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B Protein Degradation Mediate CD40 Ligand Rescue of WEHI 231 Immature B Cells from Receptor-Mediated Apoptosis1
Departments of
*
Microbiology and
Biochemistry, Boston University Medical School, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel factors plays a major role in B cell survival. Treatment
of WEHI 231 immature B cells with an Ab against the surface IgM protein
(anti-IgM) induces apoptosis that can be rescued by engagement of
CD40 receptor. The dramatic decrease in high basal levels of
NF-B/Rel activity induced by anti-IgM treatment led to cell
death. CD40 ligand (CD40L) treatment prevented the drop in NF-B/Rel
factor binding by inducing a sustained decrease in inhibitor (I)
B-
and transient decrease in IB-ß protein levels. In this
study, we have investigated the regulation of these
NF-B/Rel-inhibitory proteins. In exponentially growing WEHI 231
cells, the IB- and IB-ß proteins decayed with an approximate
t1/2 of 38 and 76 min, respectively,
which was blocked effectively upon addition of the proteasome-specific
inhibitor (benzylcarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO). Anti-IgM
treatment stabilized IB- and IB-ß proteins. CD40L treatment
resulted in a dramatic decrease in t1/2 (<5
min) for both IB molecules, which was inhibited by addition of
Z-LLF-CHO. CD40L treatment also caused a delayed increase in IB-ß
mRNA levels, most likely contributing to the observed recovery of
IB-ß levels. Microinjection of IB--glutathione
S-transferase fusion protein into nuclei of WEHI 231 cells
ablated protection by CD40L from receptor-mediated killing.
Furthermore, CD40L rescued apoptosis induced upon microinjection of a
vector expressing wild-type IB-, but not a 32A/36A mutant form of
IB-, unable to be phosphorylated and hence degraded. Thus,
control of turnover of IB proteins by CD40L plays a major role in
maintenance of NF-B/Rel and resultant rescue of WEHI 231 cells from
apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel family of transcription
factors in this process. A drop in NF-B/Rel factor activity
following treatment with anti-IgM or TGF-ß1 resulted
in induction of cell death (3, 4). Furthermore, we noted that
inhibition of NF-B/Rel induced apoptosis in a number of other B cell
lymphomas and in primary splenic B lymphocytes (3). Ectopic expression
of c-Rel led to extensive WEHI 231 cell survival (3, 4). Furthermore,
CD40 ligand (CD40L)4 rescue of WEHI 231 cells from
apoptosis induced by anti-IgM led to maintenance of NF-B/Rel
binding (5). Thus, NF-B/Rel factors play pivotal roles in control of
death and survival of B cells.
NF-B/Rel is a family of transcription factors that includes p65 (Rel
A), p50, c-Rel, RelB, p52, and the Drosophila proteins Dif
and Dorsal (reviewed in 6 . All members of this family share a
300-amino acid-residue region of homology to v-Rel, called the NRD
(NF-B/Rel/Dorsal) domain, which is involved in subunit dimerization
and binding of dimeric complexes to DNA (reviewed in Refs. 6–8). For
the most part, the members of the NF-B/Rel family can form hetero-
and homodimers that vary significantly in their transactivation
potential (6, 7, 8, 9, 10, 11). In most non-B cells, almost all NF-B/Rel
complexes are inactive, as they are sequestered in the cytosol, bound
to one or more inhibitor proteins, including IB- and IB-ß.
In contrast, NF-B/Rel activity is constitutive in B cells, but can
be further induced or modulated (11, 12, 13, 14, 15). The inducible NF-B/Rel
activity is due predominantly to release of sequestered cytoplasmic
dimeric complexes from inhibitor IB proteins (8, 9, 16, 17, 18). A large
number of genes have been found to contain one or more NF-B sites
(reviewed in Refs. 8 and 9). These include genes encoding transcription
factors, such as the c-myc gene, adhesion molecules,
cytokines, and viral proteins. In addition, the IB- gene contains
several B elements, and the induction of NF-B-binding activity
induces expression of this inhibitor, creating a feedback mechanism to
reduce the activity of these factors (19).
