(Received for publication, March 27, 1997, and in revised form, May 7, 1997)
From the § Department of Physiology, In the present study, we show that Fas receptor
ligation or cellular treatment with synthetic
C6-ceramide results in activation or phosphorylation,
respectively, of the small G-protein Rac1, Jun N-terminal kinase
(JNK)/p38 kinases (p38-K), and the transcription factor GADD153. A
signaling cascade from the Fas receptor via ceramide, Ras, Rac1, and
JNK/p38-K to GADD153 is demonstrated employing transfection of
transdominant inhibitory N17Ras, N17Rac1, c-Jun, or treatment with a
specific p38-K inhibitor. The critical function of this signaling
cascade is indicated by prevention of Fas- or
C6-ceramide-induced apoptosis after inhibition of Ras, Rac1, or JNK/p38-K.
Programmed cell death or apoptosis has been identified as a
conserved and fundamental active cellular mechanism occurring under a
range of physiological and pathological conditions (1-3). Programmed
cell death is characterized by distinct morphological changes of the
cell, including nucleus condensation and fragmentation, membrane
blebbing, or formation of apoptotic bodies (4). A variety of
stimuli have been identified to induce apoptosis in different cell
types, for example stimulation of cells via the TNF,1 CD95/Fas/ApoI, or nerve
growth factor receptor, treatment of cells with UV irradiation,
reactive oxygen intermediates, heat shock, ceramides or cytotoxic
drugs, infection by some viruses, or withdrawal of growth factors
(5-17).
Apoptosis in mature lymphocytes can be induced by cellular stimulation
via the Fas receptor (11, 13) belonging to the family of the nerve
growth factor/TNF receptors (5), which are important in the regulation
of apoptosis, proliferation or differentiation (5, 14). The major
function of the Fas receptor/Fas ligand system seems to be the
regulation of the peripheral immune response (18, 19). Thus, mutations
of the Fas receptor or its ligand result in the defects of
lpr and gld mice, respectively, characterized by
lymphadenopathy, lympho-accumulation, and autoimmune organ failure (20,
21). Recently, mutations of the Fas receptor have been suggested as a
mechanism for some human immunodeficiencies and the T-cell deficiency
of human immunodeficiency virus might be due to a pathological
stimulation via the Fas receptor (22-24). Several components of the
death machinery have been identified; in particular, proteases of the
ICE/Ced-3-family have been shown to be important in Fas triggered
apoptosis (25, 26). Recently, it was demonstrated that the Fas receptor
associates via its death domain, which exhibits significant homology
with an intracellular domain of the TNF receptor and is required for
induction of apoptosis (27), with FADD, TRADD, or RIP (11, 28, 29).
FADD in turn binds to FLICE/MACH-1 (30, 31). FLICE/MACH-1 contains an
ICE/Ced-3-like protease domain, which seems to be activated upon
ligation and trimerization of the Fas receptor by a conformational
change of FLICE/MACH-1 (30, 31). FLICE/MACH-1 then transmits the
activation signal to ICE and CPP32, finally triggering cell death (30, 31). Recent studies suggest that caspases regulate the release of
ceramide (32), a molecule that has been shown by us (9) and others (7)
to be released upon Fas receptor stimulation implying that
sphingomyelinases are downstream targets of caspases. Ceramides are
known stimuli of apoptosis and a function of Jun N-terminal kinase
(JNK) activation in the apoptotic effect of ceramides has been recently
demonstrated (16).
Several other molecules have been shown to be involved in apoptosis, in
particular the proto-oncogenes Myc, Abl, p120GAP Fos, or Ras
(9, 33-40). For example, knock-out mice of p120GAP show
dramatic apoptosis of neurons in the developing brain (35), inhibition
of Ras blocks Fas- or ceramide-induced programmed cell death in Jurkat
T-lymphocytes (9), and expression of Myc triggers cell death in
T-lymphocytes or fibroblasts (34, 37). Further molecules activated upon
Fas receptor triggering are neutral and acidic sphingomyelinases (41),
phospholipase A2 (41), NF In the present study, we investigated downstream effector molecules of
Ras and Rac proteins upon Fas receptor stimulation. Activation of
Jurkat cells via the Fas receptor or with synthetic ceramides resulted
in a Ras- and Rac1-dependent stimulation of JNK and p38-K.
The functional significance of the Fas- and ceramide-initiated signaling pathway from Ras via Rac to JNK/p38-K is indicated by an
almost complete inhibition of Fas- or C6-ceramide-mediated programmed cell death after transfection of the cells with
transdominant inhibitory Ras, Rac1, or an inhibitory Jun construct
(Tam67) and simultaneous treatment with a specific pharmacological
blocker (SB 203580) of p38-K suggesting an important function of the
described signaling pathway in the regulation of Fas-mediated
programmed cell death.
