From the Program in Cell Biology, Department of
Pediatrics, National Jewish Medical and Research Center, Denver,
Colorado, 80206 and the
Department of Biochemistry and Molecular
Genetics, ** Division of Pulmonary and Critical Care
Medicine, Department of Medicine,
Department of
Pharmacology and ¶ Department of Immunology, University of
Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, November 27, 2000, and in revised form, January 19, 2001
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ABSTRACT |
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Ligation of the tumor necrosis factor Engagement of the tumor necrosis factor- Fas ligand (Fas-L) and to a lesser extent TNF The family of Bcl-2-related proteins comprises both death-inducing
(Bax, Bak, Bcl-xS, Bad, Bid, Bik, and Hrk) and death-inhibiting (Bcl-2,
Bcl-xL, Bcl-w, Bfl-1, Mcl-1, and Boo) members (13), and, although the
mechanism of action of death-inducing Bcl-2 family members is rapidly
emerging, the mechanism underlying the death-inhibiting properties of
Bcl-2 family members is only partly understood. The ratio of death
antagonists to agonists has been proposed to regulate the death-life
rheostat within the cell (13). In addition, several Bcl-2 family
members are regulated by subcellular localization (7, 14-16). Although
Bcl-2 and Bcl-xL are essential components of the general apoptosis
regulatory machinery, they are relatively poor inhibitors of the death
signals induced by TNF The MAPKs comprise three major sub-families in mammalian cells: 1) the
p38mapk subfamily; 2) the extracellular signal-regulated
kinases (ERK) p44mapk/erk1 and p42mapk/erk2; and 3) the
c-Jun NH2-terminal kinases (JNK). In work previously reported from this laboratory, we have shown that specific members of
each MAPK sub-family are rapidly and transiently activated in response
to stimulation of mouse macrophages with TNF Recent studies have suggested that the activation of
p42mapk/erk2 in the context of other pro-apoptotic signals
confers a dominant pro-survival advantage (29, 30). For example,
activation of MEK1, an upstream activator of p42mapk/erk2, has
been shown to antagonize Fas-triggered cell death through the
up-regulation of Casper/FLIP (29). Activation of p42mapk/erk2
has also been shown to be protective against TNF Materials--
Rabbit polyclonal anti-Bcl-xL (Ab 1690),
goat polyclonal anti-CD120a (Ab 1069), anti-TRADD (Ab 1165), and mouse
monoclonal anti-Bcl-2 (Ab 7382) antibodies were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). The hamster monoclonal agonistic (Ab 80-4004-01) and antagonistic anti-CD120a (Ab 80-4005-01) antibodies and the monoclonal anti-CD120b antibody (Ab 80-4009-01) were purchased from R&D Systems (Minneapolis, MN). Anti-myc and anti-tubulin mouse
monoclonal antibodies were from Sigma Chemical Co. (St. Louis, MO).
Mouse monoclonal anti-caspase-8 (Ab 66231A) and anti-FADD (Ab 65751A)
were from PharMingen (San Diego, CA). The mouse monoclonal anti-FLAG antibody (M2) was from Kodak-Scientific Imaging
Systems (Rochester, NY). The mouse monoclonal phosphospecific anti-ERK antibody (E10) was from New England BioLabs (Beverly, MA). Fluorescent secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The expression vector encoding CD120a
has been described previously (27). All deletion and point mutants were
constructed using overlapping polymerase chain reaction (34) and
verified by restriction enzyme analysis and nucleotide sequencing. The
expression vectors for p42mapk/erk2 and MEK1.ca were a generous
gift from Dr. Lynn Heasley, University of Colorado Health Sciences
Center, Denver, CO. The vector expressing Bcl-xL was kindly provided by
Dr. Hong Zhou, Boston, MA. The vector expressing Bcl-2 in pcDNA3
was a gift from Dr. Gary Johnson, National Jewish Medical and Research
Center, Denver, CO. The vectors expressing myc-TRADD and FLAG-FADD were
provided by Dr. Hong-Bing Shu, National Jewish Medical and Research
Center, Denver, CO.
