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Mediates a Selective T Cell Survival Signal Via Phosphorylation of BAD1
Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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plays a selective and important
role in this protection. Fas triggering led to a selective
caspase-3-dependent cleavage of the enzyme and proteasome-mediated
degradation and inactivation of its catalytic fragment. These events
preceded the onset of apoptosis. Pharmacological inhibition of PKC
promoted Fas-mediated apoptosis in three different types of T cells.
Conversely, constitutively active PKC (and, to a lesser degree,
PKC
) selectively protected T cells from Fas-induced apoptosis. We
provide evidence that the distant Bcl-2 family member, BAD, is a PKC
substrate, is phosphorylated by TCR stimulation, and can mediate at
least in part the anti-apoptotic effect of
PKC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR engagement induces activation of protein kinase C (PKC), a family
of Ser/Thr kinases that consists of three subfamilies: cPKC (or
conventional PKCs: 
, 
1, 2, and 
), aPKC (or atypical PKC:
and 
), and nPKC (or novel PKCs: 
, , , and µ).
Phorbol ester stimulation, which activates PKC, protects various cells,
including T cells, from apoptosis (1, 2, 3, 4, 5). However, the PKC
isoform(s) involved in this process has not been definitively
identified in T cells.
Among T cell-expressed PKC enzymes, PKC plays a particularly
important role in TCR/CD28 signaling pathways (6). PKC
was isolated as an nPKC isoform expressed selectively in T cells
(7). It displays several unique functions in T cells,
including its ability to activate the c-Jun N-terminal kinase
(JNK)/AP-1 pathway and induce the IL-2 gene in synergy with calcineurin
(8, 9, 10), and its translocation to the site of cell contact
between Ag-specific T cells and APCs, where it colocalizes with the TCR
to form the central core of the T cell supramolecular activation
cluster (11). PKC translocation is mediated by a
Vav-regulated process that also involves Rac and the cytoskeleton
(12). In addition, we recently found that CD28
costimulation promotes TCR-induced PKC translocation and activation.
PKC then activates NF-
B and the CD28 response element in the IL-2
gene promoter (13, 14), providing evidence that PKC is
functionally coupled to TCR/CD28 costimulation. The importance of
PKC in T cell activation is indicated by the findings that
PKC-deficient mature T cells display a severe activation defect in
response to TCR/CD28 ligands (15).
In this work, we analyzed the role of PKC function in TCR- and/or
PMA-induced survival signals that protect T cells from Fas-induced
apoptosis. We show that activated PKC provides a protective survival
signal to T cells, which can be accounted for, in part, by its ability
to phosphorylate BAD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein A/G Plus-agarose was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). mAbs generated against human PKC
(aa270–427), PKC (aa114–289), PKC (aa1–175), PKC
(aa21–217), and PKC (aa94–590) were obtained from
Transduction Laboratories (Lexington, KY). The anti-actin mAb was
obtained from ICN Pharmaceuticals (Aurora, OH), and anti-human or
anti-mouse Fas mAbs were obtained from Upstate Biotechnology (Lake
Placid, NY) or BD PharMingen (San Diego, CA), respectively. PD98059 and
polyclonal Abs against native BAD or phospho-BAD
(Ser112 or Ser136) were
obtained from New England Biolabs (Beverly, MA). The anti-human
CD28 and CD3 mAbs and PE- or FITC-conjugated annexin were purchased
from BD PharMingen. Donkey anti-rabbit or sheep anti-mouse IgG
Abs and GST-Sepharose 4B were obtained from Amersham (Piscataway, NJ),
and the anti-Xpress tag mAb was purchased from Invitrogen (San
Diego, CA). An anti-14-3-3 Ab that recognizes 14-3-3
and
14-3-3
isoforms has been described previously (16).
Z-Val-Ala-Asp-CH2F (Z-VAD) was obtained from
Kamiya Biomedical (Seattle, WA). SB203580 was a gift from SmithKline
Beecham Pharmaceuticals (Philadelphia, PA). LY294002, wortmannin,
MG132, rottlerin, and Gö6976 were purchased from
Calbiochem (San Diego, CA). The IB phosphorylation inhibitor BAY
11-7082 was purchased from Biomol (Plymouth Meeting, PA). All other
reagents were obtained from Sigma (St. Louis, MO).
