and
1.5-3-fold, on stimulation with 1 nM IL-1, 6 h,
respectively. These differences primarily reflect variations in basal
levels of activity. Importantly, the variations in absolute fold
induction reflects the difference between separate experiments,
however, the relative increase and reduction induced by the various
agonists and inhibitors were consistent between experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
IL-1 induces activation of Ras in
fibronectin-attached cells. A, primary fibroblasts were
replated subconfluently on fibronectin coated 10-cm Petri dishes for
3-4 h, and labeled with [32P] (200 µCi/ml, 1 h)
prior to stimulation with 1 nM IL-1 or 40 ng/ml EGF, at
the times indicated, and subsequently lysed and immunoprecipitated at
the times indicated with primary and secondary antibody or with
20 ab alone (control lane), as described under
"Experimental Procedures." GTP and GDP-bound Ras fractions were
separated by thin layer chromatography and detected by autoradiography.
B, quantitation of autoradiographs obtained as in
A was carried out using NIH image. Data are expressed as
fold increases in GTP/GDP-bound Ras after stimulation for various
times, as indicated, relative to nonstimulated samples. Data shown are
the averages of two experiments. FN, fibronectin;
TC, tissue culture plastic.
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Fig. 2.
IL-1 induces pronounced changes in cell shape
in EGFPH-Ras transfected cells.
A, 24 h following transfection with
EGFPH-Ras, primary fibroblasts were replated subconfluently
on fibronectin-coated 8-well slides for 3-4 h as described under
"Experimental Procedures." Confocal images were taken near the base
of fluorescent cells at various times of 1 nM IL-1
stimulation, as indicated. The second row of micrographs
show a higher magnification of selected areas at the plasma membrane
for each time point. Bars represent 10 µm. B,
cells were transfected with EGFPH-Ras or
EGFPRasN17, replated as in A and stimulated with
1 nM IL-1
as indicated. The size of the cross-sectional
area at the base of cells treated as in A was determined
using NIH image, and expressed as a percentage of the original section
area. Data shown represent the averages of four or five experiments for
the various conditions. FN, fibronectin; TC,
tissue culture plastic.
B
synthetic promoter showed the same level of increase by Ras
transfection, demonstrating additive effects in the fibroblast cell
lines (Fig. 3C) but with no net effect on nuclear
translocation of EGFPRelA assessed by confocal microscopy
(data not shown). In addition, comparing the effects of NF-
B and
AP-1 using synthetic promoters showed that the negative mutant
RasAsn-17 caused a reduction of the IL-1
-mediated
responses at saturated levels of ligand of about 60% for both
transcription factors (Fig. 3D), demonstrating involvement
of Ras in IL-1-induced gene regulation through both the NF-
B and
AP-1 pathways.
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Fig. 3.
Ras activity enhances transactivation of
NF- B responsive genes by IL-1.
A, fibroblast cells (1 × 104) attached to
an extracellular matrix were transfected in triplicate wells, as
described under "Experimental Procedures," with an IL-8
promoter-luciferase reporter plasmid in the presence of Ras or
RasAsn-17 expression vectors or pcDNA3.1
(control). Forty-eight hours following transfection, cells
were left unstimulated or stimulated with various concentrations of
IL-1
, as indicated, for 6 h, 37 °C, lysed and assayed for
firefly and renilla (internal control) luciferase activity. Data shown
represent the average of five (±Ras) and two (±RasAsn-17)
experiments, and are expressed as fold induction relative to the
activity in unstimulated cells transfected with reporter alone ± S.E. B, HeLa cells (6 × 104) were
transfected in triplicate wells as described under "Experimental
Procedures" with an IL-8 promoter-luciferase reporter plasmid in the
presence of RasAsn-17 expression vectors or pcDNA3.1
(control) as indicated. Twenty-four hours following
transfection, cells were left nonstimulated or stimulated with IL-1
as indicated for 6 h, 37 °C. Data shown represent one
experiment of three identical experiments and are expressed as fold
induction relative to the activity in unstimulated cells transfected
with reporter alone ± S.D. for triplicate wells. C,
fibroblast cells were co-transfected with a synthetic NF-
B
luciferase reporter with and without Ras or with the dominant negative
RasAsn-17 and stimulated with various concentrations of
IL-1
, as indicated, 6 h, 37 °C and assayed as in
A. Data shown represent the average of two experiments, and
are expressed as relative to the activity in unstimulated cells
transfected with reporter ± S.E. D, HeLa cells were
transfected with a synthetic NF-
B (filled bars) or AP-1
(unfilled bars) luciferase reporters with and without
RasAsn-17 and stimulated with IL-1
, 6 h, 37 °C
and assayed as in B. Data shown represent one of three
identical experiments performed and expressed as fold induction
relative to the activity in unstimulated cells transfected with
reporter ± S.D. for triplicate wells.
