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J. Biol. Chem., Vol. 278, Issue 33, 30881-30888, August 15, 2003
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B
Phosphorylation
B COMPLEXES*
¶ ||
From the
Cell Biology Unit, Functional Genomics,
Division of Genomic Medicine, The Medical School, University of Sheffield,
Sheffield S10 2JF, United Kingdom and the
¶Department of Pathology, School of Medicine,
University of Washington, Seattle, Washington 98195
Received for publication, December 2, 2002 , and in revised form, March 7, 2003.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B-IB complex formation regulates the level and specificity
of NF-B activity. Quantitative analyses showed that
RelA-NF-B-induced IB
binding is regulated through
inhibitor retention and phosphorylation. RelA caused an increase in
IB phosphorylation and in degradation, which was enhanced
monotonically with inhibitor concentration. In vivo analysis
demonstrated the RelA-induced IB/RelA interactions to be
specific, saturable, and phosphorylation-dependent. In addition, it showed
that phosphorylation regulates both the level and affinity of the complexes
and demonstrated an increased average affinity to coincide with reduction in
the level of complexes during cytokine-induced pathway activation. The data
show that RelA regulation of NF-B-IB complex formation is
IB phosphorylation-dependent and that
IB/NF-B binding is dynamic and determined by
concentration of the subunits. In addition, they suggest that regulation of
both complex levels and affinities through phosphorylation, with effects on
the system steady state, participate in selective activation of the
NF-B pathway. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B transcription factors are central to inflammatory and immune
responses
(1–3).
They are composed of homo- and heterodimers of a family of at least five
related subunits, characterized by a 
300-amino acid Rel homology domain
(4). Activation is controlled
by IB (inhibitor of NF-B) proteins, the
degradation of which is a critical step in activation of NF-B
(5). The IB family
includes seven proteins containing multiple ankyrin-like repeats, which
mediate interaction with the Rel homology regions of NF-B dimers.
IB, the most extensively studied protein in this family,
interacts primarily with p50/RelA and p50/c-Rel heterodimers but also with
c-Rel and RelA homodimers (4,
6). IB inhibits
NF-B activation by masking its nuclear localization signals and thereby
sequestering the two proteins in the form of complexes in the cytoplasm
(7).
Induction of the pathway is mediated by a significant number of stimuli,
including cytokines, lipopolysaccharides, cellular stress, and cell adhesion
(4,
8,
9). During activation,
NF-B is transported into the nucleus and activates transcription of
genes bearing cognate binding motifs
(4). Transport is made possible
by dissociation of complexes following phosphorylation of IB on serines
32 and 36 by IB kinases, ubiquitination, and proteosome degradation
(10–13).
The significance of IB/NF-B interactions as a regulatory step in
pathway activation has been extensively documented
(6,
14–16).
Here we report the use of the fusion protein
IBEGFP1
to examine regulation of the function of IB by NF-B. RelA
regulation of IB and of IB/NF-B
interactions were analyzed in single living cells, using confocal microscopy,
showing that all aspects of IB function are critically dependent
on that of NF-B and that phosphorylation of the inhibitor constitutes
the basis for interaction and selection during pathway activation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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BEGFP encodes a red-shifted variant
of green fluorescent protein fused to the carboxyl terminus of
IB, constructed by cloning the IB cDNA from
pIB ctag (kind gift from Prof. Ronald Hay)
(17) digested with
BamHI plus EcoRI into pEGFP-N2 (Clontech) digested
with BglII plus EcoRI and in-filling of a SmaI plus
EcoRI digest for alignment of the reading frame. Plasmid
pIBEYFP was constructed by subcloning
the same IB fragment into pEYFP-N1 (Clontech), digested
with BglII plus EcoRI as described
(18). Plasmid
pECFPrelA was constructed by cloning the RelA cDNA from
pBluescript-relA
(19), digested with
HindIII plus BamHI, into similarly prepared
pECFP-C1 (Clontech). Plasmid pECFPp50 was constructed by
cloning the p50 cDNA from pRSV-NFB1(p50)
(19) digested with
Hin-dIII plus BglII into pECFP-C1 (Clontech) and
in-filling of the XhoI site. Site-directed mutagenesis of
pIBEGFP (Ser 
Ala at positions
32 and 36) was performed using the Muta-Gene Phagemid kit (Bio-Rad).