Plasmids and NF-B reporter assay
The cDNAs encoding Xpress epitope-tagged human wild-type PKC
and PKC, the corresponding constitutively active (A/E) mutants, and
the dominant negative (kinase-inactive; K/R) mutants of these PKCs as
well as rat PKC and mouse PKC in the eukaryotic expression vector
pEFneo have been described previously (12, 13, 17). A
green fluorescent protein (GFP) expression plasmid in the pEF
vector was obtained from Dr. G. Baier (University of Innsbruck,
Innsbruck, Austria). The cDNAs encoding wild-type or dominant negative
(SS32/36AA) IB in the eukaryotic expression vector pCMV4 were
obtained from Dr. W. C. Greene (University of California, San
Francisco, CA). The mammalian expression plasmid encoding murine BAD
(pEBG-mBAD) was acquired from New England Biolabs. The BAD-coding
region was excised by double digest with BamHI and
NotI and ligated into the corresponding sites in the
bacterial expression vector pGEX-5X-1 (Pharmacia, Kalamazoo, MI). The
resulting plasmid was used to transform Escherichia coli
BL21. Cultures were grown to log phase, induced with 0.5 mM isopropyl
-D-thiogalactoside, and GST-BAD was isolated on
GST-Sepharose 4B. The NF-B-luciferase reporter plasmid and the
method for assaying its activity have been described previously
(13).
Cell culture and transfection
Jurkat E6.1 T cells, SV40 large T Ag-transfected human leukemic Jurkat T (Jurkat-TAg), and A1.1 T hybridoma cells were grown in RPMI 1640 medium (Life Technologies, Gaithersburg, MD), supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 1x MEM nonessential amino acid solution (Life Technologies), and 100 U/ml each of penicillin G and streptomycin. Cells in a logarithmic growth phase were transfected with the indicated amounts of plasmid DNAs by electroporation as described previously (12, 17). Human PBMC were prepared from healthy volunteers by standard Ficoll-Hypaque centrifugation and cultured in the presence of an activating anti-CD3 mAb (OKT3; 1 µg/ml) plus recombinant human IL-2 (20 U/ml) for 6 days. Where indicated, the cells were pretreated with the caspase-3 inhibitor Z-VAD, PKC inhibitors (rottlerin or Gö6976), or the proteasome inhibitor MG132.
Immunoprecipitation and immunoblotting
Cells were lysed in 1 ml of lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 5 mM sodium pyrophosphate, 1 mM Na3VO4 plus 1% Nonidet P-40, and 10 µg/ml each of aprotinin and leupeptin) for 10 min on ice. After centrifugation (16,000 x g, 10 min, 4°C), the supernatants were incubated with optimal concentrations of Abs for 1 h at 4°C, followed by 30 µl of protein A/G Plus-agarose and overnight incubation at 4°C; for GST-BAD precipitation, they were incubated with GST-Sepharose 4B for 1 h at 4°C. Samples were washed four times in lysis buffer, and precipitates were dissolved in Laemmli buffer and resolved by SDS-PAGE. Electrophoresed samples were processed for immunoblot analysis as previously described (12, 17).
Induction and assessment of apoptosis
Cells were induced to undergo apoptosis by treatment with an anti-human or anti-mouse Fas mAb (50 ng/ml) or by serum deprivation. Apoptosis was assessed by staining the cells using annexin V-FITC or annexin V-PE and analyzing them on a FACScan analyzer following the manufacturer’s instructions. In experiments involving pretreatment with PKC inhibitors or Z-VAD, apoptosis was determined by propidium iodide (PI) staining (17). In experiments involving PBLs, the cellular shrinkage associated with apoptosis was also assessed by the decrease in light scatter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA-damaging agents induce selective cleavage of PKC in U937
cells, and this caspase-3-induced event has been linked to activation
of PKC and induction of apoptosis (18). We determined
whether Fas ligation also causes PKC cleavage in T cells. Treatment
of Jurkat T cells with an anti-Fas mAb induced cleavage of PKC,
as indicated by the time-dependent decrease in the expression of intact
PKC and the corresponding increase in the level of its regulatory
fragment (Fig. 1
a,
top panel). This effect was selective, because
PKC remained intact during this process (Fig. 1a,
middle panel; see also Fig. 3b), even at times
when 80% of the cells were annexin positive (data not shown; Fig. 4).