The rapid phosphorylation of exogenous IB- on serine
residues 32 and 36 has been shown to correlate with NF-B induction
in EL4 cells; if either of these serine residues was mutated, NF-B
activity was abolished (20). This phosphorylation, in turn, is believed
to target the IB- protein for ubiquitination on lysine residues
21 and 22, which marks it for degradation by the proteasome pathway
(reviewed in 21 . These lysine residues are required for NF-B
activation, as the introduction of an IB- expression vector with
mutations at the two lysine residues prevented the degradation of
IB- and inhibited the NF-B activity normally induced with
TNF-, IL-1, or PMA in Jurkat cells (22, 23). Additionally, treatment
of HeLa cells with the proteasome inhibitor,
Cbz-Ile-Glu(o-t-Bu)-Ala-leucinal (PSI), led to the accumulation of a
phosphorylated form of IB- and prevented NF-B induction (24).
In vitro translated IB- has been shown to be ubiquitinated in
HeLa cells, and remains associated with NF-B until it is degraded by
the proteasome (25). Thus, studies have demonstrated phosphorylation,
ubiquitination, and degradation of IB- in the cytoplasm lead to
release and hence induction of NF-B upon activation of cells (22, 23, 25, 26, 27). A second member of the inhibitory family, IB-ß, has
been shown to interact with p65- and c-Rel-containing dimers in a
similar fashion as IB-, retaining them in the cytoplasm (18). In
experiments similar to those described for IB-, IB-ß was
inducibly phosphorylated on serine residues 19 and 23 in HeLa cells,
presumably followed by ubiquitination thought to be on lysine residue 9
(26). Once NF-B/Rel complexes are released from their inhibitor, the
nuclear localization sequence is exposed again, allowing the dimer to
translocate to the nucleus (28, 29, 30). In B cells, the high basal rate of
IB- degradation has been proposed responsible for the large
portion of the NF-B/Rel pool found constitutively in the
nucleus (31).
CD40, a cell surface molecule found on a variety of cell types,
including B cells, is a member of the TNFR family, which includes TNFR
I and II, and Fas, among others (reviewed in 32 . Its
corresponding ligand is found on activated T cells, and is thought to
be a costimulatory molecule (33). CD40 plays a critical role in B cell
function, as evidenced by the absence of germinal centers and secondary
immune responses in CD40-deficient mice and the association of
mutations in CD40 ligand with the human disease, X-linked hyperIgM
syndrome (34, 35, 36, 37, 38). Ag receptor-mediated apoptosis in WEHI 231 B cells
is reversed by engagement of surface CD40 (39). Recently, we
demonstrated that CD40L rescue prevents the normal drop in NF-B/Rel
binding induced by anti-IgM treatment, maintaining high levels of
expression of these factors (5). Thus, following 1 h of
anti-IgM treatment of exponentially growing WEHI 231 cells, which
express high levels of p50/c-Rel, there is a transient increase in
binding levels, as well as of p50/p65 (11); by 4 to 12 h, binding
has declined precipitously and is well below levels in untreated cells
(5, 11). In contrast, costimulation with anti-IgM and CD40L for
1 h induces a much higher level of binding of c-Rel- and
p65-containing complexes and subsequently leads to maintenance of
binding above baseline levels even after 12 h, in particular of
complexes containing c-Rel (5). When the mechanism for the drop in
binding following anti-IgM treatment of WEHI 231 cells was
analyzed, an increase in the levels of IB-ß and IB- was
noted, whereas CD40L costimulation was found to lead to a sustained
decrease in IB- levels and a transient decrease in IB-ß
levels (5). As IB- and IB-ß proteins are critical for the
control of NF-B/Rel activity in WEHI 231 cells, in this study we
examined their normal regulation, as well as the effects of treatment
with anti-IgM or CD40L on their expression. Anti-IgM caused
stabilization of the proteins from decay, whereas CD40L resulted in a
rapid, dramatic increase in the rates of degradation of both IB
proteins through the proteasome pathway. The subsequent differential
patterns of IB- and IB-ß expression appeared, in part,
related to differences in control of pretranslational events.