Mutagenesis of pECFPrelA to obtain nonfluorescent fusion proteins was
performed by deletion of residues 65–67 (Thr, Trp, and Gly) within the
ECFP N terminus, critical for fluorescence, using the QuikChangeTM
site-directed mutagenesis kit (Stratagene). Oligonucleotides were synthesized
from Life Technologies. All constructs were confirmed by sequence
analysis.
Tissue Culture and Transfection—Human gingival fibroblasts
(HGFQ1 and HGFQ2, transfer 10–19) and HeLa cells were maintained in
Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf
serum (Invitrogen) and 5 mM sodium pyruvate, 100 µg/ml
penicillin and streptomycin and kept at 37 °C in a 95% O2, 5%
CO2 atmosphere. Cells were plated 24 h prior to transfection and
transiently transfected using calcium phosphate co-precipitation with glycerol
shock (60 s, 15% glycerol in phosphate-buffered saline) 4 h after
transfection, as described previously
(20,
21). Control experiments
demonstrated that transfection of mediators of the NF-B pathway, under
these conditions, had no effect on endogenous cytokine production, which was
significant for pathway activation
(20–22).
Stimulation with IL-1 (1 nM) kindly provided by Dr. Steve Poole
(National Institute of Biological Standards and Control) was carried out 24 h
after transfection, for various times, as indicated.
Confocal Microscopy—EGFP fusion proteins were visualized using a confocal laser-scanning microscope (Molecular Dynamics, CLSM 2010) fitted with a 37 °C stage incubator and coupled to a Nikon Diaphot 300 microscope and a Silicon Graphics work station. Laser power was set to 10 mW, band selection to 488 nm, photo multiplier tube voltage to 750, and varying laser attenuation to maintain pixel density below 200, within the linear range of the instrument. Emission scans were done with a x 60 Plan Apo oil immersion objective (numerical aperture of 1.4) and a 50-µm aperture generating an optical section of 0.54 µm and using a 530-nm band pass filter. To quantitate the cytoplasmic and the nuclear fusion protein level, transfected cells were scanned horizontally through the nucleus, and images were analyzed using NIH Image. Relative fluorescence was calculated by measuring mean intensity of representative areas of nucleus or cytoplasm and normalized by dividing by the attenuation and further by 2.41 for consistency with previous results obtained with a photo multiplier tube of 666 V (20).
Western Analysis—The relative levels of endogenous and
exogenous IB were determined by Western analyses as described
(20) using cells transfected
with ctag-containing, or fluocrome-tagged (pEGFP, pEYFP, pECFP,
pIBEYFP, or pECFPrelA)
NF-B signaling components to make it possible to distinguish endogenous
and exogenous proteins. Cell extracts were prepared by lysis in 1x
Laemmli sample buffer (23),
resolved on 12.5% SDS-PAGE, and transferred to polyvinylidene difluoride
membrane (Amersham Biosciences) with 
-actin used as an internal control.
Total and phosphorylated IB were detected using primary rabbit
polyclonal anti-IB and anti-phospho-IB (1:5,000;
BioLab), ECFPrelA using goat anti-RelA IgG (1:10,000; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), EGFP and its variants using rabbit
anti-GFP IgG (Santa Cruz Biotechnology), and -actin using mouse
anti-actin IgG (Sigma), followed by horseradish peroxidase-conjugated
secondary antibodies (1:2000), and visualized by ECL (Amersham Biosciences)
according to the manufacturer's instructions. Quantitation of exogenous and
endogenous expression levels of the various IB and RelA proteins
was done of cells transfected with constructs containing the untagged inserts
and those transfected with pIBEGFP or
pEGFPrelA for confocal microscopy and with
pIBEYFP or pECFPrelA for
fluorescence resonance energy transfer (FRET) analysis as well as with empty
vectors (pEGFP, pEYFP, and pECFP). Band intensity was
compared with a range of concentrations of purified EGFP, and data were
analyzed using NIH Image. These experiments validated the comparative use of
these various constructs and showed that expression levels of untagged
proteins RelA and p50 following transfection, relative to levels of the
corresponding fluorescence-tagged fusion protein were 80 ± 6 and 120
± 15%, respectively. In addition, they demonstrated that the tagged and
untagged NF-B fusions had the same effects on IL-1-induced
IB turnover.