As a control, actin also remained intact up to 24 h after
anti-Fas treatment (Fig. 1, a and b,
bottom panels). PKC cleavage (Fig. 1b,
top panel) and cell death (Fig. 1c) were blocked
in parallel by the caspase-3 inhibitor Z-VAD, suggesting that
Fas-induced PKC cleavage is mediated by caspase-3. The caspase-3
substrate poly(ADP-ribose) polymerase displayed a kinetically
similar cleavage to that of PKC under the same conditions (data not
shown).
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correlated with loss of its
catalytic fragment, as indicated by immunoblotting with a
C-terminal-specific Ab (Fig. 1
d). This loss suggested that
Fas ligation induces degradation and subsequent inactivation of the
catalytic fragment of PKC. This possibility was analyzed by
assessing the effect of a selective proteasome inhibitor, MG132, on the
Fas-mediated change in PKC expression. MG132 pretreatment inhibited
the degradation of PKC, resulting in the appearance of an intact
catalytic fragment, which was detectable even 24 h after Fas
ligation (Fig. 1d). Thus, Fas triggering appears to induce
selective, proteasome-mediated degradation of the catalytic fragment of
PKC, subsequent to the caspase-mediated cleavage of the
holoenzyme.
PKC induces a selective anti-apoptotic effect in T cells
The Fas-induced degradation and inactivation of PKC raised the
possibility that PKC mediates a survival signal that is ablated
following its degradation, thereby allowing the Fas-associated
apoptotic machinery to become fully functional. We initially used a
pharmacological approach to address this question. We previously
demonstrated that rottlerin blocked PKC activity, but not the
activity of Ca2+-dependent PKC
(17). We took advantage of this property to demonstrate
the role of PKC in regulation of FasL expression (17)
and activation of NFAT (12) or NF-B (13).
Thus, we evaluated the effects of rottlerin pretreatment on
Fas-mediated apoptosis.
In untreated cells, anti-Fas induced a relatively weak apoptosis
after short periods, which was first observed after about 3
h and reached a level of 20–25% above background at 6 h (Fig. 2a). Rottlerin pretreatment
alone induced a slight apoptotic response. However, the combination of
rottlerin pretreatment and Fas ligation induced massive apoptosis that
reached a level of 50–60% after 4 h. The concentration of
rottlerin used in this experiment was previously found to inhibit the
anti-CD3/CD28-induced activation of PKC in T cells
(13). For comparison, we tested in a similar experiment
the effects of Gö6976, a PKC inhibitor selective for
Ca2+-dependent PKCs (19), which did
not inhibit PKC. In contrast to rottlerin, Gö6976 did not have
any detectable effect on the basal or anti-Fas-induced apoptosis
(Fig. 2b), even when used at concentrations that completely
blocked the activity of PKC.
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c), which have often been used
as a model of AICD, and anti-CD3-activated human peripheral blood T
cells (Fig. 2d). We observed an effect similar to that
obtained in Jurkat cells, i.e., a synergy between anti-Fas and
rottlerin in the induction of apoptosis. The relatively low level of
apoptosis displayed by activated normal T cells (Fig. 2d) is
consistent with other studies (20). Because rottlerin was
originally found to inhibit PKC (21), we examined the
expression of this isoenzyme in the different T cells. The results
(Fig. 2e, top panel) demonstrate that Jurkat
cells of three different origins and 8-day anti-CD3-activated
(i.e., T cell-enriched) human PBLs expressed barely detectable levels
of PKC. In contrast, fresh PBLs, a B human lymphoma line (Dakiki),
or a cerebellum extract readily expressed detectable PKC. PKC
displayed a reciprocal expression pattern (Fig. 2e,
middle panel).
We also confirmed earlier findings (1, 2, 3, 4, 5) by showing that
pretreatment of Jurkat T cells with PMA (which activates cPKC and nPKC
enzymes) protected the cells from Fas-induced apoptosis, as indicated
by the 
70% reduction in the level of apoptotic cells (Fig. 2f). In contrast, a calcium ionophore, ionomycin, did not
modulate the apoptotic response in the absence or the presence of PMA.
In parallel experiments, we found that costimulation with PMA plus
ionomycin was required to induce IL-2 production by the same cells
(data not shown). Because the aPKC subfamily does not respond to PMA,
and only the cPKC subfamily requires Ca2+ for
full activation, these findings suggest that the PKC isoform involved
in T cell survival most likely belongs to the nPKC subfamily.