All reagents were purchased
from Sigma, Deisenhofen, Germany, if not otherwise cited. Human
leukemic Jurkat cells were grown in RPMI 1640 medium supplemented with
10% fetal calf serum, 10 mM Hepes (pH 7.4), 2 mM L-glutamine, 1 mM sodium
pyruvate, 100 µM nonessential amino acids, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 50 µM
Co-transfection of
Jurkat cells with transdominant inhibitory pEF-N17ras,
pCEV-N17rac1, pRc/CMV-tam67 (each 50 µg/20 × 106 cells) or vector control (pEF, pCEV) with an
expression vector for CD20 (pRc/CMV-cd20) (10 µg) was
performed as described previously (9). N17Ras, N17Rac, or Tam67 have
been shown previously to inhibit the function of Ras, Rac, or JNK,
respectively (17, 49, 50). Briefly, cells were electroporated using a
BTX electroporator at 5 pulses (99 µs) and 500 V. 12 h later,
viable cells were purified by Ficoll gradient centrifugation and
cultured for an additional 24 h. CD20+ cells were then
selected by incubation with 50 µg/ml anti-CD20 monoclonal antibody
(Dianova, Hamburg, Germany) (60 min, 4 °C), three washes, and
further incubation (60 min, 4 °C) with magnetic beads coated with a
sheep anti-mouse immunoglobulin (Dynal, Hamburg, Germany) (25). Since
the ratio of 5:1 for pEF-N17ras, pCEV-N17rac1, or
pRc/CMV-tam67:pRc/CMV-cd20 drives expression of
N17Ras, N17Rac1, or Tam67 in any CD20+ cell, the selection
of CD20+ cells permits effective sorting for N17Ras- or
N17Rac1-expressing cells. The fraction of CD20-positive and thus
N17Ras-transfected cells was determined by in vivo labeling
of the cells with [3H]thymidine as described previously
(9). Aliquots of the labeled cells were counted prior and after sorting
via magnetic beads. These experiments showed that approximately 10% of
all cells were CD20-positive. Nonspecific binding of the cells to the
magnetic beads did not exceed 0.5% of all cells, as determined by
incubation of cells with an irrelevant monoclonal mouse antibody (50 µg) and magnetic beads. Purified cells were allowed to recover for 30 min at 37 °C and then used for determination of JNK, p38-K, Rac1, or
Ras activity and apoptosis.
Inhibition of p38-K was achieved by preincubation with the specific
kinase inhibitor SB 203580 (10 µM, kindly provided by Dr.
B. MacKintosh, University of Dundee).
Untransfected Jurkat cells or Jurkat
cells co-transfected with N17Ras, N17Rac1, pCEV, or pEF and CD20 were
washed twice in phosphate-free Dulbecco's modified Eagle's medium,
resuspended in the same medium complemented with 10% dialyzed fetal
calf serum, and labeled for 4 h with 1 mCi/ml
32Pi. Cells were stimulated with anti-Fas (2 µg/ml) or C6-ceramide (5 µM) for the
indicated time or left untreated. Stimulation was terminated by lysis
in 25 mM Tris-HCl (pH 7.5), 1% Triton X-100, 0.2% SDS,
0.5% sodium deoxycholate, 20 mM MgCl2, 450 mM NaCl, and 100 µg each of aprotinin and leupeptin
(lysis buffer). Nuclei and cell debris were removed by centrifugation
(25,000 × g) at 4 °C for 15 min. Ras or Rac1 were
immunoprecipitated from the lysates at 4 °C for 45 min using the
monoclonal anti-Ras antibody Y13-259 or a polyclonal,
affinity-purified anti-Rac1 antibody (Santa Cruz Biotechnology Inc.,
Santa Cruz, CA), respectively. The anti-Rac1 antibody detects amino
acids 179-188 of the C-terminal protein domain and does not
cross-react with Rac2, Ras, or other known small G-proteins. It has
been shown previously to immunoprecipitate active Rac1 proteins (49).
The immunoprecipitates were collected by further incubation with
protein A/G-conjugated agarose beads (Santa Cruz). Precipitates were
washed seven times in lysis buffer and once in 25 mM
Tris-HCl (pH 7.5), 0.1% Triton X-100, 1 mM
MgCl2, and 125 mM NaCl; bound nucleotides were
eluted in 1 mM EDTA at 68 °C for 20 min. The samples
were then centrifuged at 15,000 × g for 5 min, and the
nucleotides in the supernatant were separated on
polyethyleneimine-cellulose plates (Machery & Nagel, Dueren, Germany)
with 0.75 M KH2PO4 (pH 3.5). The
TLC plates were analyzed by autoradiography, the spots corresponding to
GDP and GTP were scraped from the plate, and radioactivity was
determined by liquid scintillation counting. Activation of Ras or Rac1
is expressed by an increase of the GTP/(GDP + GTP) ratio.
Cell stimulation was
terminated by lysis in 25 mM Hepes (pH 7.4), 0.2% SDS,
0.5% sodium deoxycholate, 1% Triton X-100, 125 mM NaCl,
10 mM each NaF, Na3VO4, and sodium
pyrophosphate and 10 µg/ml of each aprotinin and leupeptin (RIPA
buffer), the lysates were centrifuged at 25,000 × g
for 20 min, and JNK or p38-K were immunoprecipitated from the
supernatants overnight at 4 °C using 3 µg of rabbit polyclonal
anti-JNK or anti-p38-K antiserum (Santa Cruz). All control
immunoprecipitates were performed with polyclonal rabbit
immunoglobulins. After addition of protein A/G-agarose, incubation was
continued for at least 60 min. Immunocomplexes were washed six times in
lysis buffer and resuspended in SDS-sample buffer (60 mM
Tris (pH 6.8), 2.3% SDS, 10% glycerol, 5% To measure the activity of JNK and
p38-K, cells were lysed in RIPA buffer as above after stimulation via
Fas or with C6-ceramide. Lysates were centrifuged at
25,000 × g for 20 min, and JNK or p38-K were
immunoprecipitated from the supernatants at 4 °C for 4 h using
polyclonal rabbit anti-human JNK or p38-K antisera. Immunocomplexes
were immobilized on agarose-coupled protein A/G; incubated for an
additional 60 min at 4 °C; washed twice in RIPA buffer; twice in
H/S, 1% Nonidet P-40, 2 mM Na3VO4;
once in 100 mM Tris (pH 7.5), 0.5 M LiCl; and
finally twice in kinase buffer 12.5 mM MOPS (pH 7.5), 12.5 mM To determine the
serine/threonine phosphorylation of GADD153, Jurkat cells were
metabolically labeled with 32Pi for 4 h,
stimulated with anti-Fas or C6-ceramide, and lysed in RIPA
buffer as above. GADD153 was immunoprecipitated from the post-centrifugation supernatant using a polyclonal rabbit anti-GADD153 antibody (Santa Cruz) as above, the samples were separated by 10%
SDS-PAGE and transferred to an Immobilon membrane, and phosphorylation of GADD153 was analyzed by autoradiography. After analysis, the blots
were reprobed with anti-GADD153 antibodies to test for equal amounts of
protein in all lanes. In addition, GADD153 was immunoprecipitated from
unlabeled, stimulated, or unstimulated cells and the immunoprecipitates were separated and blotted as above. The blots were blocked and incubated overnight at 4 °C with anti-phosphoserine/phosphothreonine antibodies (0.5 µg/ml; Sigma, Deisenhofen, Germany). Immunoblots were
developed using horseradish peroxidase-conjugated protein G and a
chemiluminescence kit. The blots were stripped after primary analysis
and reprobed to test for equal amounts of immunoprecipitated GADD153.