Transfections and Confocal Immunofluorescence
Microscopy--
HeLa cells were maintained in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, 100 units/ml
penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine.
Approximately 6 × 104 cells/well were seeded in
12-well plates containing 18-mm glass coverslips and grown in 5%
CO2. Cells were transfected with 250 ng of DNA the
following day using LipofectAMINE reagent (Life Technologies). 14 h after transfection, the cells were washed with PBS, fixed for 15 min
at room temperature in a solution containing 3% (w/v) paraformaldehyde
and 3% (w/v) sucrose in PBS (pH 7.5), washed again, and permeabilized
with 0.2% (v/v) Triton X-100 for 10 min. The cells were then washed,
blocked for 30 min in Hank's balanced salt solution (without
Mg2+, Ca2+, or phenol red, pH 7.2) containing
5% normal donkey serum and then incubated with the primary antibodies
(1:100) in blocking solution for 2 h. After washing with PBS, the
cells were incubated for 1 h with Cy3- and/or
fluorescein-conjugated donkey secondary antibodies (1:200). Staining
for CD120a was performed using a primary hamster monoclonal antibody
(1:200), and a Cy3-conjugated F(ab')2 goat anti-hamster IgG
in blocking solution containing 5% normal goat serum. The coverslips
were incubated overnight in PBS supplemented with 0.02% sodium azide
and mounted in a solution containing 90% glycerol, 10% Tris-HCl 0.1 M, pH 8.5, and 20 mg/ml o-phenylenediamine as an
anti-fading agent. To visualize the nuclei, cells were incubated with
10 µg/ml Hoechst 33342 together with the secondary antibodies. Cells
were observed with a Leica DMR/XA fluorescence microscope using a 100×
plan objective. Digital images were captured using a SensiCam
camera, deconvolved using the software Slidebook 2.6 (Intelligent
Imaging Innovations, Inc., Denver, CO) to remove fluorescence that was
not in focus, and processed using Adobe Photoshop 5.0 (Adobe Systems,
Inc.).
TUNEL Assay--
HeLa cells grown on coverslips and transfected
18-24 h earlier with the appropriate expression vectors were washed
with PBS, fixed and permeabilized as described above, and incubated
with terminal transferase reaction solution containing
fluorescein-conjugated dUTP for 1 h at 37 °C as recommended by
the manufacturer (Roche Diagnostics Corp., Indianapolis, IN). The cells
were washed three times with 0.03 M sodium citrate, pH 7.4, containing 0.3 M sodium chloride, to remove unbound
nucleotides, then washed with PBS. Cells were then blocked and
incubated with antibodies as above. The percentage of TUNEL-positive
cells among transfected cells was determined by counting at least 200 cells with a confocal microscope.
Caspase-8 Cleavage--
2 × 105 cells grown in
6-well plates were transfected with 500 ng of DNA using the
LipofectAMINE reagent. 18 h after transfection, the cells were
washed and lysed in Nonidet P-40 lysis buffer (50 mM Tris
buffer, pH 8.0, containing 1% Nonidet P-40, 137 mM NaCl, and protease inhibitors). Post-nuclear supernatants were subjected to
SDS-PAGE and transfer to nitrocellulose membranes. Procaspase-8 was
detected by Western blotting, and quantified by scanning densitometry using the IMAGE 1.6 software (National Institutes of Health).
NF- Co-immunoprecipitation and Western Analysis--
Approximately
1.25 × 106 HEK293GT cells/well were seeded on 100-mm
plates and grown in 5% CO2. Cells were transfected the
following day by the calcium phosphate precipitation method.
Transfected cells were lysed in 1 ml of immunoprecipitation buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100,
1 mM EDTA, 1 mM NaF, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM Na3VO4).