Importantly, CD40L was unable to rescue WEHI 231 cells upon
introduction of IB- protein into the nuclear compartment or upon
expression of a mutant form of IB- protein unable to be
phosphorylated or degraded; these results demonstrate the pivotal role
of maintenance of NF-B/Rel in CD40L-induced survival of these B
cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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WEHI 231 cells were maintained in DMEM supplemented with 10% FBS, 0.35% glucose, 0.058% glutamine, nonessential amino acids (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM ß-mercaptoethanol, and were treated with anti-IgM, as described previously (11). Sixteen hours before treatment with CD40L, cells were plated at a density of 2 x 105 cells/ml. CD40L, prepared as a soluble fusion protein, as described previously (40), and anti-CD8 reagent were generously provided by T. Rothstein (Boston University Medical School, Boston, MA). Supernatants containing CD40L and anti-CD8 were used at optimal concentrations (1:10 and 1:80, respectively) determined on the basis of proliferative assays (40). The proteasome-specific inhibitor, Z-LLF-CHO, kindly provided by D. Anderson (Signal Pharmaceuticals, San Diego, CA), was dissolved in absolute ethanol and used at a concentration of 10 µM, except where indicated (26). To inhibit protein synthesis, cycloheximide was added at a concentration of 10 µg/ml.
RNA isolation and analysis
Cytoplasmic RNA was isolated from 2 x 106
cells/sample by lysis of the cells in 400 µl cold buffer A (10 mM
HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM
PMSF, and 0.25% Nonidet P-40) and phenol-chloroform extraction (41).
RNA samples (12 µg) were denatured and separated by electrophoresis
on a 1% formaldehyde-agarose gel and transferred to GeneScreen Plus
(DuPont NEN, Boston, MA). The RNA was cross-linked by UV irradiation,
stained with ethidium bromide, and hybridized to probes, labeled by
random priming, as described previously (3). Probes for IB
expression include the human IB- clone, pMT2T-IB- (20), and
the murine IB-ß clone, pCDNA3:
KpnI-BamHI (18).
Immunoblot analysis of IB proteins
Cells were washed twice in cold PBS and resuspended in cold 10
mM Tris, pH 7.6, 10 mM KCl, 5 mM MgCl2, 0.2 mM PMSF,
and 10 µg/ml leupeptin. After incubation on ice for 10 min, cells
were lysed by addition of Nonidet P-40 to 0.5%. Nuclei were removed by
centrifugation, and protein content of cytoplasmic extracts was
quantitated using Bio-Rad Dc protein assay kit. Cytoplasmic proteins
(30–40 µg/sample) were subjected to electrophoresis on a 10%
polyacrylamide-SDS gel and electrotransferred to polyvinylidine
difluoride membrane (Millipore). Western analysis was done on the
resulting blot, as described previously (5), using an Ab specific for
IB- (SC 371) or IB-ß (SC 945) from Santa Cruz Biotechnology
(Santa Cruz, CA). The secondary Ab, horseradish peroxidase-labeled
rabbit anti-sheep (Bio-Rad, Hercules, CA), was visualized using
enhanced chemiluminescence, as published (42). Densitometry was
performed using a Molecular Dynamics 300A computing densitometer
(Sunnyvale, CA). Logarithmic regression analysis was used to calculate
the t1/2.
Microinjection analysis
WEHI 231 cells were allowed to attach to tissue culture plastic
in the presence of culture medium containing 0.4% FBS and supplemented
with 20 mM HEPES, pH 7.3. After 30-min incubation at 37°C, all cells
in a circled area (approximately 200) were microinjected with 1
µg/µl IB--glutathione S-transferase (GST) or GST protein, or
1 µg/µl wild-type or A32/A36 dominant negative mutant IB-
expression vector (26) in 130 mM KCl, 10 mM sodium phosphate, pH 7.3,
as we have described previously (3). Successful microinjection was
estimated to occur more than 90% of the time. Following
microinjection, cloning rings were placed over the microinjected areas,
and the medium was replaced with 10% FBS-DMEM. After 30-min incubation
at 37°C, cells that had survived the microinjection were detached
from the tissue culture plates by gentle trituration, transferred to
multiwell plates, and incubated at 37°C. Where indicated, cells were
treated with CD40L, anti-IgM, or anti-IgM plus CD40L, as above.
