From the Cancer Research Center and Departments of Medicine, Biochemistry, Pediatrics, Microbiology, Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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Activation of protein kinase C (PKC) can protect
cells from apoptosis induced by various agents, including Fas ligation.
To elucidate a possible interaction between Fas-mediated apoptotic signals and activation-related protective signals, we investigated the
impact of Fas ligation on PKC activity. We demonstrate that engagement
of Fas on human lymphoid Jurkat cells triggered apoptosis, and Fas
ligation resulted in partial blockade of cellular PKC activity. The
phorbol 12-myristate 13-acetate-mediated translocation of PKC The Fas/APO-1 antigen, a member of the tumor necrosis factor
receptor family, is a transmembrane molecule and is expressed by a
variety of cells, including transformed cell lines and activated T
lymphocytes (1-3). The function of Fas/APO-1 appears to be the
induction of apoptosis, and a growing number of Fas-associated molecules and signal pathways have been discovered (1, 4-6). Fas
mutation or disruption of its function in lymphoproliferation (lpr) mice leads to a progressive lymphadenopathy and
autoimmune syndrome resembling human systemic lupus erythematosus
(7-9). The cytotoxic activity of T lymphocytes is also dependent upon normal expression of Fas (10). Thus, Fas/APO-1-induced apoptosis is
required for the maintenance of at least two immunological processes
in vivo: the normal elimination of potentially autoreactive peripheral T cells and calcium-independent T cell cytotoxicity (6, 11,
12). The mechanisms of Fas-induced apoptosis have been studied
extensively (13, 14). The molecules that bind to the intracellular
domain of Fas, the death domain, have been identified as MORT1/FADD,
TRADD, and RIP (15-18). The association of MORT1/FADD in turn recruits
caspase-8/FLICE/MACH-1 to the death complex (19, 20).
Caspase-8/FLICE/MACH-1 then transmits the activation signal to ICE and
CPP32 and executes the death program (19, 20). Another signal
transduction event leading to apoptosis after Fas ligation appears to
be mediated by ceramide and involves the activation of
p21ras (21-24). Studies have shown that a
serine/threonine phosphatase is involved in ceramide signaling pathways
(25-27). This ceramide-activated protein phosphatase
(CAPP)1 is a cytosolic enzyme
and has been implicated as a specific mediator of ceramide action. CAPP
is very sensitive to low concentrations of okadaic acid and therefore
belongs to the heterotrimeric subfamily of the protein phosphatase 2A
group. Although CAPP has been demonstrated to mediate some of the
cellular actions of ceramide, the link between ceramide-induced
phosphatase activation and subsequent intracellular events is still not clear.
Protein kinase C (PKC) has been implicated as one of the critical
components of multiple signaling pathways, including T cell activation
processes (28-30). At least 11 isotypes of PKC have been discovered,
and these isotypes can be classified according to their structure and
cofactor requirements for activation (31-34). PKC A protective effect of PKC activity in apoptosis has also been
demonstrated (41-44). PKC inhibitors have been shown to block phosphorylation of Bcl-2 and lead to apoptosis, whereas activation of
PKC induced phosphorylation of Bcl-2 and abolished the apoptotic process (44-46). Individual isoforms of PKC may play specific roles in
the induction of apoptosis. In human U-937 myeloid leukemia cells,
PKC Cell surface expression levels of Fas on resting T lymphocytes can be
induced rapidly in response to T cell activation (1, 48, 49).
Activation-induced T cell death has been implicated in the termination
of immune response (6, 11, 12). Ligation of Fas significantly
suppresses TcR/CD3 complex-mediated early signal transduction events,
including inhibition of TcR/CD3-triggered tyrosine phosphorylation of
cellular proteins (50). Thus, Fas engagement may attenuate T cell
activation events, whereas T cell activation-related events, such as
activation of PKC, may reciprocally protect cells against apoptosis.
This opportunity for cross-talk between signaling pathways led us to
examine the potential interrelationship between these two seemingly
opposing processes: apoptosis and activation. Here, we demonstrate that
engagement of Fas triggers the apoptotic process in human Jurkat and
mouse thymoma cells stably expressing the fas gene (LF(+))
but not in mouse thymoma cells stably transfected with antisense
fas gene (LF( Cell Lines--
The human lymphoblastoid cell line Jurkat
(American Tissue Culture Collection, Rockville, MD) was maintained in
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated newborn calf serum (Hazelton Research Products, Inc.,
Lenexa, KA), 2 mM L-glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin. LF1210 mouse thymoma cells
were transfected with the fas gene (]LF(+)) or an antisense
fas gene (LF( Cell Viability Assays--
Jurkat, LF(+), or LF( DNA Fragmentation Assay--
The cells (1 × 106/ml) were cultured with 1.5 µg/ml anti-human Fas Ab
for 12 h and resuspended in 1 ml of 1% sodium citrate, 0.1%
Triton X-100, 50 µg of propidium iodide, and 10 µl of RNase (1 mg/ml). The stained samples were kept in the dark at 4 °C overnight before DNA fragmentation analysis by FACScan (Becton Dickinson, Mountain View, CA).