Immunofluorescence—Immunofluorescent staining of transfected
cells was carried out as described previously
(20). Briefly, incubation with
a rabbit polyclonal IB antiserum (1 µg/ml, 1 h, room
temperature; Santa Cruz Biotechnology) of cells transfected with
IBEGFP was carried out following fixation in methanol (–20
°C, 5 min) and incubation in blocking serum (5%, 1 h) and prior to
incubation with biotinylated secondary antibody (2 µg/ml, overnight at 4
°C; Santa Cruz Biotechnology) and streptavidin/Texas Red (0.2 µg/ml, 15
min; Amersham Biosciences). Data (red fluorescence) were acquired using 750 V
with excitation at 568 nm and emission at 590 nm. Excitation and emission of
green fluorescence from the fusion protein was done at 488 and 530 nm,
respectively, as above. Green and red fluorescence was plotted for individual
cells over a range of transfection levels. The level of GFP fluorescence was
corrected for minor reduction in intensity (15%) induced by methanol
fixation.
Immunoprecipitation—Biochemical analysis of complex
formation was carried out by immunoprecipitation using an anti-RelA antibody
(Santa Cruz Biotechnology), followed by Western analysis, as above, of cells
transfected with wild type and mutant
pIBEYFP, alone or together with
pCMV:relA, before and after IL-1 stimulation. Thus, HeLa cells seeded
at 3 x 106 cells/10-cm plate were transfected with the
indicated cDNAs, totaling 4.4 µg/plate, and after 24 h they were left
unstimulated or treated with IL-1 for 8 and 40 min and lysed.
Immunoprecipitation of cell lysates, as above, was performed by incubating
with 5 µg of anti-RelA antibody and protein G conjugated to Sepharose beads
(overnight, 4 °C; Sigma). Following resuspension in lysis buffer,
precipitates were separated by gel electrophoresis (20% SDS, Tris-HCl) and
transferred to polyvinylidene difluoride membranes for Western analysis, as
above.
FRET Analysis—Images of ECFP, EYFP, and FRET were obtained through a x 60 Plan Apo oil immersion objective (numerical aperture of 1.4) using a Nikon Diaphot 300 microscope and recorded by a 12-bit Hamamatsu digital camera C4742-95 driven by OpenLab software (Improvision). Complex formation was determined through measuring close association of fluorophores ECFP and EYFP by excitation of the donor, resulting in an increase in acceptor emission fluorescence and/or a decrease in donor fluorescence due to FRET (24–26) using a series of filter sets (Omega Optical): XF114 for ECFP (440DF21 excitation, 455DRLP dichroic, 480DF30 emission), XF104 for EYFP (500DF25 excitation, 525DRLP dichroic, 545DF35 emission), and XF88 for FRET (440DF21 excitation, 455DRLP dichroic, 545DF35 emission). Relative fluorescence was calculated for the various emission settings by measuring the mean intensity of representative areas of nucleus or cytoplasm and normalized by dividing by attenuation, as above. All images were corrected for background, and FRET images were further processed by subtracting overspill of ECFP (50.7%) and EYFP (30.4%) fluorescence assessed using vectors containing ECFP or EYFP only. The accuracy of the FRET signal was further confirmed by inhibition of emission at 545 nm after photobleaching the acceptor-EYFP at 500-nm excitation. In addition, control experiments included demonstration of a correlation between the reduction in yellow fluorescence and increase in cyan intensity.
Binding Analyses—The level of IB/RelA binding was
measured by FRET following co-transfection with IBEYFP (yellow
fluorescence) and ECFPrelA (cyan fluorescence), before and after IL-1
stimulation (30 min) in four independent experiments. The FRET signal from
single transfected cells expressing a range of IB or
S32A/S36A-phosphodeficient IB mutant (ligand) concentrations was
quantitated, as reported for assessment of protein/protein interactions in a
variety of systems in live cells
(27–30).