After the indicated times, one-tenth volume of a trypan blue solution
(0.04% final) was added to the well, cells were incubated for 15 min,
and cell counts were obtained.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B- and IB-ß degradation in WEHI 231
B cells
The IB- and IB-ß proteins appear to have distinct roles
in the regulation of the NF-B/Rel family of transcription factors
following CD40L treatment, as judged by the different effects on the
patterns of their expression. To begin to assess the regulation of
IB levels in WEHI 231 cells, the t1/2 of
these two proteins were first measured in exponentially growing cells.
Cultures were treated with 10 µg/ml cycloheximide, and cells were
harvested over a 60-min time period. Cytoplasmic extracts were prepared
and samples were subjected to immunoblot analysis (Fig. 1
). A fairly rapid degradation of the
IB- protein was observed (Fig. 1a). To
quantitate the t1/2 of decay, autoradiograms
were subjected to densitometric scanning and the data were analyzed by
logarithmic regression. A t1/2 for IB- of
38 min was calculated from this and five similar experiments (Table I). This value is consistent with results
of others who have shown that IB- protein has a relatively short
t1/2 in WEHI 231 cells (31). A similar time
course experiment was performed for IB-ß, and a slower rate of
decay was indicated (Fig. 1B). Analysis of data from
this and five other similar experiments yielded a value of 76 min for
the t1/2 of IB-ß in exponentially growing
WEHI 231 cells (Table I).
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B proteins are degraded through the proteasome pathway in
exponentially growing WEHI 231 cells
Recent evidence has shown that the degradation of IB-
following TNF- treatment of Jurkat or HeLa cells occurs through the
proteasome pathway (23, 26). To determine whether the proteasome
pathway is involved in degradation of IB- and IB-ß in
exponentially growing WEHI 231 B cells, Z-LLF-CHO, a specific inhibitor
of proteasome-mediated degradation, was used (43). To confirm that the
dose used by others was effective in our cells, we tested the effects
of concentrations ranging from 0.1 to 100 µM. As seen in Figure 2, a dose of 10 µM was very effective
at blocking the turnover of IB- and IB-ß. In contrast,
treatment with 100 µM Z-LLF-CHO resulted in decreased levels of
proteins, suggesting toxicity with this high dose. Therefore, 10 µM
Z-LLF-CHO was used in subsequent experiments. Cells were pretreated for
60 min with Z-LLF-CHO before cycloheximide addition, and cytoplasmic
proteins were isolated at the indicated times and subjected to
immunoblot analysis (Fig. 3). The normal
decay of both IB- and IB-ß proteins appeared completely
blocked in the presence of Z-LLF-CHO. In contrast, addition of the
calpain pathway-specific inhibitor, E64, had no significant effect on
the decay of these proteins (data not shown). Thus, the constitutive,
rapid rate of degradation of IB- and IB-ß proteins in WEHI
231 exponentially growing cells is dependent upon the proteasome
pathway, and accounts for the high levels of NF-B/Rel levels.
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B proteins
As discussed above, previously we have shown that following 1
h of anti-IgM treatment of exponentially growing WEHI 231 cells,
there is a transient increase in binding of p50/c-Rel and p50/p65
complexes, which declines precipitously by 4 h to levels at or
below those seen in untreated cells (11). Thus, we next addressed
whether the decrease in NF-B/Rel binding following 4 h of
anti-IgM treatment related to altered IB protein stability.
Cultures of exponentially growing WEHI 231 cells were incubated in the
presence of anti-IgM for 4 h and then treated with
cycloheximide. Cytoplasmic extracts were prepared at the indicated
times and analyzed by immunoblotting for IB- and IB-ß
proteins (Fig. 4). The rate of decay of
both inhibitor proteins was greatly decreased. Values of 73 and 184 min
for IB- and IB-ß, respectively, were obtained in this and a
duplicate experiment (Table I
). These findings suggest that the
decrease in NF-B/Rel binding is due in part to decreased rate of
turnover of these inhibitor proteins.