Phosphorylation of GADD153 after cellular stimulation with anti-Fas or
C6-ceramide was further analyzed by incubation of
recombinant GADD153 (0.1 µg/sample, Santa Cruz) with cell lysates
from stimulated or unstimulated samples. Cells were lysed in 12.5 mM MOPS (pH 7.5), 1% Triton, 12.5 mM
Fas-induced cell death was determined by
metabolic labeling of Jurkat cells for 12 h with 10 µCi/ml
[3H]thymidine (8.3 Ci/mmol, NEN Life Science Products)
(9). Cells were washed, aliquoted, and incubated with anti-Fas (200 ng/ml) or left untreated. Cell death was determined after 3 h by
DNA fragmentation and trypan blue staining. Briefly, cells were
disrupted by one cycle of freezing at We recently demonstrated an activation of the Ras signaling
pathway upon Fas receptor triggering or cellular treatment with ceramides (9). To identify downstream targets of this signaling pathway
initiated by Fas or ceramide, we tested the activation of JNK/p38-K.
Fas receptor triggering resulted in an approximately 15-fold
stimulation of JNK- and p38-K -activity (Fig.
1, A and B). The
stimulation of JNK or p38-K after Fas was comparable to the activation
of the kinases upon cellular treatment with C6-ceramide (Fig. 1, A and B). The activity of JNK or p38-K
was determined by phosphorylation of the substrates GST-c-Jun or
GST-ATF-2, respectively. The concentration of 5 µM
C6-ceramide used in the present study has been shown to
result in an intracellular concentration of 10-100 nmol/nmol of lipid
(51). These concentrations are physiologically relevant, since they are
also obtained upon Fas receptor ligation or serum deprivation (51). The
activation of JNK/p38-K by Fas or C6-ceramide was similar
to the stimulation of JNK or p38-K by prevoiusly reported stimuli, in
particular heat shock, sorbitol, or PMA (Fig. 1C) (16, 52
The increase of JNK or p38-K activity correlated with a phosphorylation
of the two kinases upon Fas receptor ligation or cellular treatment
with C6-ceramide (Fig. 1D). Phosphorylation was
determined by incubation of JNK or p38-K immunoprecipitates with a
phospho-JNK or phospho-p38-K specific antibody and development using
the ECL technique. Reprobing the blots with anti-JNK or anti-p38-K
antibodies revealed similar amounts of proteins in all lanes (shown in
the smaller blots below).
To elucidate the mechanism of JNK and p38-K activation by Fas and
C6-ceramide, we tested whether Ras and Rac proteins, which have been shown to be upstream regulators of JNK/p38-K (52, 56),
regulate the stimulation of JNK/p38-K after anti-Fas or C6-ceramide treatment. Rac proteins have been implied as
intermediates transmitting signals from Ras to Jun N-terminal kinase
kinase or MAP-kinase kinase, which then activate JNK or p38-K,
respectively (52, 56). A stimulation of Ras by Fas receptor ligation or cellular treatment with ceramides has been previously shown by us (9).
Further studies from our group showed a Ras-dependent activation of Rac1 and Rac2 upon Fas receptor triggering (49). Here, we
provide a detailed analysis of the kinetics of Rac activation upon Fas
receptor ligation and show that Rac is rapidly stimulated by Fas
receptor triggering peaking 10 min after stimulation (Fig. 2A). The activity of Rac1
after Fas remained increased for more than 40 min after
anti-Fas-treatment.
The activation of Rac1 upon Fas receptor ligation was mimicked by
cellular treatment with C6-ceramide (Fig. 2B);
Rac1 activation peaked approximately 10 min after addition of
C6-ceramide and remained stimulated for more than 40 min
after addition of C6-ceramide (Fig. 2B). The
specificity of C6-ceramide-mediated Rac1 stimulation is
indicated by the finding that the biologically inactive stereoisomer dihydro-C2-ceramide does not trigger Rac1 activation (data
not shown).
To analyze the involvement of Ras and Rac proteins in JNK/p38-K
activation by Fas or C6-ceramide, Jurkat cells were
transiently co-transfected with an expression vectors for transdominant
inhibitory N17Ras or N17Rac1 and CD20. The mutants have a very high
affinity to GDP (50) and bind endogenous guanine nucleotide exchange factors, preventing the activation of endogenous Ras or Rac1. N17Rac1
inhibits the activation of both Rac1 and Rac2 after Fas receptor
stimulation as demonstrated previously (49). Expression of the
B-lymphocyte antigen CD20 in transfected cell permits efficient purification of N17Ras- or N17Rac1-expressing cells (9). Inhibition of
endogenous Ras by N17Ras or Rac by N17Rac1 almost completely prevented
activation of JNK or p38-K after Fas receptor triggering (Fig.