PKC Enzymatic Assay--
Human and mouse cells (1 × 106 cells/ml) were cultured in five replicate wells of a
six-well plate with 10 ml of medium containing 1.5 µg/ml anti-human
Fas Ab or 5 µg/ml anti-mouse Fas Ab for 60 min and subsequently
treated with 100 nM PMA for 15 min. For single stimulation
experiments, the cells were exposed to either anti-Fas Ab or PMA alone
for 60 or 15 min, respectively. For PKC enzyme activity inhibitor
experiments, cells were exposed to PMA for 15 min after the addition of
0.1 µM staurosporine for 15 min. After lysing the cells
in 25 mM Tris-HCl (pH 7.5), 1% Triton X-100, 20 mM MgCl2, 150 mM NaCl (9), the
lysates were normalized for protein concentration, and 150-µg
aliquots of protein were analyzed for PKC activity using a PKC assay
kit containing a specific substrate peptide for PKC and an inhibitor
mixture that blocks the activity of PKA and calmodulin kinase (Upstate
Biotechnology Inc., Lake Placid, NY). Subsequently, the
32P-incorporating substrate from each treatment was
separated from the residual [32P]ATP using p81
phosphocellulose paper, and the radioactivity incorporated into the
substrate was measured by scintillation counting.
Immunoblotting and Cell Fractionation--
Cells (50 × 106), following the different treatments described above,
were washed twice with 1 × phosphate-buffered saline and
resuspended in 1 ml of buffer B (20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 5 mM EGTA, 10 mM
In Vitro Kinase Assay--
The cells (20 × 106) were incubated with PMA at 100 nM for 15 min prior to 60 min of exposure to anti-Fas Abs. For phosphatase inhibitor experiments, okadaic acid was added to a final concentration of 50 nM 15 min before the next treatments. The cell
lysates were then prepared with the lysis buffer (150 mM
NaCl, 50 mM Tris-HCl (pH 8.0), 1% Nonidet P-40, 0.5%
deoxycholate, 0.1% SDS, 5 mM EGTA, 10 mM NaF,
10 µg/ml leupeptin, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride) (27). The lysates were
normalized for protein concentration, and each sample (containing
approximately 500 µg of protein) was immunoprecipitated with the
corresponding anti-PKC Ab and collected by absorption to protein
A-Sepharose. The immunocomplexes bound to protein A-Sepharose were
washed with lysis buffer twice and kinase buffer twice (50 mM Tris-HCl (pH 7.4), 10 mM NaF, 1 mM Na3VO4, 0.5 mM EDTA,
0.5 mM EGTA, 2 mM MgCl2, 10 µg/ml
leupeptin, 1 mM phenylmethylsulfonyl fluoride).
Subsequently, the immunocomplexes bound to the beads were resuspended
in reaction buffer (20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 10 µM cold ATP, 0.4 mg/ml histone H1, 2.5 µCi of [ Fas-mediated Apoptotic Signals Inhibit PKC Activation--
Jurkat
cells express a significant amount of Fas antigen on their surfaces
(51). The sensitivity of these cells to treatment with anti-Fas Ab was
examined in medium containing 10% serum (Fig. 1a). The number of viable
Jurkat cells did not begin to decline until 5 h after exposure to
the Ab, and the death proportion of dead cells increased steadily from
that time on. By 24 h, approximately 65% of the Jurkat cells lost
viability. Exposure to an unrelated, isotype-matched (IgM) antibody did
not induce cell death in Jurkat cells; rather, the numbers of cells
started to increase 6 h after the addition of the unrelated Ab,
reflecting normal cell growth. The cell death observed was caused by
apoptosis, as confirmed by cytofluorometric analysis of nuclear DNA
fragmentation, as described previously (Fig. 1b). The
percentage of cells with fragmented DNA 15 h after the addition of
anti-Fas Ab was more than 20%, and by 24 h more than 30% of the
cells contained fragmented DNA. This time course of apoptosis was also
confirmed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling
(TUNEL) assay (data not shown).
We demonstrated previously that down-regulation or inhibition of PKC
could induce Jurkat cells expressing oncogenic ras to undergo apoptosis and that Fas-mediated signals may be involved in this
apoptotic process (51, 52). Several other groups have documented that
PKC can regulate or modulate both proliferative and anti-apoptotic
pathways in diverse cell types (28-30, 38, 39, 41-44, 53). The
involvement of these common messengers in signaling pathways with
disparate outcomes, cell activation or cell death, suggested that these
opposing pathways may interfere with each other or utilize cross-talk
to facilitate execution of their respective end points. T lymphocytes
express several isotypes of PKC, and the functions of the isotypes Blockade of PKC Inhibition of PKC Fas Ligation Results in Blockade of PKC Activity in Mouse
Thymocytes Overexpressing the fas Gene--
To confirm and generalize
the results obtained in human Jurkat cells, mouse thymoma cells stably
transfected with either fas (LF(+))or antisense
fas (LF(
Involvement of a CAPP-like phosphatase in this Fas-activated signaling
pathway was suggested in Jurkat cells. To determine if this observation
was generalizable, in vitro PKC kinase assays were performed
in LF(+) cells (Fig. 5c, two upper rows). Cell lysates were prepared after different treatments, and
immunoprecipitations were carried out with anti-PKC Preactivation of PKC Partially Protects from, or Delays,
Fas-induced Apoptosis--
PKC is an important component of signaling
pathways regulating cell activation and proliferation (28-30, 53).
Activation of PKC elicits a variety of cellular responses, mediated by
phosphorylation of its downstream effector proteins on serine/threonine
residues. It has been demonstrated that activation of PKC can protect
cells from apoptosis mediated by various apoptotic stimuli, such as ceramide-induced apoptosis (41-44). To determine the protective effect
of PKC on Fas-induced apoptosis, Jurkat cells were stimulated with PMA
at 100 nM for 15 min before the addition of anti-Fas Ab,
and cell viability was then examined at different time points (Fig.
6a). In those cells in which
PKC had been preactivated by PMA treatment, Fas-mediated apoptosis was
delayed significantly at all time points. Jurkat cells in which PKC had
been preactivated did not show any evidence of apoptosis until more
than 9 h after the addition of anti-Fas Ab, compared with active
apoptosis being observed by 7 h after Fas ligation in the control
cells. 50% of the control cells were dead by 15 h, in contrast to
the PMA-pretreated cultures where 50% death was not reached until
24 h. By 30 h after anti-Fas Ab treatment, there were very
few viable cells in the control cultures, whereas living cells were
still present in the preactivated cells at 36 h (data not shown).