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B- protein
Compared with the effects of anti-IgM alone, costimulation of
cells with anti-IgM and CD40L for 1 h induces a much higher
level of binding of c-Rel- and p65-containing complexes and leads to
subsequent maintenance of binding above baseline levels even after
12 h, in particular of complexes containing c-Rel (5). Since CD40L
and anti-IgM costimulation of WEHI 231 cells causes a rapid drop in
IB- protein expression, which remained depressed over a 12-h time
period (5), we first sought to determine whether CD40L treatment
altered the stability of the IB- protein. WEHI 231 cells were
treated concurrently with CD40L and cycloheximide. Cytoplasmic extracts
were made after 20, 40, and 60 min, and samples were subjected to
immunoblot analysis (Fig. 5A). After 20 min of
incubation, IB- protein was undetectable. Thus, CD40L treatment
led to a significantly more rapid decay of this inhibitory protein. To
more specifically determine the rate of decay, samples were harvested
after 2.5, 5, 7.5, and 10 min of incubation with CD40L and
cycloheximide (Fig. 5B). IB- was no longer
detectable by 10 min of CD40L treatment, and densitometric analysis
indicated a drastically reduced IB- t1/2
of approximately 2 min following CD40L treatment (average of two
experiments) (Table I). Upon costimulation of CD40L with anti-IgM
for 4 h (Fig. 5C), one finds a rate of decay
that is much more rapid than seen with the anti-IgM above (Fig. 4),
although somewhat tempered compared with CD40L alone. Thus, the effects
of CD40 signaling predominate.
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B- protein was a
transient or a long-lasting effect of CD40L treatment, cells were
pretreated with CD40L for 12 h, cycloheximide was added, and
cytoplasmic extracts were subjected to immunoblot analysis (Fig. 5D). A slight lengthening of the
t1/2 was observed. In two experiments, an
average t1/2 of approximately 8 min was measured
for the IB- protein after 12 h of CD40L treatment (Table I).
Thus, CD40L has a long-lasting effect on protein
t1/2, which most likely explains the extended
decline in levels of this inhibitory protein following engagement of
the CD40 receptor.
CD40L induces rapid turnover of IB-ß protein
We next assessed whether the early effects of CD40L treatment on
IB-ß levels could be related to increased protein turnover.
Initially, the shortest length of cycloheximide treatment used was
1 h. By this time, however, the IB-ß protein was no longer
detectable (data not shown). Therefore, samples were taken over a time
course experiment that spanned 1 h (Fig. 6A). Within 20 min,
IB-ß protein had decayed to undetectable levels. Therefore, we
harvested cytoplasmic protein between 0 and 20 min after CD40L and
cycloheximide treatment (Fig. 6B). IB-ß decayed
with a t1/2 of approximately 5 min in WEHI 231
cells upon stimulation with CD40L (Table I). To determine whether this
rapid turnover of IB-ß was maintained at the later times, the
t1/2 was measured after 12 h of CD40L
treatment, and an average t1/2 of 15 min was
obtained in two experiments (Table I). Again, the effects of CD40L
treatment were dominant over those of anti-IgM when a 4-h
costimulation assay was performed (Fig. 6C). Thus,
CD40L causes a dramatic increase in the rate of decay of IB-ß over
an extended period of time.
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B degradation through the proteasome pathway
We next assessed whether CD40L treatment further promotes
degradation through the proteasome pathway. Cells were pretreated with
10 µM Z-LLF-CHO for 1 h, and then CD40L and cycloheximide were
added concurrently and cytoplasmic extracts were analyzed for IB-
protein. Only incomplete protection was observed, and an intermediate
rate of degradation was obtained (t1/2 of
18 min) (data not shown). However, as CD40L treatment had increased the
rate of protein turnover so dramatically, we were concerned that a
higher concentration of inhibitor might be needed to completely block
the degradation. Thus, cells were pretreated with 10, 20, 30, or 40
µM Z-LLF-CHO, then CD40L and cycloheximide were added and cytoplasmic
extracts were prepared after 1.5 h. As shown in Figure 7, treatment with 30 µM allowed for
essentially complete protection of IB-, although some smaller
peptides were seen, which may represent partial degradation products.
For IB-ß, 10 µM Z-LLF-CHO protected over the 1.5-h time period.
These results demonstrate that the CD40L-induced degradation of both
proteins is through the proteasome pathway.
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B-ß mRNA and a transient
increase in IB- mRNA levels
Previously we demonstrated IB-ß protein expression decreased
well below basal values after 1 h of CD40L treatment, but then
slowly returned to basal levels by 12 h (5). The slight increase
in protein stability did not appear able to account completely for the
observed increase in protein levels. Furthermore, the stability of the
IB- protein appeared to increase without a concomitant increase
in protein levels. Thus, we measured the effects of CD40L treatment on
mRNA expression of these inhibitors using Northern blot analysis.