To more directly assess the role of PKC in mediating a T cell
survival signal, we transiently transfected Jurkat cells with
constitutively active mutants of different PKC isoforms and determined
their effects on Fas-mediated apoptosis. We previously showed that
these mutants are functional in T cells, and furthermore, they are
expressed at similar levels (12, 13, 17). The cells were
cotransfected with a GFP expression plasmid as a marker for the
selection of transfected cells. Only nPKC (i.e., and ) protected
cells from undergoing apoptosis following Fas ligation (Fig. 3a), and PKC was more
active than PKC. PKC and PKC were essentially inactive.
Moreover, in agreement with Fig. 1, Fas engagement induced a selective
loss of PKC (Fig. 3b). PKC and PKC remained intact
during the whole period of treatment, and the level of PKC started
to decrease after a long period of incubation (20 h) and, to a small
extent, by comparison with PKC. These data show that PKC protects
T cells from Fas-induced apoptosis, and that PKC is selectively
cleaved after Fas engagement.
To study the role of the catalytic activity of PKC in this
protective effect, we used different forms of PKC plasmids (Fig. 3c). Only the constitutively active mutant (A/E) conferred
effective protection. The kinase-inactive (K/R) mutant was completely
ineffective, and in fact, it increased the level of cell death. Minimal
protection was observed with wild-type PKC. However, PMA treatment
of cells transfected with wild-type PKC induced greater protection
compared with empty vector-transfected cells (data not shown). These
results suggest that PKC must be activated to protect T cells from
apoptosis. The next series of experiments was undertaken to elucidate
the protective pathway activated by PKC.
Role of NF-B in the PKC-mediated survival signal
We recently found that PKC activates the NF-B cascade in T
cells (13). This transcription factor protects different
cell types from apoptosis (22). Therefore, we studied
whether NF-B is involved in the PKC-mediated survival signal.
Jurkat cells were cotransfected with constitutively active PKC (A/E)
together with wild-type IB or a dominant negative,
unphosphorylatable form of IB (SS32/36AA or IB-S/S). However,
under the same conditions, both wild-type and IB-S/S should block
the NF-B pathway by retaining NF-B in the cytoplasm and
preventing its nuclear translocation. Neither of these IB
plasmids had a significant effect on PKC-mediated protection
following Fas engagement (Fig. 4a). However, under the same
conditions, IB-S/S completely blocked the activation of a NF-B
reporter plasmid induced by PKC-A/E (Fig. 4b). We
confirmed these results by using MG132, which blocks NF-B activation
by preventing IB degradation in the proteosome. We have
previously shown that MG132 blocks PKC-induced NF-B activation in
T cells (13). MG132 had a small effect on the protective
signal provided by PKC, reducing it by 27 and 43% at concentrations
of 1 and 10 µM, respectively (Fig. 4c). However, at these
concentrations MG132 had a toxic effect, suggesting that the increase
in cell death in PKC-transfected cells may be due to its toxic
effect rather than inhibition of a PKC-mediated survival signal
(Fig. 4c). Similar results were obtained using an IB
phosphorylation inhibitor, BAY 11-7082 (data not shown), indicating
that IB degradation and the resulting activation of NF-B do not
play a major role in mediating the protective effect of PKC.
PKC induces activation of both extracellular signal-regulated
kinase and JNK in T cells (8, 9, 10). To study the roles of
these two pathways in PKC-mediated survival, we pretreated Jurkat
cells with different inhibitors. The mitogen-activated
protein/extracellular signal-regulated kinase kinase 1 inhibitor
PD98059 and the p38/JNK inhibitor SB203580, which was found to inhibit
only p38 at 4 µM and both p38 and JNK at 40 µM (23),
were both ineffective in reversing the PKC-mediated protective
signal (data not shown). Similarly, the phosphoinositol 3-kinase
inhibitors, LY294002 and wortmannin, as well as combinations of these
drugs did not have a significant effect on the ability of PKC to
protect the cells from Fas-mediated apoptosis (data not shown). These
negative results suggest an alternative pathway through which PKC
mediates its protective effect.