Post-nuclear supernatants were precleared for 1 h with 25 µl of
protein A/G+ beads (Santa Cruz Biotechnology), then incubated for
1 h at 4 °C with 3 µg of monoclonal hamster anti-CD120a, or 3 µg of monoclonal hamster anti-CD120b or non-immune hamster antibody
as controls. Immune complexes were precipitated with 50 µl of protein
A/G+ beads for 1 h at 4 °C. The Sepharose beads were washed
four times with immunoprecipitation buffer. The precipitates were
fractionated on SDS-PAGE under non-reducing conditions, and subsequent
Western blotting analysis was performed as described previously
(27).
Phosphorothioate Oligonucleotides--
Antisense DNA
oligonucleotides with a phosphorothioate backbone were
synthesized and purified by high pressure liquid chromatography (BIOSOURCE International, Camarillo, CA). The
oligonucleotides were 3'-biotinylated to allow staining and to prevent
them from interfering with the TUNEL assay. The 20-mer antisense
oligonucleotide (2009) targeted against the coding region of the
bcl-2 gene, and the scrambled-sequence oligonucleotide with
a similar nucleotide content (sc48), were described previously (37). A
BLASTN search of the NCBI DNA data base revealed no homology of the
oligonucleotides to other human genes. The sequences were as follows:
antisense, 5'-AATCCTCCCCCAGTTCACCC-3'; scrambled,
5'-CTCATTCCTACCGACACCCC-3'. Oligonucleotides were transfected for
24 h using LipofectAMINE Plus reagent as recommended by the
manufacturer (Life Technologies). Medium was changed 7 h after the transfection.
Results shown for all experiments are representative of at least three
separate experiments.
Phosphorylation of CD120a Protects from Apoptosis--
Given the
striking changes in the subcellular distribution of CD120a that result
from its phosphorylation by p42mapk/erk2 (27), we investigated
the effect of phosphorylation on receptor function and signaling,
including CD120a-mediated apoptosis and NF-
To determine if the phosphorylation of CD120a simply rendered the
receptor non-functional we investigated the effect of phosphorylation on the ability of CD120a to activate NF- TRADD, FADD, TRAF2, Casper/FLIP, and Caspase-8 Are Not Recruited by
CD120a.4D/E--
Following the binding of TNF
To verify that we could detect the interaction of these proteins by
confocal microscopy, we tested whether caspase-8 was recruited to
"filaments" formed by the overexpression of FADD and TRADD as
previously reported (40) as well as to the CD120a-associated tubular
structures. Cells were transfected with the CD120a.4D/E mutant
receptor, and stained for endogenous caspase-8. As a control, cells
were transfected with FLAG-tagged FADD or myc-tagged TRADD, and then
co-stained for caspase-8 and either the FLAG or myc epitopes. As shown
in Fig. 3 (row e), caspase-8 exhibited a diffuse staining pattern in untransfected cells and this was not affected by
transfection of the CD120a.4D/E mutant receptor. Consistent with
previous reports (21, 40), a proportion of cells transfected with FADD
showed thick and extensive filaments in the cytoplasm as well as in (or across) the nucleus, the latter imprinting the surface of the nuclear
envelope as seen by Nomarski imaging or Hoechst staining (Fig. 3,
row g). Cells transfected with TRADD also showed cytoplasmic filaments (Fig. 3, row f). However, in contrast to what was
seen with the phosphorylation mimic CD120a.4D/E, caspase-8 was
co-localized with the filaments formed by FADD and TRADD (Fig. 3,
rows f and g). Thus, caspase-8 is recruited to
FADD and TRADD filaments but not to CD120a-associated tubular
structures. Collectively, these results suggest that the
down-regulation of apoptosis associated with phosphorylation of CD120a
does not involve the recruitment of TRADD, FADD, TRAF2, Casper/FLIP, or
caspase-8.