DNA fragmentation analysis also confirmed that activation of PKC by PMA
attenuates the Fas-mediated apoptotic process (Fig. 6b).
After 15 min of prior PMA treatment, the percentage of Jurkat cells
with fragmented DNA began to increase at 9 h after the addition of
anti-Fas Ab. By 24 h after the addition of the Ab, more than 20%
of the cells exhibited fragmented DNA compared with the more than 30%
of cells with DNA fragmentation in the absence of PMA pretreatment (as shown in Fig. 1b). These findings are consistent with the
observations of others that activation of PKC may partially protect the
cells from, or attenuate, the Fas-induced apoptotic process.
Fas-mediated apoptosis, initiated by lymphocyte activation
pathways, plays an important role in the elimination of immature autoreactive lymphocytes during thymic selection, as well as in the
regulation of the peripheral lymphocyte pool, through the engagement of
Fas antigen on the surface of lymphocytes by either anti-Fas Ab or Fas
ligand (11, 12). The PKC family consists of more than 10 isoforms, and
they are selectively engaged in various biological processes, including
critical roles in mediating lymphocyte activation signals (31-34).
Although the exact role of these different isozymes in cellular
functions remains enigmatic, it has been documented that the activation
of PKC by diacylglycerols or phorbol esters can protect cells from
apoptosis induced by various biological or chemical agents, especially
ceramide-induced apoptosis (31-34). Fas ligation, neutral
sphingomyelinase, and tumor necrosis factor- This observed difference in the mechanism by which Fas ligation
inhibits PKC Several studies have demonstrated that sphingosine or lysosphingolipids
can inhibit PKC activity, induce growth arrest at the G1
phase of the cell cycle, and down-regulate c-myc expression (26, 56). The mechanism by which C2-ceramide or
sphingomyelinase inactivates the PKC Our findings suggest a model for the integration of death-inducing
signals and survival signals. The apoptotic signals generated by Fas
ligation initiate a cascade of reactions and drive the cells to undergo
apoptosis. To ensure such a process, the signals mediated by Fas
engagement may also suppress potential protective mechanisms, such as
activation of the PKC pathway. If these protective pathways are
activated prior to Fas ligation, the process of Fas-mediated apoptosis
is delayed or is less inefficient. When the PKC pathway is activated,
subsequent Fas ligation can no longer block the phosphotransferase
activity of PKC, and the cells become more resistant to Fas-mediated
apoptotic signals. Although preactivation of the PKC pathway did, to
some extent, interfere with the apoptotic signal, it did not engender
full protection. The initial Fas signal triggers multiple diverging
pro-apoptotic pathways, with the major Fas-mediated apoptotic signal
being through the ICE protease cascade. Although the ceramide/Ras
pathway has been suggested as another result of Fas signaling which can
induce apoptosis, its ability to act independently of the ICE pathway
is unclear (21-24). PKC activation protects cells from various
apoptotic processes, including ceramide-mediated apoptosis (28-30).
The protection against apoptosis provided by PKC activity may thus only
extend to a subset of Fas-initiated signals, perhaps
ceramide/Ras-related signals. Cross-linking of cell surface Fas antigen
is known to suppress some of the TcR/CD3-mediated signal transduction
events in human T lymphocytes (50). After pretreatment with anti-Fas
Ab, CD3-stimulated production of inositol trisphosphate and tyrosine
phosphorylation of multiple cell proteins was inhibited. The inhibition
of PKC activity and translocation we observe to result from Fas
ligation may account, in part, for this suppression, and this
hypothesis is under investigation.
The data presented herein, together with the reports from other
laboratories, clearly demonstrate that anti-apoptotic mechanisms or
molecules may be induced by PKC activation. It is noteworthy that
activation-induced cell death in T lymphocytes involves the initial
activation of mitogenic (and protective) pathways (57) and subsequent
desensitization to external stimuli (58). At later times following T
cell activation, when Fas expression is up-regulated, susceptibility to
Fas-mediated apoptosis increases, and protective molecules such as PKC
are repressed, perhaps facilitating cell death. The reciprocal
interactions between protective and pro-apoptotic signals as shown here
may account for the protective (anti-apoptotic) mechanisms proposed to
be present at early times after activation (50, 59). The general
concept that activation signals in the cell might actively inhibit or
oppose apoptotic programs has been proposed in other systems, including
extracellular signal-regulated kinase activation in nerve growth
factor-differentiated PC-12 cells and CD3 cross-linking in T
lymphoblasts (50, 60). These results suggest a novel mechanism for
Fas-mediated cell death. Inhibition of PKC-mediated pathways by Fas
engagement would prevent rescue or protection from apoptosis by
coincident of subsequent PKC activation. Furthermore, the data indicate
that cross-talk between these two opposing signals (destructive
versus protective) may occur at a very early stage, and
perhaps at cellular membrane level, to determine cell fate: life or death.
from
the cytoplasm to the membrane was inhibited by treatment with anti-Fas
antibody, whereas the translocation of PKC
or
was not affected.