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Fig. 4.
The dominant negative RasAsn-17
inhibits TRAF6-dependent transcription of the IL-8
promoter. A, HeLa cells (6 × 104)
were co-transfected in triplicate wells as described under
"Experimental Procedures" with an IL-8 promoter-luciferase reporter
and with 0.125 µg of TRAF6 or MyD88 together with the indicated
amounts of RasAsn-17. Remaining DNA was made up with
pcDNA3.1 (control). Twenty-four hours after transfection, cells
were lysed and assayed for firefly and renilla (internal control)
luciferase activity. B, a similar experiment in which,
24 h after transfection, cells were stimulated with IL-1 (1 nM), 6 h, 37 °C as indicated. Cells were
subsequently lysed and assayed for luciferase activity as in
A. Data in each panel represent one of four experiments
showing the same results and are expressed as fold induction relative
to the activity in unstimulated cells transfected with reporter ± S.D. for triplicate wells.
B Regulation by Ras Is Induced through TRAF6--
Identical
experiments using a synthetic NF-
B responsive promoter demonstrated
lower fold induction due to higher base levels, and resulted in a
15-20-fold increase in activity in cells co-transfected with a TRAF6
containing construct. The dominant negative Ras construct, as
previously, caused a concentration dependent inhibition of this TRAF6
induced activation, corresponding to 60 and 80% at 0.125 and 0.25 µg
of CMVN17Ras, respectively (Fig.
5A). Again, the lower level of
induction of NF-
B activity by MyD88 was unaffected by
co-transfection with RasAsn-17, demonstrating a selectivity
of Ras regulation of NF-
B responses at the level of receptor complex
components. The correlation between the observations using the NF-
B
and the complete construct agrees with a primary regulation of this
promoter through NF-
B (32). In contrast, in similar experiments
using an AP-1 promoter, inhibition of Ras activation had a pronounced
effect on both TRAF6- and MyD88-induced responses resulting in levels
of 10 and 20% of that in the controls at higher concentrations of
RasAsn-17 (Fig. 5B). The specific involvement of
TRAF6 in Ras-mediated NF-
B responses was further confirmed by using
a constitutively active Ras mutant, RasVal-12, which gave a
weak induction of NF-
B activity (2-3-fold) (Fig. 5C).
Co-transfection in cells transfected with TRAF6 had a pronounced effect
resulting in a 25-35-fold enhancement of the response, significantly
higher that would be induced by additive effects, and indicating
synergy of the response. In contrast, the constitutively active Ras
construct had no effect on the levels induced by MyD88, supporting the
notion that Ras regulation of NF-
B involves selective interactions
between receptor complex components.
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Fig. 5.
Ras regulates TRAF6-induced transcription of
NF- B. A, HeLa cells (6 × 104) were co-transfected in triplicate wells, as described
under "Experimental Procedures," with a synthetic NF-
B
luciferase reporter and 0.125 µg of constructs containing TRAF6 or
MyD88, together with the indicated amounts of RasAsn-17
containing constructs. Remaining DNA was made up with pcDNA3.1
(control). B, cells were transfected as in A, but
with an AP-1 synthetic promoter luciferase reporter and 0.125 µg of
constructs containing TRAF6 or MyD88, together with the indicated
amounts of RasAsn-17. Remaining DNA was made up with
pcDNA3.1 (control). C, cells were transfected as in
A with 0.125 µg of RasVal-12 expression vector
in the presence of 0.125 µg of vectors containing TRAF6 or MyD88
using the NF-
B synthetic promoter as a readout. Cell extracts were
analyzed as described in the legend to Fig. 4. For each figure, data
shown represent one experiment of four giving the same results, and are
expressed as fold induction relative to the activity in unstimulated
cells transfected with reporter ± S.D. for triplicate
wells.
B Pathway Is Induced Downstream of
TRAF6--
IL-1 stimulation of TRAF6-transfected cells resulted in a
more than additive effect on induction of NF-
B activity (Fig.