Analysis was limited to including cells expressing levels of fusion protein of
up to 8–10 times that of the endogenous levels, corresponding to
fluorescence of 2.5–3, to ascertain reliable cytokine responses
(20,
21). Readings were ranked
according to yellow fluorescence (IBEYFP), and to increase
accuracy, they were pooled in consecutive groups of three for data analysis.
The specificity of the interaction was demonstrated by competition of the
IBEYFP/ECFPrelA binding, carried out by co-transfecting with
increasing levels of pCMV:relA or pCMV:p50, or using a
mutant pECFPrelA lacking the cyan fluorescent site. Saturated binding
and apparent dissociation constants were estimated by nonlinear least squares
fitting of a noncooperative one-site equilibrium-binding model of the FRET
versus IB (yellow fluorescence). All data were analyzed
using MLAB for the Macintosh (Civilized Software, Silver Spring, MD).
Cellular concentrations of the various fusion proteins were determined by
Western analysis of transfected cells as above and were included in
calculations of binding affinity (Ka) using the
equation,
 | (Eq. 1) |
B/cell determined by FRET; Receptors/cell or Binding protein is
RelA concentration (molecules/cell); and [Ligand] is IB
concentration (molecules/cell).
Controls included calculations using various size pools for data analysis
(groups of 3–6), as well as using larger data sets allowing up to 12
individual cell readings for each IB concentration, all
demonstrating the same results as regards specific binding sites and affinity
values. In addition, the single cell analysis was assessed by determining the
R2 values. These were found to be of the same order as
that obtained from conventional binding analysis, varying by 15 ± 5%,
demonstrating a high level of accuracy, and further revealing an underlying
Gaussian distribution as regards cell/cell variation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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BEGFP degradation by
co-transfection with RelA alone or together with p50, increasing IL-1-induced
turnover from 60 to 90–95% (Fig. 1,
A and B). In contrast, levels of a serine 32/36
phosphorylation-deficient IB mutant
(31–33)
were unaffected by IL-1 stimulation, either when transfected alone or
following co-transfection with NF-B subunits. In addition, there was a
positive correlation between the magnitude of the effect and the initial
cytoplasmic levels of IB
(Fig. 1C).
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The RelA-mediated effect on turnover was confirmed by Western analysis,
which, in addition, demonstrated an enhanced phosphorylation of
IBEGFP in IL-1-stimulated cells following co-transfection
(Fig. 2, A and
B). The RelA-mediated effect on phosphorylation of the
inhibitor protein resulted in a 2-fold increase in non-IL-1-stimulated levels
and caused a 50–100% enhancement of the peak value at 5–10 min of
IL-1 stimulation. In addition, these experiments showed similar but less
pronounced effects on the endogenous protein.
|
The dependence of the RelA-induced effects on IB
concentration, demonstrated by single cell analysis, prompted quantitation of
inhibitor levels. Immunocytochemical staining of transfected cells and
monitoring of red and green fluorescence demonstrated a linear relationship
between total and transfected levels of the protein over a wide range of
concentrations (Fig. 3, A and
B). A mean transfection level of 1 fluorescent unit
corresponded to an increase in cytoplasmic IB to about 3-fold
the average endogenous levels (Fig.
3B). Quantitation by Western analysis of cells
transfected with pIBEYFP and
pECFPrelA showed average concentrations of 400,000 and 370,000
molecules/cell for IBEYFP and ECFPrelA
(Fig. 3C),
respectively, and revealed endogenous IB levels to be about
135,000 molecules/cell, 2.5-fold higher than that estimated for the average
levels of endogenous RelA, as expected
(20,
22), with all values close to
the ranges demonstrated for the endogenous proteins
(22).
|
Confocal microscopy further demonstrated retention of IB in
the cytoplasm by RelA (Fig. 4).