In vitro kinase assay of PKC
or
phosphotransferase activity demonstrated that Fas ligation inhibited the ability of PKC
to phosphorylate histone H1 as substrate but did not inhibit
isozyme activity. This inhibition of PKC
activity mediated by Fas
ligation was reversed by okadaic acid, a phosphatase inhibitor, suggesting the involvement of a member of the protein phosphatase 2A
subfamily in this component of Fas signaling. Identical patterns of PKC
isozyme inhibition were obtained using mouse thymoma cells overexpressing the fas gene (LF(+)). These results suggest
that the selective inhibition of a potentially protective, PKC-mediated pathway by Fas activation may, to some extent, contribute to
Fas-induced apoptotic signaling.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
1,
2, and
are dependent upon calcium
for activity, whereas PKC
,
,
, and
are not. PKC
and
/
cannot be activated by phorbol esters or diacylglycerols,
although they belong to the PKC family by structure (33). The
differential expression of PKC isotypes in mammalian tissues and the
different substrate specificities of different PKCs indicate that
distinct PKC isotypes may have precise cellular localizations and
substrate preferences. T cells express at least seven different
isotypes of PKC:
,
1,
,
,
,
, and
.
PKC isotypes are regulated by phosphorylation and binding of various
cofactors. Upon activation, the enzyme is redistributed among different
cellular compartments (35-37). The roles of PKC
,
, and
have
been implicated in T cell activation by transfection experiments
(28-30, 38, 39). These three isotypes of PKC regulate T cell
activation via control of transcriptional factors such as AP-1, NFAT,
and nuclear factor-
B, which, in turn, modulate the activity of the
interleukin-2 gene promoter/enhancer (28-30, 38, 39). PKC
translocates from the cytoplasm to the membrane in response to
antigen-specific activation (30, 39). The
isoform of PKC interacts
with 14-3-3
, and overexpression of 14-3-3
blocks the
activation-mediated translocation of PKC
(39). Other studies have
demonstrated that the human immunodeficiency virus Nef protein inhibits
the translocation of PKC
after phorbol ester stimulation (40).
PKC
activity is inactivated by ceramide, perhaps through the action
of CAPP (27).
is cleaved in the third variable region by caspase-3 during
apoptosis induced by various agents, and overexpression of cleaved
PKC
fragment resulted in cell death (47). Furthermore, cleavage of
another isoform, PKC
, by caspase-3-cysteine protease caused cells to
undergo apoptosis, whereas overexpression of the anti-apoptotic factors
Bcl-2 or Bcl-XL blocked the cleavage of PKC
(44-46).
)). Fas ligation selectively inhibits the
activation of different isotypes of PKC in both Jurkat and LF(+) cells.
The translocation of PKC
in response to phorbol 12-myristate
13-acetate (PMA) stimulation is inhibited by prior Fas ligation.
PKC
-mediated phosphorylation of histone H1 is blocked by prior Fas
activation. This inhibition of PKC activity by Fas activation could be
prevented by pretreatment with okadaic acid, indicating an involvement
of a protein phosphatase in Fas signaling. The activity of PKC
was
not affected by Fas stimulation. Therefore, these data suggest that the
integration of multiple pro- and anti-apoptotic signals resulting from
Fas activation may be required to execute the apoptotic program successfully.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)) (generous gifts from Dr. S.-T. Ju, Boston
University) and cultured in RPMI 1640 medium containing 10%
heat-inactivated newborn calf serum plus 0.7 mg/ml Geneticin.
) cells
(0.5 × 106 cells/ml) were cultured in six-well plates
with 5 ml of medium containing 10% newborn calf serum plus 1.5 µg/ml
anti-human Fas Ab (Pan Vera Corp., Madison, WI) for Jurkat and PH1
cells or 1.5 µg/ml IgM Ab as control. The mouse thymoma cells (LF)
were cultured under the same conditions with the addition of 5 µg/ml
mouse anti-mouse Fas Ab (Pharmingen, San Diego). Cells were collected
at the time points indicated and enumerated using trypan blue dye
exclusion to assess viability. Error bars represent the S.D. over five
independent experiments.
-mercaptoethanol, 10 µg/ml leupeptin, 10 µg/ml aprotinin) (39).
The cell suspensions were transferred to a 1-ml syringe and sheared by
being passed 40 times through 25-gauge needle. The lysates were
centrifuged at 280 × g for 10 min to precipitate
nuclei, and the supernatants were collected. One-third of the whole
cell extract was saved, and the remainder was centrifuged at
16,000 × g for 30 min. The supernatant (cytosol) was
collected, and the pellet was washed in buffer B containing 1% Nonidet
P-40 for 1 h on ice and centrifuged again at 16,000 × g. The supernatant representing the membrane fraction was
saved. Each fraction (whole cell lysate, cytosol fraction, and membrane fraction) was normalized and separated on 8% of acrylamide gel. Subsequently the gel was immunoblotted with the anti-mouse (Santa Cruz
Biotechnology, Santa Cruz, CA) or human (Transduction Laboratories, Lexington, KY) PKC Abs, which recognize the isoforms of PKC
,
,
and
. The blot was developed with an anti-mouse Ig alkaline phosphatase reagent (Oncogene Science, Uniondale, NY).
-32P]ATP (6,000 Ci/mmol)) and incubated at 30 °C for 10 min. The reactions were
terminated by the addition of protein loading buffer. Proteins were
separated on 10% SDS-polyacrylamide gel, and the gel was subjected to autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cell growth kinetics and DNA fragmentation in
response to Fas ligation. Panel a, growth curves of
Jurkat cells in the presence of either anti-Fas Ab or an irrelevant IgM
Ab. Viable cells were enumerated at the time points indicated.
Error bars represent the S.D. over five independent
experiments. Panel b, percent of Jurkat cells with DNA
fragmentation at different time points after the addition of anti-Fas
Ab. Error bars represent the S.D. over five independent
experiments.
,
, and
have been studied extensively in T cell activation
(28-30). PMA mobilizes PKC, and together with increasing cytoplasmic
free calcium, mimics some aspects of T cell activation. To examine the
interrelationships proposed above, PKC enzymatic activity was measured.