6). Furthermore, similar to the effect on
the non-IL-1 induced levels, co-transfection with RasAsn-17
resulted consistently in a 75-80% reduction of the NF-
B activity. In contrast, the IL-1-mediated response induced following MyD88 transfection was no more than additive and consistent with results using the IL-8 promoter, no significant inhibition was observed on
co-transfection with RasAsn-17. In comparison to the
pronounced effects induced on the TRAF6-mediated response by blocking
Ras activity, a dominant negative TRAF6,
TRAF6-(289-522) (15), had
no effect on Ras induced activation of NF-
B. Thus, co-transfection
with
TRAF6-(289-522), failed to inhibit the 3-fold enhancement in
NF-
B transcription mediated by the constitutively active
RasVal-12 (data not shown), suggesting that while Ras
involvement is mediated through TRAF6 at the level of the receptor, its
subsequent regulation of the NF-
B pathway is induced downstream of
the adaptor proteins.
View larger version (14K):
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Fig. 6.
Ras and IL-1 co-regulate transcription of
NF- B through TRAF6. Cells transfected
with 0.125 µg of TRAF6 or MyD88 together with various amounts of the
dominant negative mutant RasAsn-17 and a construct
containing the synthetic NF-
B promoter were stimulated with 1 nM IL-1
. Cell extracts were harvested as in Fig.
5A. Data are presented as fold induction of promoter
activity over nonstimulated levels in cells transfected with control
DNA. Shown is one of four experiments yielding identical results, ± S.D. for triplicate wells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activity by Ras is induced through TRAF6.
View larger version (22K):
[in a new window]
Fig. 7.
Mechanism of Ras-mediated regulation of IL-1
induced responses. The data presented show that structural
regulation of IL-1 mediated responses is coordinated through the Ras
GTPase following its activation. Furthermore, they support a mechanism
where the IL-1 induced Ras activity is matrix-dependent and
mediated through TRAF6 as regards effects on NF- B activity, and
furthermore, that subsequent to activation it affects gene regulation
downstream of TRAF6 in the signaling pathway using: AcP,
accessory protein; HSPG, heparan sulfate proteoglycan;
integrin; IRAK1, IRAK2, IRAK3/M; interleukin-1 regulated kinases 1, 2, and 3 or M, MyD88, Tollip, TRAF6; TRAF6, Ras, and NF-
B.
Our data further suggest that the IL-1 effect involves an increase in the concentration of the GTPase at the plasma membrane during activation. This is likely, to a significant extent, to be a consequence of the reduction in cell shape. However, Ras isoforms have been shown to have the ability to rapidly diffuse through the plasma membrane (34) and an increase in local concentration at the cell surface could reflect recruitment to specific areas to facilitate signal transduction. This could involve a mechanism similar to that demonstrated for Ras, Rac, and Rho during integrin and growth factor signaling, when localization to caveolin-rich regions in pre-assembled complexes is a prerequisite for signal transduction (35-37).
The rapid induction of Ras activation suggests that it is induced through an immediate, receptor-associated event, and could be a direct consequence of the early IRAKs and TRAF6 association. This likely involves receptor-associated regulators of the GTPases such as the putative Rap/GTPase activating protein (GAP), IIP-1 (38). Furthermore, the attachment dependence suggests that activation could be induced as a consequence of recruitment of the matrix-dependent, accessory receptor component to the IL-1 receptor complex (17) and thus result from co-regulation through integrin mediated activities. Such effects could be induced following selective activation of signaling components by structural events, as suggested by our data demonstrating specificity for the activities mediated through receptor-associated proteins MyD88 and TRAF6. This type of collaboration would thus be similar to that reported for MyD88 and Tollip during recruitment to the IL-1 receptor (14).
We show that IL-1-induced NF-B mediated transcription of IL-8 is
dependent on Ras activity, demonstrated by using wild type and dominant
negative mutant forms of Ras in transient transfection reporter assays.
This agrees with previous findings from this laboratory, showing a
direct dependence of IL-1 induced inflammatory genes (19) and NF-
B
activation (18, 20) on cell architecture and attachment. These effects
of Ras could be mediated through MEKK (39) with subsequent effects on
the NF-
B pathway (40-42) resulting from induction of I
B
phosphorylation (43). However, the lack of effect of nuclear
translocation of RelA could reflect that Ras, as has been suggested for
Rac1 (24), may regulate subsequent transactivation events such as
phosphorylation of nuclear NF-
B (44). In addition, the burgeoning
evidence of complexity of NF-
B activity, involving association of
nuclear I
B
·NF-
B complexes subsequent to independent
translocation (45) and regulation through RelA
shuttling2 suggests that
significant activation of the pathway may be induced without any net
effect on the level of nuclear RelA. This is also supported by results
from studying the endogenous protein demonstrating that activation of
the pathway does not necessarily correlate with an absolute increase in
nuclear NF-
B.3 Finally, it
is possible that the lack of RelA translocation reflects that
structural induction activates only a subset of
NF-
B-dependent genes, as has been shown for glycogen
synthase kinase-3
(46).