Recruitment of Bcl-2 but Not of Bcl-xL by Phosphorylated
CD120a--
Because the mechanism underlying the protection from
apoptosis by phosphorylated CD120a did not appear to involve
sequestration of the pro-apoptotic adapter molecules TRADD, FADD, or
caspase-8, we next explored the hypothesis that protection may be
mediated by the recruitment of pro-survival factors of the Bcl-2
family, especially because Bcl-2 is associated with intracellular
membranes especially mitochondria and the endoplasmic reticulum (41). HeLa cells were transfected with the CD120a.4D/E mutant receptor and
stained for endogenous Bcl-2 and CD120a. Endogenous Bcl-2 was detected
in the cytoplasm and was associated with small granular structures
(Fig. 4, a and b),
consistent with previous reports (42). This pattern was not
significantly affected by transfection of wild type CD120a (Fig. 4,
e-h). However, transfection of the CD120a.4D/E mutant
receptor induced a dramatic redistribution of Bcl-2 to cytoplasmic
tubular structures (Fig. 4, c and d). Double-staining experiments showed precise co-localization of the
phosphorylation mimic CD120a.4D/E and Bcl-2, indicating that endogenous
Bcl-2 is recruited to the tubules formed by phosphorylated CD120a (Fig.
4, i-l). Cotransfection of vectors encoding Bcl-2 and
CD120a.4D/E also resulted in the recruitment of Bcl-2 to
CD120a-containing tubules (data not shown). Bcl-2 was also recruited to
the tubular structures formed when wild type CD120a was phosphorylated
by cotransfection with MEK1.ca and p42mapk/erk2 (Fig. 4,
m-p). Colocalization of Bcl-2 and CD120a.4D/E was also observed in cells fixed and permeabilized with methanol instead of
paraformaldehyde and Triton X-100, respectively, excluding the
possibility that this association could be the result of the use of
detergents (43).
We next tested whether the interaction between Bcl-2 and phosphorylated
CD120a could be detected as a protein complex by
co-immunoprecipitation. CD120a or the CD120a phosphorylation mutants
were co-expressed with Bcl-2 in HEK293 cells. As can be seen in Fig.
4q, Bcl-2 was co-immunoprecipitated with the CD120a.4D/E
mutant receptor but not with wild type CD120a.wt. Bcl-2 was also
co-immunoprecipitated with CD120a when phosphorylated by co-expression
with MEK1.ca and p42mapk/erk2, a point confirmed by the gel
shift of the phosphorylated receptor upon SDS-PAGE (Fig.
4q). No interaction was observed between Bcl-2 and the death
domain-inactive CD120a.L351N, suggesting that different mechanisms
account for the inability of this receptor mutant and the
phosphorylated CD120a to protect against the induction of apoptosis.
To determine if the recruitment of Bcl-2 by phosphorylated CD120a was
specific, we also stained for Bcl-xL. Bcl-xL was mainly expressed in
cytoplasmic structures (Fig. 5) (mostly
mitochondria, as assessed by co-localization with Mitotracker orange,
data not shown), but was not recruited to the tubular structures
containing CD120a.4D/E (Fig. 5). Bcl-xL was also absent from
CD120a-containing tubules in HeLa cells that had been cotransfected
with CD120a.wt, MEK1.ca, and p42mapk/erk2 (Fig. 5). These
findings thus indicate that phosphorylated CD120a is targeted to the
endoplasmic reticulum where it preferentially interacts with Bcl-2.