BAD is a substrate, and mediates the survival signal, of PKC
The finding that rottlerin synergizes with an anti-Fas Ab to
induce rapid and massive cell death suggested that this inhibitor
affects a post-transcriptional event mediated by PKC. The Bcl-2
family member, BAD, is phosphorylated by different kinases on three Ser
residues, i.e., Ser112,
Ser136, and Ser155. This
phosphorylation inhibits apoptosis by preventing BAD from translocating
to the mitochondria, where it binds to Bcl-2 or
Bcl-xL and inhibits their anti-apoptotic
activity (24). Therefore, we studied whether PKC could
play a role in the phosphorylation of BAD. These experiments were
performed using serum-starved cells to reduce the basal phosphorylation
of BAD. In unstimulated cells, basal BAD phosphorylation was observed
at both Ser112 and Ser136
(Fig. 5a). Costimulation with
anti-CD3 plus anti-CD28 Abs increased phosphorylation at both
residues, and this effect was essentially abolished by pretreatment
with the PKC inhibitor, rottlerin (Fig. 5a). That this
phosphorylation occurs at sites relevant for the anti-apoptotic
effect of phospho-BAD (24) is also supported by the
finding that BAD immunoprecipitated from anti-CD3-stimulated cells
was associated with increased levels of endogenous 14-3-3 (Fig. 6b).
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), we determined whether rottlerin
affects the basal phosphorylation of BAD in unstimulated cells. Fig. 5b shows that rottlerin pretreatment caused
dephosphorylation of endogenous BAD, mainly at
Ser136. This dephosphorylation was first detected
at 1 h and preceded cell death, consistent with the idea that this
event predisposes T cells to apoptosis. These results suggest that
basal PKC activity promotes cell survival by maintaining basal BAD
phosphorylation.
Next, we determined the effect of transfected constitutively active
PKC on the phosphorylation of BAD in serum-starved Jurkat
leukemia or A1.1 hybridoma cells. PKC-A/E induced BAD
phosphorylation in both Jurkat and A1.1 (Fig. 5c) cells.
This phosphorylation occurred on both Ser112 and
Ser136, and it was blocked by rottlerin.
We also tested the abilities of different PKC isoforms, i.e., and
, to phosphorylate BAD in vitro. For comparison, we also examined
the phosphorylation of myelin basic protein (MBP), a common PKC
substrate. PKC was 3-fold more effective than PKC in inducing
MBP phosphorylation (Fig. 5d, top panel).
Conversely, PKC was about 8-fold more effective than PKC in
phosphorylating recombinant GST-BAD (second panel from top).
The two kinases did not phosphorylate GST (data not shown). Thus, by
comparison with PKC, PKC appears to have considerable selectivity
for BAD. PKC phosphorylated BAD mainly at
Ser136 and, to a lesser degree, at
Ser112. In contrast, PKC did not induce
detectable phosphorylation of these serine residues. Therefore, it can
be concluded that PKC preferentially phosphorylates BAD at sites
that are relevant to the anti-apoptotic function of phospho-BAD.
The bottom panel shows that equal amounts of BAD were
present in all kinase reactions. We further studied this kinase
selectivity in vivo by comparing the effect of rottlerin with that of
Gö6976. Consistent with the results shown in Fig. 5, a
and c, rottlerin blocked the phosphorylation of
Ser136 and Ser112 in BAD
induced by anti-CD3/CD28 Abs, but it had no effect on serum-induced
phosphorylation (Fig. 5e). Gö6976, in contrast, was
incapable of blocking the same phosphorylation event. In agreement with
these results, Fig. 6a shows that a dominant negative PKC
mutant (PKC-K/R) blocked BAD phosphorylation induced by
anti-CD3.
Phosphorylated BAD is sequestered in the cytosol by binding to 14-3-3
proteins, so we next decided to study whether CD3 stimulation increases
the amount of BAD-associated endogenous 14-3-3 protein in T cells.
Under resting conditions, a small amount of 14-3-3 coimmunoprecipitated
with BAD. This amount was increased 8-fold by -CD3 (Fig. 6b). Similar amounts of BAD were present in all
immunoprecipitates (bottom panels).