A PKC-specific enzymatic assay for detecting total PKC activity was
used to measure the phosphotransferase activity of PKC in Jurkat cells
after different combinations of Fas ligation or PMA treatment (Fig.
2a). Adding anti-Fas Ab alone
for 60 min did not elicit PKC activation in Jurkat cells. The
phosphotransferase activity of PKC was increased 15 min after treatment
with PMA alone, at 100 nM, or exposure to the combination
of PMA plus calcium ionophore. After incubation of the cells in medium
containing anti-Fas Ab to activate Fas signaling for 60 min, however,
induction of total cellular PKC activity by PMA was attenuated
substantially (by 60%). In reversing the sequence of the treatments,
it was found that ligation of Fas after prior stimulation of the cells
with PMA for 15 min did not suppress or interfere with PKC activation.
Staurosporine, a PKC inhibitor, dramatically inhibited PKC activity in
response to PMA, as expected. Because Fas-generated signals blocked
PMA-mediated PKC activation, we next determined whether this blockade
of PKC activation by Fas signaling was sustained at later time points after Fas ligation. PKC enzymatic activity was examined at various time
points after exposure to anti-Fas antibody (Fig. 2b). Up to
6 h after Fas ligation, PMA treatment still could not elicit PKC
activity. Because of the very large fraction of the cells undergoing
apoptosis after that time point, it was impossible to collect enough
cells to assay PKC enzymatic activity. The levels of PKC protein,
measured by immunoblotting, at different time points after Fas
engagement were similar, excluding the possibility that the effects of
Fas ligation on PKC activity were the result of changes in the
expression PKC proteins (Fig. 2b, inset).
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Fig. 2.
Inhibition of PKC activity after Fas
ligation. Panel a, relative 32P
incorporation into a PKC-specific substrate peptide after Fas ligation,
with or without subsequent PMA stimulation, in lysates of Jurkat cells.
Error bars represent the S.D. over five independent
experiments. Control, untreated cells; PMA + IONO, cells treated with 100 nM PMA plus 2 µM calcium ionophore for 15 min; PMA, cells
treated with 100 nM PMA for 15 min; anti-Fas Ab,
cells treated with 1.5 µg/ml anti-human Fas Ab alone; anti-Fas
Ab + PMA, cells treated with 1.5 µg/ml anti-Fas Ab for 60 min
before exposure to 100 nM PMA for 15 min; stauro. + PMA, cells treated with 0.1 µM staurosporine for 15 min before exposure to 100 nM PMA for 15 min; PMA + anti-Fas Ab, cells treated with 100 nM PMA for 15 min
before anti-Fas Ab treatment (1.5 µg/ml). Panel b,
PMA-induced relative 32P incorporation into a PKC-specific
substrate peptide at different time points after preincubation with
anti-Fas Ab (1.5 µg/ml). Cells were harvested 15 min after the
addition of PMA and lysates used for PKC activity assay. Error
bars represent the S.D. over five independent experiments.
Control, untreated cells; PMA, cells treated with
100 nM PMA for 15 min; anti-Fas 2 h, 4 h, or 6 h + PMA, cells treated with 1.5 µg/ml
anti-Fas Ab for 2, 4, or 6 h, respectively, before exposure to 100 nM PMA. The levels of total cellular PKC protein in
response to Fas ligation at the various time points indicated above
were also determined (inset). Proteins in lysates from the
cells were separated on a SDS-polyacrylamide gel and immunoblotted with
an anti-PKC Ab that recognizes a region common to all isotypes of
PKC.
Translocation from Cytoplasm to Membrane by Fas
Ligation--
One of the phenomena associated with PKC activation is
the redistribution of the enzyme among different cellular compartments (35-37). It has been proposed that receptors for activated protein kinase C (or 14-3-3
) or Nef oncoprotein may mediate PKC
translocation (35, 39, 40). The major isoforms of PKC (
,
, and
) undergo translocation from the cytoplasm to the cellular membrane
in T cells stimulated with PMA (30, 35, 39, 40). To examine whether the
inhibitory effect of Fas ligation on PKC activation might affect enzyme
or isozyme translocation, cells were treated with PMA, anti-Fas Ab, or
a combination of them (Fig.
3a). The levels of PKC
,
, and
proteins in the nuclear-free whole cell, membrane, or
cytoplasmic fractions from stimulated or unstimulated cells were
assessed by immunoblotting with the corresponding antibodies. (Because
the Fas-mediated cell death program is executed at the cytoplasmic
level (10, 21, 51), isozyme data from the nuclear fraction were not
included in this study.) Comparable amounts of the three isozymes were
expressed in the cytoplasm of unstimulated Jurkat cells and underwent
translocation to the membrane fraction upon PMA stimulation. The
addition of anti-Fas Ab alone did not mobilize the enzyme isoforms to
redistribute. After the addition of the anti-Fas antibody for 1 h,
PKC
no longer translocated from the cytoplasm to the membrane in
response to PMA stimulation, whereas PKC
and PKC
translocated
normally. The translocation of PKC
, a PMA-insensitive isoform, was
also examined under the same stimulatory conditions. Neither treatment
with PMA nor anti-Fas Ab mobilized this isozyme from the cytoplasm to
the cellular membrane.
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Fig. 3.
Selective inhibition of different PKC
isoforms by Fas ligation. Panel a, effect of Fas
ligation on the intracellular localization of PKC isoforms. Jurkat
cells were treated with PMA, anti-Fas Ab, or a combination of the two.
Nuclear-free whole cell lysates (WC), membrane fractions
(M), and cytoplasmic fractions (C) were prepared.
After immunoprecipitation with isotype-specific antibodies ( ,
,
,
), immunoblotting was performed with the corresponding Abs.