Thus, an increase in nuclear localization of tagged
(Fig. 4A,
1–4) or untagged
(Fig 5A, 5 and
6) inhibitor at higher expression levels
(Fig. 4A, 3 and
5) was not observed in the presence of a comparable
amount of the NF-B subunit (Fig.
4A, 4 and 6). In addition, quantitation over a
wide range of IBEGFP expression levels showed the effect to
correlate with increasing levels of (pCMV) relA in a
concentrationdependent manner (Fig. 4,
B and C).
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The RelA/IB interdependence was further analyzed by
simultaneous observations of RelA and IB using cyan (ECFP) and
yellow (EYFP) variants of GFP, respectively (pECFPrelA and
pIBEYFP)
(Fig. 5A). These
experiments demonstrated that in the unstimulated state, both subunits were
located in the cytoplasm. Quantitation showed that, similarly to experiments
using EGFP containing constructs
(20), IL-1 stimulation caused
a successive increase in nuclear RelA but had no appreciable effect on
cytoplasmic levels (Fig.
5B). Similarly, the rate of reduction of cytoplasmic
inhibitor levels during activation was the same as that using the green
fluorochrome, (compare Figs.
5B and
2B) and coincided with
a reduction in the much lower levels of nuclear IB. The levels
of RelA-IB complexes were assessed by measuring FRET using
ECFPrelA as a donor and IBEYFP as the acceptor
(34). FRET was observed in
cells expressing both fusion proteins demonstrating in vivo
association of IBEYFP and ECFPrelA
(Fig. 5A, 6 and
9), not seen in control cells, transfected with
constructs containing EYFP and ECFP only
(Fig. 5A, 3). A
reduction in FRET signal (compare 6 and 9) following
cytokine stimulation correlated with nuclear translocation of RelA (compare
4 and 7) and a decrease in cytoplasmic IB
(compare 5 and 8). Both the level of FRET and of
IB showed a steady state at about 50% after 30 min of IL-1
stimulation (Fig. 5B).
The addition of RelA caused a concentration-dependent increase in the level of
complexes over a range of inhibitor concentrations, reaching successively
higher levels of saturation (Fig.
5C).
Analysis by immunoprecipitation of transfected cells demonstrated that RelA
caused an increase in IB-containing complexes, both in control
and IL-1-stimulated cells. Using a phosphospecific antibody, the experiments
similarly demonstrated a significant increase in the level of complexed
phosphorylated inhibitor in the presence of RelA
(Fig. 6A).
Transfecting the Ser32/Ser36-phosphodeficient mutant,
however, resulted in a significant reduction in IB/RelA
interactions, in both control and IL-1-stimulated cultures
(Fig. 6B).
Quantitation further demonstrated a correlation between effects on complex
levels (1.96 ± 0.4) and phosphorylation (2.35 ± 0.58) using
tagged or untagged RelA, before and after stimulation, with unaffected low
levels using the phosphodeficient IBEGFP mutant
(Fig. 6C).
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The NF-B/IB interaction was further characterized
using FRET by measuring signals from transfected cells expressing a range of
IB levels and modeling the data by noncooperative binding of
ligand (IB) to a single class of sites (RelA). Specificity of
the interaction was demonstrated by competition by increasing amounts of
pCMVrelA or pCMV p50, or of a non-fluorescing mutant of
ECFPrelA (ECFPrelAmut) (Fig.
7A). This showed a dose-dependent inhibition of the FRET
signal, which corresponded to about 70% at 4-fold overexpression of either of
the RelA constructs, whereas co-transfection with p50 had no effect. Further,
in agreement with biochemical data, shown in
Fig. 6C, using the
phosphorylation site-deficient IB-mutant resulted in about a 50%
reduction in specific RelA binding (Fig.
7A).
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In addition, these experiments showed that IB/RelA
interaction (FRET) is dynamic and dependent on IB concentration
(EYFP fluorescence), demonstrating saturation before and after incubation with
IL-1 (Fig. 7B). A
pronounced fall in the FRET signal after 30 min of IL-1 stimulation
corresponded to a reduction in complex concentration to 38 ± 8%,
correlating with the decrease determined by immunoprecipitation (see
Fig. 6). In comparison, using
the phosphonegative mutant resulted in a much decreased level of complexes,
which were unaffected by IL-1 stimulation
(Fig. 7B).