3, A and B) or
cellular treatment with ceramides (Fig. 3, C and
D). The crucial role of Ras and Rac proteins for Fas-or C6-ceramide-initiated JNK and p38-K activation is also
indicated by an inhibition of the phosphorylation of JNK and p38-K upon Fas receptor or C6-ceramide triggering by transfection of
N17Ras or N17Rac1 (data not shown). The efficiency of N17Ras or N17Rac1 expression and the purification process has been shown previously (49)
and is demonstrated in Fig. 3E. These data indicate an approximately 90% inhibition of Ras or Rac1 activation after anti-Fas triggering (Fig. 3E and Ref. 49). In summary, these data
suggest that Ras and Rac proteins are upstream regulators of JNK/p38-K upon Fas receptor stimulation or treatment with
C6-ceramides.
To determine the significance of the observed signaling cascade for
Fas- or ceramide-induced programmed cell death, we measured the effect
of an inhibition of endogenous JNK/p38-K, Rac1, or Ras by transfection
of transdominant inhibitory constructs or treatment with
pharmacological inhibitors on apoptosis after anti-Fas or
C6-ceramide (Fig. 4). Cells
were co-transfected with either Tam67, N17Rac1, or N17Ras and CD20 or
treated with the specific p38-K inhibitor SB 203580, metabolically
labeled with [3H]thymidine, purified by sorting via CD20,
and stimulated with anti-Fas or C6-ceramide, and apoptosis
was determined as described above. In all experiments apoptosis was
also detected by typical morphological changes after trypan blue
staining. The experiments show that inhibition of JNK and p38-K,
endogenous Rac1, or Ras prevents Fas- and
C6-ceramide-induced apoptosis by more than 80%, indicating
the significance of the signaling cascade from Ras via Rac proteins to
JNK/p38-K for Fas- and ceramide-triggered programmed cell death. Single
inhibition of JNK or p38-K did not change Fas- or ceramide-induced
apoptosis. Control experiments with SB 203580 showed that the inhibitor
reduced p38-K activity after Fas receptor stimulation by approximately
90% (data not shown). The efficiency of Tam67 transfection was tested
by measuring the up-regulation of c-Jun and IL-2 production upon
treatment of the cells with PMA (10 nM) and ionomycin (500 ng/ml). Both activation markers have been previously shown to be
inhibited by expression of Tam67 (45). Induction of c-Jun or release of IL-2 were almost completely inhibited in cells transfected with Tam67
and sorted via CD20, whereas control transfected cells responded normally with c-Jun expression and IL-2 synthesis (data not shown). These results indicate a sufficient transfection and expression of
Tam67 in CD20-sorted Jurkat cells.
To gain insight into the function of JNK/p38-K activation upon Fas
receptor stimulation, we measured the phosphorylation of the
transcription factor GADD153. GADD153 is a known target of JNK and has
been implied in the regulation of gene expression and/or cell cycle
regulation (57-59). Fas receptor ligation (Fig. 5A) or ceramide treatment
(Fig. 5B) induced a strong phosphorylation of GADD153, which
was almost completely inhibited by transfection of transdominant
inhibitory N17Rac1 or of the transdominant inhibitory c-Jun construct
Tam67 and simultaneous pretreatment with the p38-K inhibitor SB 203580 (Fig. 5, C and D). Similar results were obtained using an anti-phosphoserine antibody showing an approximately 10-fold
stimulation of GADD153 phosphorylation (data not shown).
The in vivo phosphorylation of GADD153 could be mimicked by
incubation of recombinant GADD153 with cell lysates obtained from cells
stimulated via the Fas receptor or with C6 ceramides (Fig. 5E).
The data show that the phosphorylation of GADD153 is regulated by a
signaling cascade via Rac proteins and JNK/p38-K.
The results of the present and previous studies (9, 49) show a
signaling cascade from the Fas receptor via the small G-proteins Ras
and Rac to JNK/p38-K and the transcription factor GADD153. Activation
of this cascade seems to be necessary for Fas- or
C6-ceramide-mediated cell death, since genetic or
pharmacological inhibition of Ras, Rac, or JNK/p38-K prevents the
induction of apoptosis. Furthermore, the results identify GADD153 as a
new, Ras-, Rac-, and JNK/p38-K-regulated downstream effector of Fas and
synthetic ceramide. This notion is supported by the finding that
incubation of recombinant GADD153 with cell lysates in vitro from Fas or C6-ceramide stimulated cells results in a
phosphorylation of GADD153.
In the present study, inhibition of Ras and Rac proteins was achieved
by transient transfection of transdominant inhibitory constructs. These
mutants, N17Ras or N17Rac1, have a very high affinity to GDP and
therefore bind the endogenous GDP/GTP-exchange factors, preventing the
activation of endogenous Ras or Rac1/2 (49, 50). To identify
transfected cells, we performed a co-transfection with an expression
vector for CD20, which is a B-cell antigen not expressed in
T-lymphocytes. This co-transfection technique permits efficient sorting
of transfected cells as described previously (9). The blockade of
JNK/p38-K stimulation by genetic inhibition of Ras and Rac indicates
that Fas receptor-induced JNK and p38-K activation is mediated by a
sequential activation of Ras and Rac proteins. Our data show that Rac1
is longer (more than 40 min) active than JNK/p38-K. Since Rac1 is able
to interact with different downstream effector molecules linking Rac1
to cytoskeleton regulation (60, 61), JNK/p38 activation (52, 56), or
reactive oxygen release (62, 63), it is likely that JNK/p38-K are not
the only target molecules of Rac1 upon Fas receptor ligation. Thus, it
might be possible that Rac determines in a temporally organized way the
activity of different downstream molecules, and therefore the
activation time of these molecules might be shorter than of Rac1.