Control, untreated cells; PMA, cells treated with
100 nM PMA for 15 min; anti-Fas Ab, cells
treated with 1.5 µg/ml anti-Fas Ab for 60 min; anti-Fas Ab + PMA, cells treated with 1.5 µg/ml anti-Fas Ab for 60 min before
exposure to 100 nM PMA for 15 min. Panel b,
effect of Fas ligation on the kinase activity of PKC isozymes. Jurkat
cells were treated with anti-Fas Ab, PMA, or a combination of the two
in the absence or presence of 10 nM okadaic acid
(OA) or 0.1 µM staurosporine
(Stau.). The lysates were immunoprecipitated with either
anti-PKC
or
Ab, and the immunoprecipitates were collected on
protein A-Sepharose beads for in vitro kinase assay of
phosphotransferase activity. Nuclear free whole cell lysates were also
separated by SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and immunoblotted with either anti-PKC
or
Ab to
determine isozyme protein levels (bottom two rows). The
results presented are representative of three independent
experiments.
Activity by Fas-mediated CAPP--
It has
been reported that ceramide generated intracellularly can activate
CAPP, a member of the protein phosphatase 2A subfamily, which may then
play an important role in the regulation of ceramide-induced apoptosis
in various types of cell lines, or of ceramide-mediated down-regulation
of c-Myc in myeloid leukemia cells (25-27). Activation of CAPP in
Molt-4, a human leukemia cell line, and in Jurkat cells blocked the
activity of PKC
at the downstream level of enzyme translocation
(27). Because ceramide has also been reported to play a role in
Fas-induced apoptosis (21-24), we tried to determine the potential
involvement of CAPP in Fas-mediated signaling and whether this
phosphatase may interfere with PKC activity (Fig. 3b,
two upper rows). The cells were treated with PMA, anti-Fas Ab, or a combination of the two, with or without exposure to okadaic acid or staurosporine (a serine/threonine protein kinase inhibitor), and the lysates were subsequently prepared for in vitro PKC
kinase assay. After immunoprecipitation with either anti-PKC
or
antibody, the immunocomplexes were mixed with histone H1 as an
exogenous substrate for the kinase assay. Histone H1 was not
phosphorylated by PKC immunocomplexes from unstimulated cells or from
cells treated with anti-Fas Ab alone. In response to PMA treatment, the
histone H1 protein substrate was phosphorylated by both PKC
and
immunocomplexes, and this phosphotransferase activity was suppressed by
staurosporine. After the addition of anti-Fas Ab for 1 h, PKC
immunocomplexes from cells stimulated with PMA could no longer
phosphorylate histone H1. PMA-stimulated enzymatic activity of PKC
immunocomplexes was similarly suppressed after Fas Ab treatment (not
shown), consistent with the suppression of translocation of this
isotype to the cell membrane (see panel a). In the presence
of okadaic acid, a phosphatase inhibitor, at 10 nM,
however, histone H1 was again phosphorylated by PKC
from
PMA-activated, anti-Fas Ab-treated cells. PMA-stimulated PKC
isozyme
activity was unaffected by Fas ligation or by okadaic acid. These
results indicated that a CAPP-like phosphatase might be involved in the
Fas-mediated signals that inactivate the PKC
isoform. In addition,
the lack of an effect of Fas ligation on PMA-mediated PKC
activity
may define discrete regulatory pathways for the different isotypes of
PKC and may also explain the incomplete inhibition of PKC activity by
Fas ligation (as demonstrated in Fig. 2a). Together, these
data suggest that the signals generated by Fas engagement which
interfere with the activity of certain PKC isotypes may function
selectively at different steps along PKC pathways. The protein levels
of the PKC isoforms under the same conditions were also determined, to
exclude the possibility that changes in protein levels could account
for the observed differences in enzymatic activities (Fig.
3b, two lower rows). The levels of PKC
and
proteins did not change after the different treatments.
)) genes were studied (Figs.
4 and 5).
The level of cell surface expression of Fas on LF(+) cells was
comparable to that on Jurkat cells (51). After the addition of
anti-mouse Fas Ab, the numbers of viable LF(+) cells started to decline
at 12 h, and about 80% of the cells had died by 36 h (Fig.
4a). In comparison, the viability of LF(
) cells was not affected by Fas engagement; rather, by 10 h after the addition of
anti-Fas Ab, the numbers of LF(
) cells were seen to increase. The
occurrence of apoptosis in LF(+) cells after Fas ligation was confirmed
by an assay of DNA fragmentation (Fig. 4b). By 15 h
after the addition of anti-Fas Ab, more than 15% of LF(+) cells had
fragmented DNA, and the percentage of DNA fragmentation was increased
further at 24 h after Fas ligation (to more than 25%), which is
in good agreement with the cell viability assay results seen in Fig.
4a. The percentage of DNA fragmentation in LF(
) cells did
not increase at any time point after Fas engagement and remained
comparable to that of the control (untreated) cells. Cell viability
assays and DNA fragmentation assays were also performed in the parental
LF1210 cells. Because there was only a very low level of Fas antigen
expressed on the surface of LF1210 cells, apoptosis in response to Fas
ligation was minor (data not shown). The effect of Fas ligation on PKC
activity in these cell lines was then defined. A PKC enzymatic assay
was performed in LF(+) and LF(
) cells after treatment with anti-Fas
and/or PMA (Fig. 5a). PKC activity was not elicited by
exposure to anti-Fas Ab for 60 min in either LF(+) or LF(
) cell
lines, whereas 15 minutes of treatment with PMA activated PKC in both
cells. 1 h after the addition of anti-Fas Ab, PKC activation in
LF(+) cells was inhibited significantly (by 60%) but not in LF(
)
cells. The levels of PKC protein were assayed in LF (+) and LF (
)
cells by immunoblotting following the same treatments, and no changes
in the protein levels were induced by PMA or anti-Fas Ab. To define
further how the signals generated through Fas ligation may block the
PMA-elicited PKC activation, the translocation status of PKC
,
,
and
in response to different treatments was examined by
immunoblotting protein lysates from the LF(+) cells (Fig.