Exponentially growing WEHI 231 cells were either left untreated or
treated with CD40L for 1, 3, 5, and 12 h. Total mRNA was extracted
and subjected to Northern blot analysis (Fig. 8). A dramatic increase in IB- mRNA
levels was seen at 1 h, which dropped slowly over the duration of
the time course (Fig. 8a). The rapid increase in
IB- mRNA most likely reflects enhanced transcription of the
IB- gene due to the significant increase in NF-B/Rel activity
seen by 1 h of CD40L stimulation (5), as the IB- promoter
has been shown to be transcriptionally activated by NF-B (19). The
effects of this increase are clearly offset by the dramatic increase in
the rate of degradation of the protein. Furthermore, the later decrease
in mRNA levels most likely compensates for the slight increase in
protein stability seen at the later times. IB-ß mRNA levels were
unchanged after 1 h of treatment, and increased following 3 h
of treatment, staying at this level for the remainder of the time
course (Fig. 8B). Thus, the delayed increase in
expression of IB-ß protein following CD40L treatment can be
accounted for, in part, by concurrent changes in mRNA levels, in
conjunction with a slight increase in protein turnover.
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B modulation in CD40L-mediated rescue
If the increase in the rate of degradation of IB proteins in
the cytoplasm by the proteasome mediates rescue by CD40L of
receptor-mediated apoptosis, then one would predict that cell death
induced by introduction of IB- protein directly into the nucleus
would not be similarly overridden. Thus, we next tested directly the
ability of CD40L to rescue WEHI 231 cells from apoptosis induced by
microinjection of IB--GST protein into the nuclear compartment
(Fig. 9A). As expected,
a significant level of apoptosis was induced in cells 20 h
following microinjection of IB--GST, as judged by trypan blue
positive staining, but not following microinjection with GST, or in
cells that were not microinjected. Costimulation with CD40L was unable
to protect WEHI 231 cells that had been microinjected with
IB--GST protein from apoptosis (Fig. 9A).
Furthermore, microinjection of nuclei with IB- protein promoted
receptor-mediated apoptosis of WEHI 231 cells, and CD40L was similarly
unable to protect these cells from death signals (Fig. 9B). In contrast, CD40L could afford significant
protection from anti-IgM-induced cell death alone, as expected
(Fig. 9B). Thus, overexpression of IB- protein
within the nucleus abrogates CD40L-mediated protection from
apoptosis.
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B- protein
if present in the cytoplasmic compartment and in a
proteasome-degradable form, WEHI 231 cells were microinjected with
vectors expressing IB- protein in either a wild-type or a mutant
form, i.e., with serine to alanine conversions at positions 32 and 36,
such that the protein cannot be phosphorylated. In the case of an
introduced expression vector, the resultant IB- protein is
synthesized on polyribosomes, and therefore localized to the cytoplasm;
in this compartment, the wild-type protein can be phosphorylated and
degraded by the proteasome pathway, but the 32A/36A mutant IB-
cannot (26). Costimulation with CD40L provided significant survival
signals to WEHI 231 cells microinjected with a vector leading to
expression of wild-type IB- protein (Fig. 9C).
In contrast, cell death upon expression of the mutant IB- protein
could not be rescued by CD40L costimulation. Thus, the effects of CD40L
on IB protein can account for much of the survival signals mediated
by this factor. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel expression due, at least in part, to an early and sustained
drop in IB- and a transient drop in IB-ß protein levels (5).
In this study, we demonstrate these changes in IB protein levels are
due to a coordinate decrease in protein stability through the
proteasome pathway and differential regulation of pretranslational
events. Furthermore, CD40L-mediated induction of IB protein
degradation is pivotal to its ability to rescue WEHI 231 cells from
apoptosis. Treatment with anti-IgM for 4 h led to
stabilization of the labile IB- and IB-ß proteins, which is
most likely responsible for the drop in NF-B/Rel binding seen after
these later times. (A similar increase in stability was also seen at
6 h of anti-IgM treatment; data not shown.) In contrast, CD40L
treatment of WEHI 231 cells caused a rapid dramatic increase in the
rate of proteasome-mediated degradation of both IB-
(t1/2 = 38 min in untreated cells vs 2
min following CD40L treatment) and IB-ß
(t1/2 = 76 min vs 5 min). This dramatic
increase in the rate of IB protein turnover overrode the effects of
anti-IgM and was presumably preceded by hyperphosphorylation,
although changes in phosphorylation of murine IB species cannot be
readily detected in one-dimensional gel electrophoresis (20).