Inhibition of JNK was achieved by transfection of a transdominant
inhibitory c-Jun construct, TAM67, which has been used previously to
inhibit cell death triggered by the nerve growth factor-p75 receptor
(17). The Tam67 c-Jun mutant lacks the N-terminal transactivation domain, which includes the JNK binding site, and functions as a
transdominant interfering mutant. p38-MAP kinase was inhibited by
incubation of the cells with the pharmacological inhibitor SB 203580, which is considered to be a specific inhibitor of p38-K (64). Tam67
transfection prevented the up-regulation of c-Jun or release of IL-2
after T-cell stimulation. Likewise, SB 203580 almost completely
inhibited the activity of p38-K upon Fas receptor triggering. This
demonstrates the efficiency of the Tam67 transfection and the kinase
inhibitor treatment. Since, in accordance with other studies (45, 65),
single inhibition of JNK or p38-K did not prevent Fas- or
ceramide-triggered programmed cell death, whereas combined inhibition
of JNK and p38-K significantly reduced Fas- or ceramide-mediated
apoptosis, the activation of both kinases seems to be required for
induction of apoptosis in Jurkat cells. Likewise, ceramide-initiated
JNK activation has been demonstrated to be essential for the induction
of apoptosis after ionizing radiation, UV light, heat shock, ceramide,
TNF Our notion of an activation of JNK and p38-K by Fas receptor ligation
is supported by previous studies showing that Fas stimulates these
kinases (16, 43-45, 65, 66). The time course of JNK activation after
Fas seems to depend on the dose of the antibody used for stimulation,
since treatment with 1-2 µg/ml anti-Fas results in JNK activation
after already 10 min (Ref. 43 and present study), whereas lower
concentrations (30-100 ng/ml) require longer incubation times (60-90
min) to induce JNK stimulation.
In summary, the studies suggest an important function of JNK/p38-K for
Fas- or ceramide-mediated programmed cell death.
The activation of a signaling cascade from the Fas receptor via Ras or
Rac proteins and JNK/p38-K to GADD153 may have several functions
important for Fas- or ceramide-triggered programmed cell death;
JNK/p38-K-regulated phosphorylation of GADD153 may result in a change
of GADD153 activity and in an altered regulation of gene expression
and/or the cell cycle. GADD153, also known as CHOP or growth arrest and
DNA damage-inducible gene 153, has been recently shown to be
phosphorylated upon stress via a pathway involving p38-K (57, 59).
GADD153 forms heterodimers with members of the C/EBP family
transcription factors (57-59), which either inhibit C/EBP binding to
DNA or direct the GADD153-C/EBP heterodimers to other DNA sequences
resulting in a change of gene expression or of the cell cycle (58, 59).
The function of GADD153 phosphorylation upon Fas receptor triggering is
unknown. However, since several reports indicate an important role of
the cell cycle in the regulation of cellular apoptosis and cells may undergo apoptosis only during a certain phase of the cell cycle (36,
67-69), it might be possible that regulation of the cell cycle is the
major role of Fas-initiated GADD153 regulation via Ras and Rac1. In
this context, the phosphorylation of GADD153 may have one of the
following functions. First, if phosphorylation of GADD153 upon Fas
receptor ligation inhibits the activity of GADD153 to induce cell cycle
arrest, the cells will progress faster through the cell cycle. If cells
are sensitive to Fas only during a specific phase of the cell cycle, a
faster cell cycle will result in a higher percentage of Fas sensitive
cells per time, which might be the function of GADD153. This hypothesis
is supported by the effects of N17Ras or N17Rac on Fas- or
ceramide-induced death. Ras or Rac may be necessary for cell cycle
progress and inhibition of the cell cycle by N17Ras or N17Rac
transfection may therefore block the cell cycle preventing Fas- or
ceramide-induced cell death. Second, the phosphorylation of GADD153 may
promote its ability to induce cell cycle arrest. In this case, the cell cycle might be arrested during a Fas-sensitive phase permitting Fas
receptor ligation to trigger apoptosis. Finally, it might be possible
that ceramide and Fas induce growth arrest and/or DNA damage, which may
result in an altered activity of GADD153. Thus, the phosphorylation of
GADD153 would be secondary to the induction of apoptosis. This
possibility seems to be unlikely since the phosphorylation of GADD153
was also observed in cell lysates lacking nuclei. Therefore, the
phosphorylation of GADD153 does not depend on the presence of a
nucleus. Furthermore, GADD153 phosphorylation is already observed 10 min after Fas receptor ligation or C6-ceramide treatment.
This time seems to be rather short to mediate a significant DNA damage
resulting in the regulation of GADD153. However, since the
physiological function and regulation of GADD153 is only poorly
characterized, an alternative function is certainly possible and
elucidation of the exact role of GADD153 in Fas-induced apoptosis has
to be performed in future studies.
It has been shown that Fas-induced cell death does not require new
mRNA or protein synthesis (70). Our experiments do not exclude the
possibility that cells already in a certain state of the cell cycle are
able to undergo apoptosis upon Fas receptor ligation, whereas in a cell
population with heterogenous cell cycle status the cell cycle
progression regulated via Ras, Rac, JNK/p38-K, and GADD153 might be
necessary to achieve complete induction of apoptosis by Fas.