Bcl-2 Is a Critical Determinant in the Regulation of Apoptosis by
Phosphorylated CD120a--
We next examined whether the protection
against apoptosis afforded by phosphorylated CD120a was mediated by
Bcl-2. HeLa cells were transfected for 24 h with a 3'-biotinylated
Bcl-2 antisense phosphorothioate oligonucleotide, or an
oligonucleotide of the same nucleotide content but with a scrambled
sequence as a control. Staining with Cy5-streptavidin confirmed the
efficient nuclear delivery of the oligonucleotides in 100% of the
cells (Fig. 6a). Western blot
analyses showed decreased Bcl-2 protein levels in cells treated with
the Bcl-2 antisense oligonucleotide at concentrations of at least 300 nM, whereas treatment with the control oligonucleotide had
little effect (Fig. 6b). Protein levels of Bcl-xL and
CD120a, a death receptor of the TNF receptor superfamily, induces
apoptosis in certain cell types upon ligation by TNF Recently reported studies have established the concept that activation
of p42mapk/erk2 confers survival advantages to cells in the
face of activation of apoptotic pathways. For example, Xia et
al. (32) showed that apoptosis induced by growth factor withdrawal
from PC12 cells could be overcome by dominant activation of
p42mapk/erk2. Similarly, the activation of T-cells by
concanavalin A was shown by Yeh et al. (29) to antagonize
Fas-triggered cell death through the MEK1-dependent
up-regulation of Casper/FLIP, indicating that cross-talk between
p42mapk/erk2 and the pro-apoptotic signals induced by Fas
ligation results in a dominant survival response. Therefore, to
distinguish between (i) direct inhibition of CD120a-induced apoptosis
by the phosphorylated receptor and (ii) potential secondary (or
additional) CD120a-independent pro-survival effects of activated
p42mapk/erk2 we studied the mechanism of protection against
apoptosis by phosphorylated CD120a with a mutant receptor in which the
p42mapk/erk2 phosphorylation sites were mutated to Glu and Asp
residues (CD120a.4D/E) to mimic the effects of phosphorylation on
receptor function (27). The findings from the present study reveal a
novel mechanism by which p42mapk/erk2 protects against
CD120a-mediated apoptosis, namely, redistribution of the
phosphorylated receptor to tubular structures associated with the
endoplasmic reticulum wherein it serves as a platform (or signal) for
the co-recruitment of Bcl-2.
The mechanism underlying the protective effect conferred by the
phosphorylated receptor may be attributable to the loss of the
dephosphorylated receptor from its primary site of apoptotic signaling, and/or to a pro-survival effect mediated by Bcl-2. We
propose that both mechanisms contribute a role in the protection from
apoptosis by the phosphorylated receptor. First, work reported by
Schutze et al. (45) has shown that the endocytic inhibitor monodansylcadaverine inhibits the induction of death following cross-linking of CD120a, and studies initially reported by Bradley et al. (46) and confirmed by other groups including our own have conclusively shown that an extensive pool of CD120a exists in the
trans-Golgi network (27, 47). Likewise, Bennett and colleagues (48) have reported that Fas is localized within the Golgi
complex and that disruption of the Golgi complex with brefeldin A
inhibits the ability of Fas to induce apoptosis. Thus, although ligation of the cell surface receptor would intuitively seem likely to
be necessary for the initiation of apoptosis, these recent studies
support the contention that the initiation of apoptotic signaling by
death receptors may involve assembly of the signaling complex within
the broad spatial confines of the Golgi complex. In contrast, other
functions of CD120a, such as the activation of proline-directed protein
kinases, occur independently of receptor internalization (45). Because
phosphorylation of CD120a by p42mapk/erk2 promotes its
translocation from the Golgi complex to the endoplasmic reticulum while
preserving a level of receptor expression at the cell surface, we
speculate that the inability of the phosphorylated receptor to signal
apoptosis may result in part from selective receptor loss from the
Golgi complex while the fraction preserved at the cell surface may be
responsible for signaling NF- Second, although initial reports suggested that Bcl-2 was incapable of
protecting against apoptosis induced by death receptors, recent studies
have clarified the mechanisms and conditions under which Bcl-2 is
protective against death receptors ligation (17). In particular, in
type II cells, of which HeLa cells are a representative (49, 50), Bcl-2
inhibits apoptosis by blocking the release of cytochrome c
from mitochondria (51, 52). Our findings also show that Bcl-2 is
involved in the protection against apoptosis that results from CD120a
phosphorylation, because antisense oligonucleotide-mediated down-regulation of Bcl-2 protein reversed the protection against apoptosis afforded by the CD120a.4D/E expression. In addition, bulk
cleavage of pro-caspase-8 in type II cells has been shown to occur as a
consequence of cytochrome c release from mitochondria and
the ensuing activation of caspase-9 (17). We also observed an absence
of bulk pro-caspase-8 processing in response to CD120a.4D/E expression,
a finding that is consistent with the conclusion that Bcl-2 serves to
inhibit the activation of the mitochondrial amplification pathway in
these cells. Thus, Bcl-2 is involved in the protection against
apoptosis conferred by CD120a phosphorylation, and this likely is
mediated by the prevention of cytochrome c release from mitochondria.