5b). After different combinations of stimulants, cytoplasmic
and membrane fractions and the whole cell lysates of LF(+) cells were
prepared for the detection of PKC
,
, and
. All three PKC
isoforms were expressed in the cytoplasmic fraction of unstimulated
LF(+) cells but not in the membrane fraction. In response to treatment
with PMA, the majority of the PKC
and
proteins translocated from
cytoplasm to membrane, whereas PKC
did not translocate. Fas
engagement by itself did not cause redistribution of any of the three
enzymes. After exposure of the cells to anti-Fas Ab for 1 h, PMA
treatment no longer induced the translocation of PKC
to membrane.
However, the translocation of PKC
in response to PMA treatment was
not affected by prior Fas ligation. Analysis of PKC
subcellular
location by immunoblotting was also conducted; and as found in Jurkat
cells, Fas-initiated signals did not block the translocation of the
enzyme (data not shown).
View larger version (18K):
[in a new window]
Fig. 4.
Fas-induced apoptosis in mouse thymocytes
overexpressing the fas gene. Panel a,
growth kinetics of LF(+) and LF( ) cells in the presence or absence of
anti-Fas Ab. The error bars represent the S.D. of five
independent experiments. LF(
), LF(
) cells cultured under
normal growth conditions; LF(
) Fas, LF(
) cells cultured
in medium containing 5 µg/ml anti-Fas Ab; LF(+), LF(+)
cells cultured under normal growth conditions; LF(+) Fas,
LF(+) cells cultured in medium containing 5 µg/ml anti-Fas Ab.
Panel b, DNA fragmentation of LF(+) or LF(
) cells at
different time points after anti-Fas Ab treatment. The error
bars represent the S.D. of five independent experiments.
Control, untreated cells; anti-Fas Ab 15 h or
24 h, cells were treated with 5 µg/ml anti-Fas Ab for 15 or 24 h, respectively.
View larger version (37K):
[in a new window]
Fig. 5.
Inhibition of PKC activity in murine LF cells
by Fas ligation. Panel a, relative 32P
incorporation into a PKC-specific substrate peptide after Fas ligation,
with or without subsequent PMA stimulation, in LF(+) or LF( ) cells.
Error bars represent the S.D. over five independent
experiments. Control, untreated cells; anti-Fas
Ab, cells treated with 5 µg/ml anti-Fas Ab for 60 min;
PMA, cells treated with 100 nM PMA for 15 min;
anti-Fas Ab + PMA, cells treated with 5 µg/ml anti-Fas Ab
for 60 min before exposure to 100 nM PMA. Panel
b, effect of Fas ligation upon the intracellular localization of
PKC isoforms in LF(+) cells. The notations are as described in the Fig.
3a legend. Panel c, effect of Fas ligation on the
phosphotransferase activity of specific PKC isozymes in LF(+) cells.
The notations are as described in the Fig. 3b legend.
or
Ab, and a
PKC kinase assay was performed by mixing the immunocomplexes with
histone H1 to serve as a substrate. Histone H1 was not phosphorylated
by PKC isozyme immunocomplexes from control samples or from cells
treated with anti-Fas Ab. However, histone H1 protein phosphorylation activity was present in PKC
and
immunocomplexes from cells treated with PMA and was suppressed in the presence of staurosporine. 1 h after the addition of anti-Fas Ab, PKC
could not be
activated by exposure to PMA, as assayed by histone H1 kinase activity, but PKC
isozyme activity was intact. Treatment with okadaic acid prevented the blockade of PKC
activity by Fas ligation. The protein levels of the PKC isoforms under the same conditions were also determined, to exclude the possibility that these different treatments may affect the expression of the isozymes (Fig. 5c,
two lower rows). The different treatments did not alter the
levels of PKC
or
. These data were in good agreement with the
findings generated in Jurkat cells and suggested again that
Fas-generated signals may interfere selectively with the activities of
different isotypes of PKC.
View larger version (18K):
[in a new window]
Fig. 6.
Protective effect of PKC activation on the
Fas-induced apoptotic process. Panel a, Jurkat cells,
with or without pretreatment with PMA for 15 min, were cultured in
medium containing anti-Fas Ab, and the numbers of viable cells were
enumerated at the time points as indicated in the figure. The
error bars represent the S.D. of five independent
experiments. Panel b, DNA fragmentation in Jurkat cells was
analyzed after Fas ligation in response to pretreatment with PMA. The
error bars represent the S.D. of five independent
experiments. Control, untreated cells; 100 nM
PMA + anti-Fas Ab 7 h, 9 h, 15 h,
24 h, cells treated with 100 nM PMA for 15 min
before exposure to anti-Fas Ab for 7, 9, 15, or 24 h,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
can activate
intracellular ceramide, which can then mediate apoptosis (21-24). This
study examined the possible interactions between apoptotic signals
elicited by Fas activation and protective events generated by PKC, both
of which can be the direct results of T cell activation stimuli. We
demonstrate herein that Fas engagement in human Jurkat cells and in
mouse thymoma cells overexpressing Fas antigen (LF(+) cells) partially
blocked phorbol ester-induced PKC activation, and this inhibition
persisted for at least 6 h after Fas ligation. Fas ligation was
not able to suppress PKC activity when the PMA stimulus was delivered
before Fas ligation, however. The suppression of PKC activity by Fas ligation involved certain T cell activation-related isotypes of PKC
(
and
) and blocked them at different steps of the enzyme activation pathway. The translocation of PKC
in response to PMA was
blocked by ligation of Fas antigen. PKC
-mediated translocation was
not blocked, but its activity, as assayed by phosphorylation of histone
H1, was inhibited after Fas ligation. This inhibition may be mediated
by a member of the protein phosphatase 2A subfamily. Overall, these
data suggest that blockade of certain isoforms of PKC, at different
levels of the enzyme activation depending on the specific isozyme, may
be a part of Fas signaling. PKC
and
activation may be suppressed
by Fas signals or Fas-related inhibitory proteins so as to ensure rapid
execution of the apoptotic process. In addition, loss of PKC
activity may itself directly promote apoptosis, as shown recently in an
experimental system where a dominant negative PKC
transgene induced
programmed cell death in COS cells (54).