This type of specificity is also in agreement with the suggested
divergence of the pathway upstream of TRAF6, and indicate that MyD88
can activate NF-B via a TRAF6 independent pathway. The effect on
both TRAF6 and MyD88 induced AP-1 regulation indicate that Ras may be
able to influence activation of specific genes through selectivity of
adaptor protein function, reflected in distinct sets of upstream
regulators. The total lack of effect of the dominant negative TRAF6 on
NF-
B activation induced through transfection with
RasVal-12 suggests that structural regulation through Ras
feeds into the NF-
B pathway downstream of TRAF6 or is induced
through second messengers totally separate from that regulated by
TRAF6. Thus, activation through TRAF6 via TAB-2 and TAK-1 (47)
constitutes an IL-1-regulated pathway controlling NF-
B, while other
regulators, such as NIK and NAK (48) have been shown not to directly
mediate IL-1 induced NF-
B activity. The indicated synergy in the
observed response suggests, however, that whatever the upstream
discrepancies in signaling, the Ras-induced effect on the NF-
B
pathway is not totally distinct from the IL-1 mode of activation but
rather that, the two activities converge upstream of induction of transcription.
This type of receptor proximal divergence of TRAF6/MyD88-regulated
pathways has been reported in in vitro studies on Toll signaling (49). Studies on TRAF6 and MyD88 knockout mice have also
shown a degree of compensation and/or redundancy and partial overlapping at the level of the adaptor proteins in transcriptional activation by IL-1 and lipopolysaccharide (50, 51). Other examples are
signaling proteins ECSIT (evolutionarily conserved signaling
intermediate in toll signaling) (52) and glycogen synthase kinase-3
(46) which provide selective options for cross-talk between kinase
pathways in increasingly complex regulatory networks. By analogy with
these systems, the type of model applied to our data suggests that IL-1
mediated responses may be "tailored" by GTPase activation and
regulated by events such as cell attachment induced by selective
involvement of receptor complex components (see Fig. 7).
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ACKNOWLEDGEMENTS |
---|
We thank Prof. Alan Hall, UCL, Dr. David
Goeddel, Tularik Inc., Dr. Julian Downward, ICRF, and Dr. Filipo Volpe,
Glaxo, for the kind gifts of expression vectors. We thank the Immunex
Corporation and Dr. Steve Poole, NIBSC, for the gift of the
IL-1.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the British Heart Foundation, the Special Trustees, Sheffield Hospitals, and the Medical Research Council (to E. E. Q.). The confocal microscopy facility was co-funded by the Wellcome Trust and the Medical Research Council.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: Functional Genomics, Division of Molecular and Genetic Medicine, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, United Kingdom.
Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M006772200
2 F. Carlotti, S. K. Dower, and E. E. Qwarnstrom, submitted for publication.
3 E. E. Qwarnstrom and S. K. Dower, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear factor-
B;
IL-1R, interleukin 1 receptor;
TRAF, tumor
necrosis factor receptor-associated factor;
IRAK, IL-1R associated
kinase;
CMV, cytomegalovirus;
EGF, epidermal growth factor;
EGFP, enhanced green fluorescent protein.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Lukashev, M. E., and Werb, Z. (1998) Trends Cell Biol. 8, 437-441[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Hall, A.
(1998)
Science
279,
509-514 |
3. | Lim, L., Manser, E., Leung, T., and Hall, C. (1996) Eur. J. Biochem. 242, 171-185[Abstract] |
4. | Nobes, C. D., and Hall, A. (1999) J. Cell Biol. |
5. |
Scita, G.,
Tenca, P.,
Frittoli, E.,
Tocchetti, A.,
Innocenti, M.,
Giardina, G.,
and Di Fiore, P. P.
(2000)
EMBO J.