The mechanism by which the phosphorylated CD120a·Bcl-2 complex is
formed upon CD120a phosphorylation and how it specifically protects
against CD120a-induced apoptosis remains to be investigated. We
speculate that the recruitment of Bcl-2 to the CD120a-associated endoplasmic reticulum tubules serves to recruit other pro-apoptotic Bcl-2 family members thereby preventing these molecules from
interacting with mitochondria in initiating cytochrome c
release. A similar mechanism has recently been proposed to explain the
pro-survival effect of the adenoviral protein E1B 19K, a distantly
related Bcl-2 homolog that has been shown to inhibit FADD-induced death upstream of caspase-8 (21) and to interact with tBid-activated Bax,
thereby preventing Bax from promoting cytochrome c release from mitochondria (50). Other pro-apoptotic molecules such as Bak, Bim,
and Bid are also potential candidates that could potentially be
sequestered by the phosphorylated CD120a·Bcl-2 complex. Although an
attractive hypothesis, we consider it less likely that the phosphorylated CD120a·Bcl-2 complex would recruit non-Bcl-2 family pro-apoptotic proteins such as Apaf-1 or effector caspases, because it
has been quite difficult to detect interactions between these proteins
in mammalian cells (53, 54). Our findings also raise the question of
the possible role of the endoplasmic reticulum in the protection
against apoptosis conferred by CD120a phosphorylation. Previous work
has shown the endoplasmic reticulum to be a major site of Bcl-2
localization (41) and protection against apoptosis. Deletion of the
C-terminal 20-residue hydrophobic membrane insertion domain abrogates
or diminishes the death-inhibitory effects of Bcl-2 (42), indicating
that the subcellular localization of Bcl-2 may contribute to its
anti-apoptotic function. Similarly, the pro-survival effect of Bcl-2 is
affected by its localization to either the endoplasmic reticulum or
mitochondria in a cell-dependent fashion (55). Furthermore,
Hacki and colleagues (56) have recently shown that the endoplasmic
reticulum-targeted expression of Bcl-2 also prevented the release of
cytochrome c from mitochondria thus emphasizing the
potential of cross-talk between these two organelles in the regulation
of apoptosis. Unexpectedly, although Bcl-2 was recruited to
phosphorylated CD120a, Bcl-xL was not. The significance of this finding
is currently unclear. However, while in many situations Bcl-2 and
Bcl-xL exhibit similar activities, differences in their activities have
previously been noted. For example, Bcl-xL, but not Bcl-2, has been
found to redistribute from the cytosol to intracellular membranes upon
induction of thymocyte apoptosis in response to dexamethasone (57),
whereas FK506 and cyclosporin-induced apoptosis in WEHI-231.7 cells is prevented by enforced expression of Bcl-xL but not by Bcl-2 (58).