activation compared with
is of interest. The process of regulation of PKC by lipids such as phosphatidylserine and
diacylglycerol includes several discrete events, such as membrane translocation and phosphorylation of these enzymes (28-30, 35-37). PKC participates in signal transduction pathways through mobilization from the cytoplasm to the cellular membrane, where it is then activated
by membrane-associated stimulatory molecules. All isozymes of PKC
contain the conserved regulatory regions, C1 and
C2 (31-34, 55). Cleavage of certain isozymes, like PKC
,
in the third variable (V3) region deletes the two
regulatory regions and results in a catalytically active fragment that
appears to be associated with the induction of apoptosis. It has been
demonstrated that PKC
can be cleaved by caspase-3. One explanation
for blockade of PKC
translocation by Fas ligation may be that the
enzyme is a dual function molecule. Activation of the caspase protease
family by Fas ligation may result in the generation of the
pro-apoptotic fragment of PKC
, and therefore the enzyme could
function as a regulator in the death program. Alternatively, it is
possible that PKC
activation (in the absence of cleavage) would
normally serve an anti-apoptotic, protective function, like PKC
.
Experiments employing ectopic or overexpression of this enzyme isoform
will be necessary to determine the role of PKC
in pro-
versus anti-apoptotic processes.
isoform, in particular, remains
controversial (27, 57). In mouse epidermal cells (HEL-37) or human skin fibroblasts (SF3155), the phorbol ester-induced translocation of
PKC
, but not of PKC
, was inhibited by ceramide treatment (57).
Inhibition of translocation did not occur, however, in MOLT-4 human
leukemia cells and Jurkat cells (27). Instead, the inactivation of
PKC
by ceramide MOLT-4 and Jurkat cells appears to be through
activation of CAPP. In our studies, we demonstrated that exposure to
PMA could still induce the translocation of PKC
, even after
engagement of Fas by anti-Fas Ab. Yet, the activity of the enzyme, as
measured by the phosphorylation of histone H1 substrate, was suppressed
dramatically, and this inhibition could be reversed by okadaic acid at
concentrations in the range required for inhibition of protein
phosphatase 2A in vivo. Because activation of the
ceramide/Ras pathway is one component of Fas-induced signaling, the
possible existence and activation of CAPP in Fas-mediated pathways may
account for the subsequent intracellular events we observe, including
the inhibition of PKC
activity. The activity of PKC
in the cells
remained inhibited after Fas ligation even after the enzyme was
immunoprecipitated, suggesting post-translational modification of the
enzyme as a result of Fas ligation or, less likely, the association of
an inhibitory protein that coprecipitates in an inhibitory complex with
the enzyme. Activation of PKC by phorbol esters is associated with new
phosphorylation of the PKC molecule itself. New phosphorylation of
PKC
, but not of the
isoform, by PMA treatment was partially
inhibited in the presence of anti-Fas
Ab.2 The finding that Fas
ligation subsequent to prior PMA treatment does not inhibit PKC
activity suggests that inhibitory process activated by the Fas signal
may prevent activation of PKC
rather than inhibiting its enzymatic
activity directly. Although a CAPP-like phosphatase may play a role in
the inhibition of PKC
activity by Fas, there are no data to support
a direct interaction between PKC
and a phosphatase. Rather, the data
cumulatively suggest that activation of CAPP-like phosphatase by Fas
ligation may indirectly regulate this isozyme of PKC. It is possible
that CAPP may activate a PKC
-specific, inhibitory protein. In
preliminary studies, we have found that cytoplasmic extracts from cells
in which Fas-initiated signals had been induced contain an activity
that inhibits PKC activation in vitro.2 The lack
of responsiveness of PKC
to Fas ligation demonstrates the discrete
and specific nature of Fas-generated signals and lends support to the
general concept that there are distinct functions for the different
isozymes of PKC and that these isoforms can themselves be regulated
differentially by networks of kinases and phosphatases. A study has
shown that down-regulation of c-myc expression in human
myelogenous leukemia HL-60 cells could be regulated by an okadaic
acid-insensitive, PMA-mediated pathway or an okadaic acid-sensitive,
dephosphorylation mechanism (26).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Shyr-Te Ju for providing the LF cells and for very helpful comments and Drs. C. Terhorst and D. Sherr for helpful discussions.
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FOOTNOTES |
---|
* This project was supported in part by Research Grant CA50459 from the NCI, National Institutes of Health (to D. V. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a cardiovascular biology training grant from the
NHLBI, National Institutes of Health.
§ To whom correspondence should be addressed: Cancer Research Center, Boston University School of Medicine, 80 E Concord St. K-701, Boston, MA 02118. Tel.: 617-638-4173; Fax: 617-638-4176; E-mail: dfaller{at}bu.edu.
2 C. Y. Chen and D. V. Faller, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: CAPP, ceramide-activated protein phosphatase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; Ab, antibody.
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REFERENCES |
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