Alternatively, JNK and p38-K may have cytosolic targets involved in the
apoptosis of lymphocytes upon Fas receptor ligation. In particular,
p38-K has been implied in the phosphorylation of heat shock protein HSP
25/27 (71, 72). HSP 25/27 is phosphorylated by a MAP kinase-activated
protein kinase, which is a substrate of p38-K during cellular stress
responses (72, 73). Since constitutive expression of HSP 25/27 has been
shown to prevent Fas-induced apoptosis (73), it might be possible that
phosphorylation of HSP 25/27 inhibits the function of HSP 25/27,
permitting the cell to undergo apoptosis after Fas receptor
ligation.
The effects of Fas receptor ligation on Ras, Rac, and JNK/p38-K are
mimicked by synthetic ceramides, and inhibition of the signaling
cascade from Ras to JNK also prevents Fas- as well as ceramide-triggered programmed cell death. Further data from our group
show that the consumption of sphingomyelin, the release of ceramide,
activation of the acidic sphingomyelinase, and stimulation of JNK/p38-K
are prevented by inhibition of caspases by transfection with CrmA or
after treatment with pharmacological caspase
inhibitors.3 This notion is
supported by recent findings showing that the activation of JNK and
p38-K upon Fas receptor ligation requires caspases (45, 65). The data
suggest a signaling cascade from Fas via caspases, the acidic
sphingomyelinase, release of ceramide, and activation of Ras and Rac to
a stimulation of JNK/p38-K. However, a recent study using transfection
of FADD, RIP, or TRAF-2 into MCF-7 cells proposed that JNK activation
and apoptosis after TNF receptor ligation are separate responses (74).
Therefore, JNK/p38-K activation may not be sufficient by itself to
trigger apoptosis; however, under physiological conditions it might be
necessary as a positive signal for the pathway signaling programmed
cell death. In situations with a very high activation level of the pathway triggering apoptosis, e.g. after transfection of
genes inducing cell death, this positive feedback may not be absolutely required.
Ras, Rac, and JNK/p38-K are not only involved in apoptosis but are also
important in the regulation of cell proliferation. For example, Ras and
JNK are also activated upon CD28 or CD40 receptor stimulation, which
induce cell proliferation or differentiation (75-78). A similar dual
function for the regulation of cell proliferation/differentiation or
programmed cell death has been shown for c-Myc (34, 37), c-Fos (33),
Cdc-2 (36), or p120GAP (35). Therefore, the actual function
of Ras, Rac, JNK/p38-K, and GADD153 may depend on co-signals provided
from, e.g., growth factor receptors, oncogenes, or receptors
inducing programmed cell death. In this concept, Ras and Rac proteins
and JNK/p38-K may control apoptosis and may be necessary for but not
sufficient to induce apoptosis.
We thank Dr. S. Gutkind, Dr. R. Davis, and
Dr. B. MacKintosh for valuable reagents. We gratefully acknowledge the
excellent technical help of A. Behyl, K. Baltzer, and Caroline
Müller.
Department of Pediatrics,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B (42), Jun kinases (43-45),
tyrosine and serine/threonine kinase (46, 47), the tyrosine phosphatase
FAP (48), and Rac proteins (49).
Cell Culture and Stimulation
-mercaptoethanol (all from Life Technologies, Inc., Eggenstein,
Germany.) For activation, cells were washed twice in Hepes-buffered
saline (H/S: 132 mM NaCl, 20 mM Hepes, 5 mM KCl, 1 mM CaCl2, 0.7 mM MgCl2, 0.8 mM MgSO4)
and incubated at 37 °C with 2 µg/ml monoclonal anti-human Fas
antibody (clone CH-11, Dianova-Immunotech, Hamburg, Germany) or 5 µM C6-ceramide (Biomol, Hamburg, Germany) for
the indicated times.
-mercaptoethanol). Separation of proteins was performed by 10% SDS-PAGE, followed by an
electrophoretic transfer to Immobilon polyvinylidene difluoride filters
(Millipore, Eschborn, Germany). Blots were blocked and incubated
overnight at 4 °C with anti-phospho-JNK or anti-phospho-p38-K antibodies (New England Biolabs, Schwalbach, Germany). Each antibody was diluted to 0.5 µg/ml in Tris-buffered saline supplemented with
0.1% Tween 20. Immunoblots were developed by incubation with horseradish peroxidase-conjugated protein G (Bio-Rad) and a
chemiluminescence kit (Amersham). All blots were stripped by a 45-min
incubation in 20 mM Tris (pH 6.8), 2% SDS, 70 mM
-mercaptoethanol at 70 °C after primary analysis
and reprobed to test for equal amounts of immunoprecipitated protein.
Alternatively, an aliquot of the immunoprecipitates was separated by
SDS-PAGE and analyzed by Western blotting for equal amounts of protein
in all immunoprecipitates.
-glycerophosphate, 0.5 mM EGTA, 7.5 mM MgCl2, 0.5 mM NaF, 0.5 mM Na3VO4. After washing, the
immunoprecipitates were resuspended in kinase buffer supplemented with
10 µCi/sample [
-32P]ATP (6000 Ci/mmol, NEN Life
Science Products), 10 µM ATP, and 1 µg/ml GST-c-Jun
(amino acids 1-79) or GST-ATF-2 (amino acids 1-96). The samples were
incubated at 30 °C for 15 min and stopped by addition of 5 µl of
boiling 5 × reducing SDS sample buffer. Samples were separated by
10% SDS-PAGE and analyzed by autoradiography and laser densitometry.