19,
2393-2398 |
6. | Coso, O. A., Chiariello, M., Yu, J. C., Teramoto, H., Crespo, P., Xu, N. G., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146[Medline] [Order article via Infotrieve] |
7. | Minden, A., Lin, A. N., Claret, F. X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157[Medline] [Order article via Infotrieve] |
8. | Perona, R., Montaner, S., Saniger, L., SanchezPerez, I., Bravo, R., and Lacal, J. C. (1997) Genes Dev. 11, 463-475[Abstract] |
9. | Devary, Y., Rosette, C., Didonato, J. A., and Karin, M. (1993) Science 261, 1442-1445[Medline] [Order article via Infotrieve] |
10. |
Greenfeder, S. A.,
Nunes, P.,
Kwee, L.,
Labow, M.,
Chizzonite, P. A.,
and Ju, G.
(1995)
J. Biol. Chem.
270,
13757-13765 |
11. |
Muzio, M.,
Ni, J.,
Feng, P.,
and Dixit, V. M.
(1997)
Science
278,
1612-1615 |
12. | Wesche, H., Henzel, W. J., Shillinglaw, W., Li, S., and Cao, Z. D. (1997) Immunity 7, 837-847[Medline] [Order article via Infotrieve] |
13. |
Wesche, H.,
Gao, X.,
Li, X. X.,
Kirschning, C. J.,
Stark, G. R.,
and Cao, Z. D.
(1999)
J. Biol. Chem.
274,
19403-19410 |
14. | Burns, K., Clatworthy, J., Martin, L., Martinon, F., Plumton, C., Maschera, B., Lewis, A., Ray, K., Tschopp, J., and Volpe, F. (2000) Nat. Cell Biol. 2, 346-351[CrossRef][Medline] [Order article via Infotrieve] |
15. | Cao, Z. D., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D. V. (1996) Nature 383, 443-446[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Qwarnstrom, E. E.,
Page, R. C.,
Gillis, S.,
and Dower, S. K.
(1988)
J. Biol. Chem.
263,
8261-8269 |
17. |
Valles, S.,
Tsoi, C.,
Huang, W. Y.,
Wyllie, D.,
Carlotti, F.,
Askari, J. A.,
Humphries, M. J.,
Dower, S. K.,
and Qwarnstrom, E. E.
(1999)
J. Biol. Chem.
274,
20103-20109 |
18. |
Qwarnstrom, E. E.,
Ostberg, C. O.,
Turk, G. L.,
Richardson, C. A.,
and Bomsztyk, K.
(1994)
J. Biol. Chem.
269,
30765-30768 |
19. | Ostberg, C. O., Zhu, P., Wight, T. N., and Qwarnstrom, E. E. (1995) FEBS Lett. 367, 93-97[CrossRef][Medline] [Order article via Infotrieve] |
20. | Zhu, P., Xiong, W. S., Rodgers, G., and Qwarnstrom, E. E. (1998) Biochem. J. 330, 975-981[Medline] [Order article via Infotrieve] |
21. | Qwarnstrom, E. E., Macfarlane, S. A., Page, R. C., and Dower, S. K. (1991) Proc. Natl. Acad. Sci. U. S.A. 88, 1232-1236[Abstract] |
22. |
Palsson, E. M.,
Popoff, M.,
Thelestam, M.,
and O'Neill, L. A. J.
(2000)
J. Biol. Chem.
275,
7818-7825 |
23. |
Singh, R.,
Wang, B.,
Shirvaikar, A.,
Khan, S.,
Kamat, S.,
Schelling, J. R.,
Konieczkowski, M.,
and Sedor, J. R.
(1999)
J. Clin. Invest.
103,
1561-1570 |
24. |
Jefferies, C. A.,
and O'Neill, L. A. J.
(2000)
J. Biol. Chem.
275,
3114-3120 |
25. |
Puls, A.,
Eliopoulos, A. G.,
Nobes, C. D.,
Bridges, T.,
Young, L. S.,
and Hall, A.
(1999)
J. Cell Sci.
112,
2983-2992 |
26. | Brown, P. J., and Juliano, R. L. (1985) Science 228, 1448-1451[Medline] [Order article via Infotrieve] |
27. | Duckett, C. S., Perkins, N. D., Kowalik, T. F., Schmid, R. M., Huang, E. S., and Baldwin, A. S. (1993) Mol. Cell. Biol. 15, 2413-2419[Abstract] |
28. |
Winitz, S.,
Russell, M.,
Qian, N. X.,
Gardner, A.,
Dwyer, L.,
and Johnson, G. L.
(1993)
J. Biol. Chem.
268,
19196-19199 |
29. |
Carlotti, F.,
Chapman, R.,
Dower, S. K.,
and Qwarnstrom, E. E.