The ability of PKC to phosphorylate BAD at
Ser136 and Ser112 and the
reported anti-apoptotic effect of phosphorylation at these sites by
other kinases (24, 25, 26, 27, 28, 29, 30) prompted us to examine whether
transient PKC overexpression can inhibit the ability of
overexpressed BAD to promote apoptosis. Compared with
vector-transfected cells, transfection with BAD induced a slight
increase in the basal or anti-Fas-induced apoptosis of the cells
(Fig. 6c). Although this increase was small, it was
consistently observed in three of three experiments. This increase was
reversed by coexpressed PKC-A/E; in addition, PKC inhibited by
70% the anti-Fas-induced apoptosis in cells that were not
transfected with BAD. We further analyzed the effect of PKC on death
induced by serum deprivation (Fig. 6d). Under these
conditions, the promoting effect of BAD on cell apoptosis was more
prominent. Thus, BAD increased the basal or serum starvation-induced
apoptosis of Jurkat cells by 160 and 97%, respectively. The
BAD-induced increase in cell death was still reversed by PKC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we demonstrate that, in T cells, PKC provides a survival
signal that protects the cells from Fas-mediated apoptosis. We also
show that Fas ligation causes caspase-mediated PKC cleavage and
proteasome-mediated degradation of the catalytic fragment. Finally, our
results show that the proapoptotic protein, BAD, is phosphorylated
under stimulation conditions that also activate PKC, i.e., combined
CD3/CD28 costimulation and, moreover, that BAD is a substrate for
PKC. These results are consistent with a very recent report showing
that PKC induces BAD phosphorylation through activation of Rsk
(33). It is possible that two alternative pathways, which
lead to BAD phosphorylation, are activated by PKC, but, depending on
BAD, Rsk, and PKC localization, one pathway would be predominant
over the other. This is consistent with the recent finding that BAD is
a PKC target in nonhemopoietic cells (29), although the
identity of the relevant PKC isoform(s) has not been established
in this study. Our results show that PKC transduces signals from
TCR/CD28 to BAD. Various studies demonstrated that CD28 costimulation
provides a survival signal that protects T cells from AICD
(34, 35, 36, 37, 38, 39, 40). This survival signal can be mediated by several
mechanisms, e.g., up-regulation of the anti-apoptotic protein
Bcl-xL and increased production of IL-2
(34, 35). The protective role of PKC revealed by the
current study implies an additional protective pathway associated with
CD28 costimulation and is consistent with our earlier findings that
PKC integrates signals from the TCR/CD3 complex and CD28
(13).
Several findings support the conclusion that PKC is the major PKC
isoform that protects T cells from Fas-mediated apoptosis and most
likely accounts for the anti-apoptotic effect of phorbol ester in T
cells. First, the protective effect of phorbol ester was not modulated
by ionomycin (Fig. 2f), suggesting that a member of
the novel Ca2+-independent PKC
subfamily is involved. Second, rottlerin, which selectively inhibits
PKC activity in vitro and in intact T cells (12, 13, 17), synergized with anti-Fas to induce cell death (Fig. 2, a, c, and d), whereas the cPKC
inhibitor, Gö6976, did not have any effect under similar
conditions (Fig. 2b). Third, PKC was more effective than
other PKC isoforms in protecting cells from Fas-mediated apoptosis
(Fig. 3a); although PKC also displayed a significant
protective effect. Fourth, a dominant negative PKC construct blocked
CD3-mediated BAD phosphorylation (Fig. 6a). Nevertheless, we
cannot rule out the possibility that other
Ca2+-independent PKCs, e.g., PKC, which is
relatively abundant in T cells, also play a protective role. This idea
is consistent with our observation, and in agreement with others
(33), that constitutively active PKC also protected T
cells from apoptosis, albeit less potently than PKC (Fig. 3a). However, PKC, which is also inhibited by rottlerin
(21), most likely has a minor, if any, role in this
process, because it was barely expressed in the cells used (Fig. 2e) (17).
Activated NF-B can protect cells from apoptosis (22).
Our experiments revealed that blocking of IB function by the
corresponding dominant negative mutant (Fig. 4a) or
inhibition of the proteasome machinery (Fig. 4c) did not
greatly interfere with the protective effect of PKC on Fas-mediated
apoptosis, suggesting that NF-B is not the major pathway involved in
this effect.