Furthermore, microinjection analysis indicated that, in large part,
CD40L survival signals were mediated through the decreased expression
of IB protein levels. Recent studies have demonstrated a profound
role of NF-B/Rel activity in promoting survival of cells from
apoptosis induced by anti-IgM (3), TGF-ß1 (4, 44),
IB- (45), or TNF- (46, 47, 48). The findings presented in this
work suggest that the dramatic decrease in IB protein stability
induced by the engagement of CD40 receptor is responsible for the
maintenance of high levels of NF-B/Rel activity, and thus the
continued expression of signals crucial for promoting survival of these
B cells.
Overall, the changes in stability for both inhibitory proteins occurred
in an essentially equivalent fashion. At later times, an increase in
the rate of protein degradation was still apparent, although turnover
was somewhat attenuated after 12 h of CD40L treatment
(t1/2 = 8 min and 15 min, respectively,
for IB- and IB-ß). Importantly, for IB- expression,
the later restoration of protein stability was counterbalanced by a
decrease in the early activation of mRNA levels, resulting in
maintenance of low IB- levels over the entire time course. In
contrast, a delayed and sustained increase in IB-ß mRNA levels was
observed. Thus, enhanced stability and rate of synthesis promote a
return to basal levels of expression of this inhibitory protein.
The CD40L molecule has been shown to associate with a number of
intracellular molecules, including TRAF-2, TRAF-3, and TRAF-5 (49, 50, 51, 52).
Overexpression of TRAF-2 or TRAF-5 has been shown to activate NF-B,
although the intermediate effectors have not been identified (51, 52).
While the precise nature of association remains unclear, the evidence
suggests that they are involved in the recruitment and activation
(either directly or indirectly) of downstream messengers. Some of these
messengers found to be activated in various human B cell lines after
CD40L treatment include the src kinase lyn,
phospholipase C-
1, phosphatidylinositol-3 kinase, and
protein kinase C (13, 53). CD40 stimulation has also been shown to
activate the c-jun kinase (JNK), a member of the
mitogen-activated protein kinase family, Bcl-XL, and
members of the STAT family (54, 55, 56, 57). Our results, however, indicate
that the ability of CD40L to induce degradation of IB protein is
critical to rescue of WEHI 231 cells from receptor-mediated apoptosis.
Furthermore, the inability of CD40L to override cell death mediated by
the mutant IB- protein indicates the importance of
phosphorylation of serine residues at positions 32 and 36. A large,
multisubunit kinase, containing IKK- and IKK-ß, has been
implicated in the phosphorylation of serine residues 32 and 36 of
IB- and 19 and 23 of IB-ß proteins, and is a likely
candidate as the kinase involved in the targeting of either one or both
IB molecules for degradation in the WEHI 231 cell line (58, 59, 60, 61, 62, 63).
Clearly, there is at least some portion of the proteasome pathway that
is common to the degradation of IB- and IB-ß, given their
sensitivities to the proteasome inhibitor. Additionally, recent data
suggest that at least a portion of the signaling path leading to the
degradation of IB-, IB-ß, and IB-
in 70Z/3 cells is
common to all three, and most likely includes the multisubunit kinase
IKK, or the upstream mitogen-activated protein 3 kinase-like molecule
NF-B-inducing kinase (NIK) (64, 65). A number of proteins have been
shown to be dependent upon the proteasome pathway, including cyclins
and p53 (66, 67). Yet, the degradation of these proteins are highly
regulated, carefully timed events, each displaying diverse patterns of
activation and degradation. Differences must clearly exist between
these proteins; the work shown in this study demonstrates this for the
two IB proteins, IB- and IB-ß, each with different rates
of degradation. This could be due to a number of factors, including
phosphorylation and/or ubiquitination, or even the use of different
subunits in the make-up of the proteasome. Additionally, the position
and sequence surrounding the sites of phosphorylation and
ubiquitination of the proteins themselves are dissimilar and may also
contribute to the differences in degradation. This is substantiated by
recent evidence that IKK- phosphorylation of IB-ß is less
efficient than IB- (61). The data in this study demonstrate that
for B cell survival, engagement of another member of the TNFR family,
CD40L, uses the mechanism of protein degradation to affect IB
protein levels, thereby allowing the activation of NF-B/Rel required
for rescue of WEHI 231 cells from apoptosis.