Characterization of the interaction by Scatchard analysis demonstrated a
pronounced (4.2 ± 1.0-fold) increase in affinity
(Ka) of RelA binding to the wild type inhibitor
following stimulation with the cytokine, relative to that calculated for
control cultures (Fig.
7C). Quantitation of fusion protein levels, as in
Fig. 3C, gave
estimated affinity constants (Ka) for these
interactions in the range of 107 to 5 x 107
M–1 in unstimulated cells and between 5 x
107 and 5 x 108 M–1
following IL-1 stimulation, respectively. In comparison, the affinity of RelA
for the phosphonegative mutant was only 25% of that for the wild type and, in
addition, was unaffected by cytokine activation
(Fig. 7C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B/IB interdependence and regulation
through IB phosphorylation. The real time GFP measurements on
single cells, in addition, show that the IB/NF-B
interaction is specific, saturable, and dynamic; that the RelA-induced
increase in binding relies on trafficking; and that regulation of complex
formation and of the affinity of the interaction are IB
phosphorylation-dependent.
Our data underscore the reciprocal nature of NF-B/IB
regulation, coupled with a system in which free components successively
accumulate in the nucleus with increasing levels, and bound complexes largely
remain in the cytoplasm, in the resting state
(20). This is reflected in the
inverse correlation between the inhibitor cytoplasmic concentration and the
ratio of cytoplasmic to nuclear levels and by the cytoplasmic accumulation of
IB in the presence of excess RelA. Regulation is probably
influenced by the level of the endogenous proteins and of de novo
synthesis through autoregulation
(35), since the low
transfection levels of the GFP fusions, corresponding to 2–3-fold that
of the endogenous protein, are well within the physiological range of
variation (22). Thus, the
successive increase in nuclear levels at higher concentrations of
IB-EGFP probably reflects titering out of endogenous NF-B.
Further, competition for the endogenous pool of NF-B could account for
the enhanced level of breakdown of IBEGFP and the limited effect
of co-transfection at low levels of exogenous inhibitor
(22).
Enhanced cytoplasmic retention of IB, such as that induced by
RelA, has significant effects on pathway regulation
(36–41).2
This is supported by mathematical modeling, which shows that the B
system is dissipative and far from equilibrium and is at a steady state
determined by dissociation of complexes, intercompartmental trafficking, and
synthesis and breakdown of components
(21).3
Further, simple kinetics (Briggs-Haldane) show that saturable
NF-B/IB binding, under steady state conditions, can occur
with apparent affinity constants much lower than those observed with pure
components in vitro, in agreement with the lower affinities measured
in our study, compared with those observed using Biocore
(42).
The involvement of basal cycling in regulation
(21), indicated by the
slightly delayed decrease in FRET (47%) compared with that of the inhibitor
fluorescence (56%) (significant at 5%), suggests a reassociation of released
ECFPrelA with free IBEYFP prior to nuclear entry. The biological
significance of transport is not likely to enhance the level of activation,
since the kinetics of inhibitor reduction in the two compartments is identical
(60–70%). Rather, it may constitute the basis for recruiting specific
subsets of NF-B intermediates for distinct facets of pathway
regulation, such as complexes activated subsequent to nuclear translocation
(36). The significance of
selectivity during pathway activation is supported by the burgeoning evidence
of diversity and complexity of NF-B induction
(2,
36,
41,
43–47).
This further supports the notion of a system where perturbations in
concentrations of signaling components and in the system steady state, such as
that induced by increased levels of RelA, can have marked effects on both the
extent and mechanism of NF-B-induced gene transcription.
Enhanced inhibitor phosphorylation in the presence of exogenous RelA is
probably to some extent due to an increase in NF-B-IB
complexes and reflects preferential phosphorylation of complexed inhibitor by
IB kinases (48).