The function of BAD was mostly studied in systems in which apoptosis is
induced by growth factor deprivation of hemopoietic or neuronal cells
(25, 26, 41). The role of BAD in protecting lymphoid cells
from apoptosis induced by the interaction of death receptors with their
ligands (e.g., Fas/FasL) is less clear, particularly because these
cells express low levels of BAD (42). In addition, BAD
appears to regulate apoptotic events associated with mitochondria, and
it has been proposed that Fas engagement induces a cascade of events
that is independent of mitochondria (43). Thus, the idea
that phosphorylation of BAD is a potentially important mechanism for
suppression of apoptosis by Fas is antithetical to data suggesting that
Fas can induce apoptosis independent of the mitochondria-dependent
pathway governed by BAD and other Bcl-2 family proteins. However, it is
becoming increasingly clear that cross-talk exists between
mitochondria-dependent and death receptor-mediated apoptosis pathways
(43). Furthermore, BAD can cooperate with a Fas-mediated
signal to promote T cell death. For example, T cells derived from
BAD-transgenic mice are highly susceptible to apoptotic
stimuli, including Fas ligation (44). Moreover, the same
study demonstrated that Fas ligation induces marked up-regulation of
BAD expression in thymocytes. In addition, BID, a BH3
domain-containing proapoptotic Bcl-2 family member, is a specific
proximal substrate of caspase8 in the Fas and TNF- apoptotic
signaling pathways. Although full-length BID is localized in cytosol,
truncated BID (tBID) translocates to mitochondria and thus transduces
apoptotic signals from cytoplasmic membrane to mitochondria. tBID
induces first the clustering of mitochondria around the nuclei and
release of cytochrome c independently of caspase activity,
and then the loss of mitochondrial membrane potential, cell shrinkage,
and nuclear condensation in a caspase-dependent fashion. Coexpression
of Bcl-xL inhibits all the apoptotic changes
induced by tBID. Therefore, BID is a mediator of mitochondrial damage
induced by caspase-8 (45, 46, 47, 48). The finding that BAD acts
as a key regulator of T cell apoptosis and T cell development
(44) coupled with the selective expression of PKC in T
cells and its unique role in TCR signaling (6, 15) suggest
that our findings and those of others (33) are
biologically relevant in the context of the balance between
TCR/CD28-mediated survival signals and Fas-mediated apoptosis.
The outcome of PKC activation vis-à-vis cellular apoptosis, as
well as the contributions of distinct PKC isoforms to protection from
apoptosis, are likely to be stimulus and cell type specific, as
indicated by several examples. Thus, pharmacological PKC inhibition was
found to rescue neuronal cells from apoptosis induced by growth factor
deprivation (49), and this effect probably reflects the
promoting role of PKC in neuronal apoptosis (50).
Furthermore, another PKC isoform, i.e., PKC, was found to protect
COS cells from apoptosis (51). Lastly, a nPKC isoform
(probably PKC) was reported to promote, rather than inhibit, the
glucocorticoid-induced apoptosis of thymocytes (52). These
examples strongly suggest that the nature of survival pathways is
dependent on the cellular context and/or the particular triggering
stimulus. In this regard, leukemic or activated T cells are more
susceptible than resting cells to Fas-mediated apoptosis (53, 54). Based on our findings (Fig. 2), it would be interesting to
determine whether strategies designed to selectively inhibit the
function of PKC can synergize with death receptor agonists to
facilitate the elimination of autoreactive T cells in vivo, consistent
with a recent study (55).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martin Villalba or Dr. Amnon Altman, Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121.
3 Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas ligand; PKC, protein kinase C; cPKC, conventional PKC; aPKC, atypical PKC; nPKC, novel PKC; JNK, c-Jun N-terminal kinase; GFP, green fluorescent protein; PI, propidium iodide; MBP, myelin basic protein; tBID, truncated BID; Z-VAD, Z-Val-Ala-Asp-CH2F
Received for publication November 30, 2000. Accepted for publication February 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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: a new essential superstar on the T cell stage. Immunol. Today 21:567.[Medline]
, a human novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells. J. Biol. Chem. 268:4997. to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17:3101.[Medline]
, a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T-cells. Eur. J. Immunol. 29:132.[Medline]
are required for TCR-induced T cell activation. Immunity 12:151.[Medline]
B activation induced by CD28 costimulation is mediated by PKC. Proc. Natl. Acad. Sci. USA 97:3394. participates in NF-B/Rel activation induced by CD3/CD28 costimulation through selective activation of IB (IKK). Mol. Cell. Biol. 20:2933. is required for TCR-induced NF-B activation in mature but not immature T lymphocytes. Nature 404:402.[Medline]
cooperates with calcineurin to induce Fas ligand expression during activation-induced T cell death. J. Immunol. 163:5813. in induction of apoptosis. J. Biol. Chem. 272:20317.B or die?. Curr. Biol. 7:R94.[Medline]
and promote T cell survival by a Rsk-dependent phosphorylation and inactivation of BAD. J. Biol. Chem. 275:37246. signals apoptosis through a Bid-dependent conformational change in Bax that is inhibited by E1B 19K. Mol. Cell 6:53.[Medline]
in cerebellar granule cells apoptosis. NeuroReport 9:2381.[Medline]
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