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Acknowledgments |
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B- protein
and expression vectors. |
Footnotes |
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2 Current address: Harvard Medical School, 181 Longwood Avenue, MCP 8, Boston, MA 02115.
3 Address correspondence and reprint requests to Dr. Gail E. Sonenshein, Department of Biochemistry, Boston University Medical School, 80 East Concord Street, Boston, MA 02118-2394. E-mail address:
4 Abbreviations used in this paper: CD40L, CD40 ligand; GST, glutathione S-transferase; IB, inhibitor B; IKK, IB kinase; TRAF, tumor necrosis factor receptor-associated factor; Z-LLF-CHO, (benzylcarbonyl)-Leu-Leu-phenylalaninal.
Received for publication June 26, 1997. Accepted for publication December 29, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel induces apoptosis of murine B cells. EMBO J. 15:4682.[Medline]
B/Rel activity inducing apoptosis of B cells: transcriptional activation of IB. Immunity 5:31.[Medline]
B/Rel and c-myc expression during CD40 ligand rescue of WEHI 231 early B cells from receptor-mediated apoptosis through modulation of IB proteins. J. Immunol. 157:81.[Abstract]
B: structure-function relationship of its protein subunits. Biochem. J. 290:297.
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immunoglobulin enhancer-binding protein NF-B by a posttranslational mechanism. Cell 47:921.[Medline]
B-like activity. Cell 65:1281.[Medline]
Bß regulates the persistent response in a biphasic activation of NF-B. Cell 80:573.[Medline]
B controls expression of inhibitor IB: evidence for an inducible autoregulatory pathway. Science 259:1912.B proteolysis by site-specific signal-induced phosphorylation. Science 267:1485.B phosphorylation marks the NF-B inhibitor for degradation via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA 92:10599.B requires site-specific ubiquitination. Proc. Natl. Acad. Sci. USA 92:11259.B and stabilizes a newly phosphorylated form of IB that is still bound to NF-B. EMBO J. 13:5433.[Medline]
B to the ubiquitin-proteasome pathway. Genes Dev. 9:1586.B phosphorylation sites that signal its ubiquitination and degradation. Mol. Cell. Biol. 16:1295.[Abstract]
B interacts with the nuclear localization sequences of the subunits of NF-B: a mechanism for cytoplasmic retention. Genes Dev. 6:1899.B p50 precursor are controlled by a small region in its C-terminus. EMBO J. 10:4159.[Medline]
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B degradation is responsible for constitutive NF-B activity in mature murine B-cell lines. Mol. Cell. Biol. 14:3276.B, AP-1 and NF-AT during B cell stimulation through the CD40 receptor. Int. Immunol. 7:151.B blocks transforming growth factor ß1-induced apoptosis of murine hepatocyte cell lines. Cell Growth Differ. 8:1049.[Abstract]
B activity induces apoptosis in murine hepatocytes. Am. J. Pathol. 151:891.[Abstract]
B in preventing TNF--induced cell death. Science 274:782.-induced apoptosis by NF-B. Science 274:787.B. Science 274:784.B by TNF receptor 2 and CD40. Science 269:1424. activation site. J. Immunol. 157:39.[Abstract]
B by a novel ubiquitin-dependent protein kinase activity. Cell 84:853.[Medline]
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B kinase. Cell 90:373.[Medline]
B kinase complex (IKK) contains two kinase subunits, IKK and IKKß, necessary for IB phosphorylation and NF-B activation. Cell 91:243.[Medline]
B in response to multiple stimuli. Mol. Cell. Biol. 17:1441.[Abstract]
B induction by TNF, CD95 and IL-1. Nature 385:540.[Medline]
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