Conversely, the pronounced reduction in RelA/IB binding at
steady state, observed with the 32/36 mutant, demonstrated dependence of
complex formation and affinity on IB phosphorylation, as found
for interactions through the PEST domain of the protein
(49). In the absence of a
direct involvement of the IB signal-receiving domain in
NF-B interaction (42),
the effects on specific binding, observed using the mutants, could be
explained by changes in inhibitor conformation in vivo. A decrease in
binding of IB proteins lacking the phosphorylation sites
necessary for proteosome degradation
(6,
31,
32), is consistent with a
critical role for RelA in controlling IB turnover. Of the three
components of the classical B complex, RelA is present at the lowest
concentration (22). Linking
IB turnover to RelA levels will therefore optimize signals
through the IB kinases. In addition, ongoing studies suggest that
further amplification through positive feedback is induced by increased
activation of upstream kinases in the presence of excess
RelA.4 This is in
agreement with a system based on reutilizing a limited amount of RelA through
repeated cycles of IB binding, phosphorylation, and degradation
and with continuous enhanced effects with increasing RelA concentrations.
Alterations in the level and affinity of IB-NF-B
complexes correlated with the rate of inhibitor degradation. With only 20% of
NF-B free of IB
(20), a 4-fold change in
complex affinity is likely to have a significant role in regulation of
inhibitor turnover, subsequent to effects on basal shuttling and reduction in
free NF-B. The identical results obtained in the blocking experiments,
using tagged and untagged RelA, show that the lower affinities we observe
in vivo compared with that measured with purified proteins
(41) are not artifacts of GFP
tagging (50). The increase in
NF-B/IB affinity correlates with the enhanced cytoplasmic
FRET/IBEYFP ratio following IL-1 stimulation, suggesting a
selective process. This is also supported by the lack of a simple inverse
correlation between the affinity and the level of complexes and indicates that
low affinity complexes are specifically targeted for degradation during
cytokine activation. The biological significance of this selection could be to
facilitate a rapid release in response to a transient signal through the low
affinity complexes, characterized by a high dissociation rate. The
heterogeneity of NF-B-IB complex affinity may thus
constitute a buffer to incoming signals.
In summary, the data show that translocation, phosphorylation, and complex
formation are all critical for regulation of IB steady state
levels by NF-B. They demonstrate that IB/NF-B
binding is dynamic, saturable at steady state, and phosphorylation-dependent.
They also show that both the extent and the affinity of the
IB/NF-B interaction play a role in system regulation,
affecting both basal levels and those induced in response to incoming signals.
The multifaceted effects of RelA on the concentration-dependent limitation of
inhibitor turnover thus constitute a complex feedback loop, with effects on
the system steady state and with expected significance for the specificity and
level of NF-B activity.
|
FOOTNOTES |
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Present address: Dept. of Pathology, Brigham and Women's Hospital, Harvard
Medical School, 221 Longwood Ave., Boston, MA 02115.
|| To whom correspondence should be addressed: Cell Biology Unit, Div. of Genomic Medicine, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, UK.
1 The abbreviations used are: EGFP, enhanced green fluorescent protein; IL-1,
interleukin-1; FRET, fluorescence resonance energy transfer; GFP, green
fluorescent protein; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced
yellow fluorescent protein.
2 L. Yang and E. E. Qwarnstrom, unpublished observations.
3 S. K. Dower, L. Yang, and E. E. Qwarnstrom, unpublished observations.
4 I. Evans, K. P. Ray, and E. E. Qwarnstrom, unpublished observations.
|
ACKNOWLEDGMENTS |
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B (pGEX.IBctag) was a generous gift
from Prof. Ronald Hay. pRSV-NF-B1 (p50) and
Bluescript-RelA (p65) were obtained through the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID National Institutes of
Health, from Dr. Gary Nabel and Dr. Neil Perkins. We also thank Dr. Steve
Poole for the kind gift of IL-1 and Prof. Steve Dower for useful
discussions. The confocal microscopy analyses were carried out using the
facility in the Section of Functional Genomics, University of Sheffield,
UK. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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), pCMV:p50 (
), or a mutant,
pECFPrelA, lacking the cyan fluorescent site (
). One of two
experiments for each condition is shown. B, an analysis of binding of
I
, control cultures;