The substrates GST-c-Jun and GST-ATF-2 were expressed in DH5
bacteria after transformation, growth for 12 h, and incubation
with isopropyl-1-thio-
-D-galactopyranoside (200 µM) for 4 h. Bacteria were lysed in 25 mM Hepes (pH 7.4), 0.2% SDS, 0.5% sodium deoxycholate,
1% Triton X-100, 125 mM NaCl, and 10 µg/ml each of
aprotinin and leupeptin; GST fusion proteins were purified by binding
to glutathione-agarose and eluted in kinase buffer with 20 mM glutathione. Purity of the preparations was tested by
SDS-PAGE and Coomassie staining.
-glycerophosphate, 0.5 mM EGTA, 7.5 mM
MgCl2, 0.5 mM NaF, 0.5 mM
Na3VO4, centrifuged, and recombinant GADD153
and 10 µCi/sample [
-32P]ATP were added to the
supernatants. The samples were then incubated at 37 °C for 20 min,
and the reaction was stopped by addition of 5 µl of boiling SDS
sample buffer and 5%
-mercaptoethanol. The samples were separated
by 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane,
and analyzed by autoradiography and laser densitometry.
20 °C and thawing, after
which unfragmented genomic DNA was collected by filtration through
glass fiber filters (Pharmacia, Freiburg, Germany) and counted by
liquid scintillation (9). Results are expressed as % DNA
fragmentation ± S.D. compared with control samples. Experiments
were done in triplicate and repeated three times. In all experiments an
aliquot of the cells was analyzed for cell death by trypan blue.
Apoptosis was identified by the typical morphological changes, in
particular membrane blebbing, condensation, and fragmentation of
nuclei. Comparison of the DNA fragmentation method and the morphologic
changes permits exact determination of apoptosis versus
necrosis. In all experiments initial rates of apoptosis did not exceed
5% of all cells.
55). The inactive stereoisomer dihydro-C2-ceramide did
not induce stimulation of JNK or p38-K, showing the specificity of the
activation.
Fig. 1.
A and B, JNK (A)
and p38-K (B) are activated upon cellular stimulation via
the Fas receptor or with synthetic C6-ceramide. Jurkat
cells were stimulated for the indicated time with anti-Fas antibodies
(2 µg/ml) or C6-ceramide (5 µM) and JNK or
p38-K were immunoprecipitated. Washed immunoprecipitates were subjected
to in vitro kinase assays using GST-c-Jun or GST-ATF-2
fusion proteins as substrates. Samples were separated by 10% SDS-PAGE,
blotted, and analyzed by autoradiographies. The blots show an
approximately 15-fold stimulation of JNK and p38-K after Fas receptor
ligation or treatment of the cells with C6-ceramide. After
primary analysis, all blots were reprobed with anti-JNK or anti-p38-K
antibodies to determine the level of JNK or p38-K protein in the
samples (small blots). The analysis shows similar protein levels in all lanes. C, cellular treatment with heat shock, sorbitol, or
PMA (known stimuli of JNK or p38-K) induces a similar activation of JNK
and p38-K as observed after stimulation with anti-Fas (2 µg/ml) or
C6-ceramide (5 µM). The inactive stereoisomer
dihydro-C2-ceramide (5 µM) does not induce
the stimulation of JNK or p38-K showing the specificity of the observed
activation event. Cells were treated at 42 °C for 30 min, with 400 mM sorbitol, 10 ng/ml PMA, anti-Fas (2 µg/ml), and
C6-ceramide or dihydro-C2-ceramide (each 5 µM). The activity of JNK and p38-K was determined as
described above. The reprobed blots show similar amounts of JNK or
p38-K in all lanes. D, activation of JNK and p38-K
correlates with a phosphorylation of the kinases after Fas receptor
ligation or cellular treatment with C6-ceramide (5 µM). Cells were stimulated as indicated, and JNK or p38-K
were immunoprecipitated from the lysates. The immunoprecipitates were
blotted and analyzed for phosphorylation using a phospho-JNK or
phospho-p38-K specific antibody, followed by ECL-development. The blots
were stripped and reprobed with an anti-JNK or anti-p38-K antibody
showing similar amounts of all proteins in all lanes. Control
(c) obtained from Fas- or ceramide-stimulated samples using
an unspecific, irrelevant rabbit antibody did not show any cross-reacting kinase activity. All experiments were repeated three
times.
[View Larger Version of this Image (39K GIF file)]
Fig. 2.
Fas (A) or
C6-ceramide (B) induce an activation of
Rac1. Jurkat cells were metabolically labeled for 4 h and
stimulated with anti-Fas (2 µg/ml) or C6-ceramide (5 µM) for the indicated time. Rac1 was immunoprecipitated
from the lysates, and bound guanine nucleotides were eluted from the
immunoprecipitates, separated by TLC, and analyzed by autoradiography.
The results show a long lasting activation of Rac1 upon ligation of the
Fas receptor, which is comparable with the stimulation of Rac1 after
cellular treatment with C6-ceramide. Activation of Rac1 was
repeated three times. Immunoprecipitates using an irrelevant polyclonal
control rabbit antiserum did not reveal any unspecific
immunoprecipitation of a small G-protein (c = control
immunoprecipitates).
[View Larger Version of this Image (81K GIF file)]
Fig. 3.