(1999)
J. Biol. Chem.
274,
37941-37949 |
30. | KissToth, E., Guesdon, F. M. J., Wyllie, D. H., Qwarnstrom, E. E., and Dower, S. K. (2000) J. Immunol. Methods 239, 125-135[CrossRef][Medline] [Order article via Infotrieve] |
31. | Marshall, C. J. (1995) Cell 80, 179-185[Medline] [Order article via Infotrieve] |
32. |
Mukaida, N.,
Mahe, Y.,
and Matsushima, K.
(1990)
J. Biol. Chem.
265,
21128-21133 |
33. | Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992) Cell 70, 401-410[Medline] [Order article via Infotrieve] |
34. |
Niv, H.,
Gutman, O.,
Henis, Y. I.,
and Kloog, Y.
(1999)
J. Biol. Chem.
274,
1606-1613 |
35. | Roy, S., Luetterforst, R., Harding, A., Apolloni, A., Etheridge, M., Stang, E., Rolls, B., Hancock, J. F., and Parton, R. G. (1999) Nat. Cell Biol. 1, 98-105[CrossRef][Medline] [Order article via Infotrieve] |
36. | Wary, K. K., Mariotti, A., Zurzolo, C., and Giancotti, F. G. (1998) Cell 94, 625-634[Medline] [Order article via Infotrieve] |
37. |
Michaely, P. A.,
Mineo, C.,
Ying, Y. S.,
and Anderson, R. G. W.
(1999)
J. Biol. Chem.
274,
21430-21436 |
38. | Sims, J. E., Bird, T. A., and Mitcham, J. L. (1995) Cytokine 324 |
39. | Minden, A., Lin, A., McMahon, M., Lange-Carter, C., Derijard, B., Davis, R. J., Johnson, G. L., and Karin, M. (1994) Science 266, 1719-1723[Medline] [Order article via Infotrieve] |
40. |
Hirano, M.,
Osada, S.,
Aoki, T.,
Hirai, S.,
Hosaka, M.,
Inoue, J.,
and Ohno, S.
(1996)
J. Biol. Chem.
271,
13234-13248 |
41. |
Meyer, C. F.,
Wang, X.,
Chang, C.,
Templeton, D.,
and Tan, T. H.
(1996)
J. Biol. Chem.
271,
8971-8976 |
42. |
Karin, M.,
and Delhase, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9067-9069 |
43. | Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88, 213-222[Medline] [Order article via Infotrieve] |
44. | Zhong, H. H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell 1, 661-671[Medline] [Order article via Infotrieve] |
45. |
Johnson, C.,
VanAntwerp, D.,
and Hope, T. J.
(1999)
EMBO J.
18,
6682-6693 |
46. | Hoeflich, K. P., Luo, J., Rubie, E. A., Tsao, M. S., Jin, O., and Woodgatt, J. R. (2000) Nature 406, 86-90[CrossRef][Medline] [Order article via Infotrieve] |
47. | Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., and Matsumoto, K. (1999) Nature 398, 252-256[CrossRef][Medline] [Order article via Infotrieve] |
48. | Tojima, Y., Fujimoto, A., Delhase, M., Chen, Y., Hatakeyama, S., Nakayama, K., Kaneko, Y., Nimura, Y., Motoyama, N., Ikeda, K., Karin, M., and Nakanishi, M. (2000) Nature 404, 778-782[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Muzio, M.,
Natoli, G.,
and Saccani, S.
(1998)
J. Exp. Med.
187,
2097-2101 |
50. |
Lomaga, M. A.,
Yeh, W. C.,
Sarosi, I.,
Duncan, G. S.,
Furlonger, C.,
Ho, A.,
Morony, S.,
Capparelli, C.,
Van, G.,
Kaufman, S.,
vanderHeiden, A.,
Itie, A.,
Wakeham, A.,
Khoo, W.,
Sasaki, T.,
Cao, Z. D.,
Penninger, J. M.,
Paige, C. J.,
Lacey, D. L.,
Dunstan, C. R.,
Boyle, W. J.,
Goeddel, D. V.,
and Mak, T. W.
(1999)
Genes Dev.
13,
1015-1024 |
51. | Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999) Immunity 11, 115-122[Medline] [Order article via Infotrieve] |
52. |
Kopp, E.,
Medzhitov, R.,
Carothers, J.,
Xiao, C. C.,
Douglas, I.,
Janeway, C. A.,
and Ghosh, S.
(1999)
Genes Dev.
13,
2059-2071 |