In conclusion, we have shown that phosphorylated CD120a recruits Bcl-2
to tubular structures associated with the endoplasmic reticulum and
that Bcl-2 contributes to the protection against apoptosis that arises
as a consequence of CD120a phosphorylation. Phosphorylation of CD120a
may thus contribute to the promotion of resistance to death
receptor-induced apoptosis in tumor cells while at the same time
preserving the ability of the receptor to activate NF- receptor CD120a initiates responses as diverse as apoptosis and the
expression of NF-
B-dependent pro-survival genes. How
these opposing responses are controlled remains poorly understood. Here
we demonstrate that phosphorylation by p42mapk/erk2 inhibits
the apoptotic activity of CD120a while preserving its ability to
activate NF-
B. Phosphorylated CD120a is re-localized from the Golgi
complex to tubular structures of the endoplasmic reticulum wherein it
recruits Bcl-2. Antisense-mediated down-regulation of Bcl-2 antagonized
the localization of CD120a to tubular structures and reversed the
protection from apoptosis conferred by receptor phosphorylation. We
propose that phosphorylation of CD120a represents a novel,
Bcl-2-dependent mechanism by which the apoptotic activity of the receptor may be regulated. Thus, oncogenic activation of p42mapk/erk2 may serve to inhibit the apoptotic activity of
this death receptor while preserving NF-
B-dependent
responses and may thus indirectly contribute to a failure to eliminate
cells bearing oncogenes of the Ras-Raf-MEK-p42mapk/erk2 pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF
)1 receptor CD120a
(p55) sets in motion the activation of signaling pathways that lead to
apoptosis in certain cells (1). Interaction with TNF
leads to
trimerization of the receptor and to the binding of the adapter
molecule TRADD through death domain·death domain interactions
(2). The complex then recruits several signaling molecules, including
TRAF2 and RIP, which stimulate MAPK and NF-
B activation pathways
(3-5), and FADD, which mediates activation of apoptosis by recruitment
of caspase-8 (3). The requirement of the FADD-caspase-8 pathway in
CD120a-mediated apoptosis has been elegantly demonstrated by the
resistance of cells derived from FADD-knock-out mouse embryos to
TNF
-induced apoptosis (6). Activation of caspase-8 leads to the
activation of caspase-3 and the cleavage of Bid to generate a
truncated, pro-apoptotic fragment (tBid) capable of inducing the
release of cytochrome c from mitochondria (7) and thereby
promoting apoptosis.
-mediated apoptosis
have been shown to be negatively regulated by several molecules, including Casper/FLICE-inhibitory protein (FLIP) (8, 9), Toso (10), the
inhibitor of apoptosis family of proteins (IAP), including c-IAP1,
c-IAP2, XIAP, and survivin/TIAP (11), as well as several members of the
Bcl-2 family (12). Casper/FLIP, a homolog of caspase-8, interacts with
FADD (8) and prevents the recruitment and activation of caspase-8,
thereby inhibiting the signaling cascade initiated by death receptors
(9), whereas Toso inhibits Fas-dependent apoptosis through
the induction of Casper/FLIP expression (10). In contrast, IAP proteins
have been shown to interact with, and inhibit, caspases 3, 7, and 9. In
addition, c-IAP1 and c-IAP2 are capable of binding to TRAF1 and TRAF2, although the mechanism by which they promote survival is
poorly understood (11).
and Fas-L in cells that efficiently activate
caspase-8 at the death-inducing signaling complex (DISC) (so-called
"type I" cells), but effectively block apoptosis in cells that
poorly activate caspase-8 at the DISC ("type II" cells) (13,
17-20). When present, the survival influence of Bcl-2 family proteins
in Fas-L or TNF
-induced apoptosis generally occurs downstream of the
activation of caspase-8 through the inhibition of cytochrome
c release from mitochondria (12, 21-23).
(24-26). Moreover,
whereas the targets of these kinases include transcription factors
involved in the inflammatory response, recent studies have also shown
these targets to include CD120a itself (27). In addition, we have shown
that activation of p42mapk/erk2 by other receptors,
e.g. growth factor receptors, also promotes phosphorylation
of CD120a. (28). As a result of phosphorylation, CD120a expression,
especially in the Golgi complex, is down-regulated and the receptor is
redistributed to tubular structures associated with the endoplasmic
reticulum (27). Thus, the phosphorylation of CD120a by
p42mapk/erk2 may represent a broad mechanism by which TNF
,
growth factors, and other stimuli may regulate the activity of CD120a.
-induced apoptosis of L929 cells (31), serum deprivation-induced apoptosis in PC12 cells (32), and UV-induced apoptosis in human primary neutrophils (33).