A-D, inhibition of Ras or Rac prevents
activation of JNK (A and C) or p38-K
(B and D) after Fas (A and
B) or C6-ceramide (C and
D) stimulation pointing to a regulation of the two kinases by Ras and Rac. Jurkat cells were co-transfected with transdominant inhibitory N17Ras or N17Rac-1 and CD20; CD20-positive cells were sorted
and stimulated via the Fas receptor (2 µg/ml) or with
C6-ceramide (5 µM); and JNK or p38-K activity
was determined as described above. Transfection of the vector controls
(pEF or pCEV) did not affect Fas- or C6-ceramide-induced
stimulation of JNK or p38-K. E, transfection of N17Rac1
prevents the activation of Rac1 after Fas receptor ligation, whereas
transfection of the vector did not alter the stimulation of Rac1.
Transfection of N17Rac1 also prevents the activation of Rac2 by Fas as
demonstrated previously (9). The autoradiographies were also analyzed
by laser scanning and the results are presented as % increase of the
GTP/(GDP + GTP) ratio. The data show the efficiency of the transfection
and sorting process almost completely blocking the activation of
endogenous Rac1. Control immunoprecipitates (c) using a
rabbit antiserum do not show the precipitation of a cross-reacting
kinase. All experiments were repeated at least two times.
[View Larger Version of this Image (47K GIF file)]
Fig. 4.
Inhibition of Rac proteins, JNK, and p38-K
prevents Fas- or C6-ceramide-induced programmed cell
death. Cells were labeled for 12 h with
[3H]thymidine prior to induction of apoptosis. Rac
proteins or JNK were inhibited by transient co-transfection of
transdominant inhibitory N17Rac1 or Tam67 and CD20. CD20+
cells were sorted and treated with anti-Fas (2 µg/ml) or
C6-ceramide (5 µM) for 4 h. p38-K was
inhibited by preincubation with the specific p38-K inhibitor SB 203580 (10 µM). DNA fragmentation was determined by binding of
intact DNA to glass fiber filters, followed by liquid scintillation
counting (9). Apoptosis was also measured by staining an aliquot of the
cells with trypan blue and determining typical morphological changes.
The two methods measuring apoptosis or DNA fragmentation showed very
similar results.
[View Larger Version of this Image (30K GIF file)]
Fig. 5.
A and B, GADD153 is
phosphorylated after stimulation via the Fas receptor ligation
(A) or treatment with C6-ceramide
(B). Jurkat cells were metabolically labeled with
32Pi, stimulated with anti-Fas (2 µg/ml)
(A) or C6-ceramide (5 µM) (B) for the indicated time, GADD153 was immunoprecipitated,
samples were separated by 10% SDS-PAGE and blotted, and
phosphorylation was analyzed by autoradiography. Reprobing of the blots
revealed similar amounts of GADD153 in all lanes. C and
D, inhibition of Rac or JNK and p38-K prevents
phosphorylation of GADD153 upon Fas receptor (C) triggering
or C6-ceramide (D) application. Jurkat cells
were cotransfected with N17Rac1 or Tam67 and CD20 and sorted for
CD20+ cells. Tam67-transfected cells were also incubated
with the p38-K inhibitor SB 203580. Cells were stimulated with anti-Fas
(2 µg/ml) (C) or C6-ceramide (5 µM) (D), and the phosphorylation of GADD153 was determined as above. E, cell lysates from Fas- or
ceramide-activated cells phosphorylate GADD153 in vitro.
Jurkat cells were stimulated via the Fas receptor (2 µg/ml) or with
C6-ceramide (5 µM) and lysed, and the lysates
were incubated with recombinant GADD153 in the presence of
[-32P]ATP. Lysates from stimulated cells contain a
kinase activity phosphorylating GADD153. Control immunoprecipitates
(c) using irrelevant rabbit antibodies show the specificity
of GADD153 immunoprecipitation. The experiments were repeated three
times.
[View Larger Version of this Image (43K GIF file)]
, or H2O2 (16). However, in this study,
Tam67-transfected U937 cells were almost completely resistant to
induction of apoptosis by the mentioned stimuli. Therefore, it might be
possible that the expression levels of JNK versus p38-K
determine whether both kinases are required for induction of apoptosis
or only one of the two kinases. Thus, in some cell types Tam67 might be
sufficient to prevent apoptosis by ceramide, Fas, or stress, whereas in
other cells, e.g. Jurkat, inhibition of both JNK and p38-K
is required to prevent apoptosis. JNK/p38-K do not seem to be
important in all forms of apoptosis, since transfection with Tam67,
treatment with SB 203580, or combination of the two inhibitors did not
prevent apoptosis in Jurkat cells incubated with thapsigargine (1 µM), a microsomal Ca2+ ATPase
inhibitor.2
*
This study was supported by Deutsche Forschungsgemeinschaft
Grants Gu 335-2/2 and La 315-6/1, Association of International Cancer
Research Grant 94-194, Sandoz Grant 95-1-005, the
Mildred-Scheel-Stiftung (Grant 10-0983), and an Else Übelmesser
grant (all to E. G.), and a grant from the University of
Heidelberg (to B. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-7071-2972196; Fax: 49-7071-293073.
1
The abbreviations used are: TNF, tumor necrosis
factor; p38-K, p38 kinase; ICE, interleukin I-converting enzyme; GAP,
GTPase-activating protein; JNK, Jun N-terminal kinase; PAGE,
polyacrylamide gel electrophoresis; GST, glutathione
S-transferase; MOPS, 4-morpholinepropanesulfonic acid;
IL, interleukin; PMA, phorbol 12-myristate 13-acetate.
2
B. Brenner, U. Koppenhoefer, C. Weinstock, O. Linderkamp, F. Lang, and E. Gulbins, unpublished data.
3
Brenner, B., Koppenhoefer, U., Weinstock, C.,
Linderkamp, O., Lang, F., and Gulbins, E. (1997) Cell Death
Differ., in press.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.