However, the p42mapk/erk2 substrates involved in the survival
responses are not known. Given the finding that CD120a itself is
phosphorylated upon activation of p42mapk/erk2 (27), we have
investigated the influence of CD120a phosphorylation on TNF
-induced
apoptosis. We report herein that the phosphorylation of CD120a by
p42mapk/erk2 inhibits TNF
-induced apoptosis in HeLa cells
through a Bcl-2-dependent mechanism. In contrast, the
ability of the receptor to activate NF-
B is unaffected by
phosphorylation. As we will show, these findings shed new light on how
phosphorylation of CD120a may contribute to the regulation of the
multiple functions of this death receptor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B Reporter Gene Assays--
Activation of NF-
B was
determined by a reporter gene assay. Briefly, 2.1 × 105 HEK293GT cells/well were seeded in 6-well cluster
plates. The following day, the media were replaced 1 h prior to
transfection with 0.5 µg/ml NF-
B-luciferase reporter gene plasmid
and varying amounts of CD120a constructs using the calcium phosphate
method as previously described (35). The cells were then maintained for
18-24 h prior to stimulation in fresh media with 0.5 µg/ml agonist
hamster anti-CD120a monoclonal antibody or an irrelevant IgG as control
for 6 h prior to lysis and determination of luciferase activity
(36).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation. To address
this question, we used HeLa cells, because previous studies have shown
these cells to undergo apoptosis through the endogenous receptor (38)
and hence they express the necessary signaling pathways to mediate this
response. In previous studies we induced receptor phosphorylation by
co-transfection with constitutively active MEK1 and
p42mapk/erk2. However, these kinases have been shown to affect
the balance between apoptosis and survival (29-32) and thus the
activation of p42mapk/erk2, per se, may affect the
ability of CD120a to induce apoptosis in a CD120a
phosphorylation-independent fashion in cells co-transfected with
p42mapk/erk2 and MEK1.ca. Therefore, to address the role of
phosphorylation of CD120a on TNF
-induced apoptosis in the absence of
potential artifacts introduced by activated p42mapk/erk2, HeLa
cells were transfected with expression vectors encoding wild type
CD120a and CD120a.4D/E, a mutant receptor that we have previously shown
to mimic the phosphorylated receptor by being redistributed to the
endoplasmic reticulum in the absence of p42mapk/erk2 (27).
14 h later, the cells were stimulated with TNF
and
cycloheximide (50 ng/ml and 10 µg/ml, respectively) for 4 h,
fixed, permeabilized, and stained for CD120a under conditions that did
not permit the detection of the endogenous receptor. Apoptotic cells
were quantified on the basis of characteristic changes in the
morphology of the nucleus stained with the Hoechst dye 33342 (Fig.
1a). As expected, transfection
of wild type CD120a induced a robust increase in the proportion of
apoptotic cells, both in the presence and in the absence of TNF
and
cycloheximide. In contrast, transfection of the CD120a.4D/E mutant
receptor completely abolished apoptosis as compared with that induced
by the transfected wild type receptor. To confirm these results,
apoptotic cells were also identified by TUNEL staining. Similar results
were obtained when the percentage of TUNEL-positive cells among
CD120a-transfected cells was quantified by confocal fluorescence
microscopy (Fig. 1b). In addition, the inability of the
transfected CD120a.4D/E mutant to induce apoptosis was similar to that
of CD120a.L351N, a receptor bearing a point mutation in the death
domain known to abolish the apoptotic activity of the receptor (39). In
contrast, the CD120a.4A (T236A, S240A, S244A, S270A) mutant receptor,
in which the four p42mapk/erk2 consensus sites had been mutated
to Ala residues, did not protect the cells from the induction of
apoptosis (data not shown). To further confirm the effect of
phosphorylation of CD120a on the activation of the apoptotic process,
transfected HeLa cells were lysed and equal amounts of proteins from
post-nuclear lysates were resolved by SDS-PAGE and subjected to Western
blotting for procaspase-8 using an antibody that specifically
recognizes uncleaved and inactive procaspase-8. As shown in Fig.
1c and as expected, expression of wild type CD120a induced
the processing of procaspase-8 (as did TRADD, which was used as an
alternative positive control). In contrast, expression of the
CD120a.4D/E mutant receptor did not induce the processing of
procaspase-8.
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Fig. 1.
Phosphorylation of CD120a protects from the
induction of apoptosis. HeLa cells were transfected with wild-type
CD120a or mutants of CD120a as indicated. 14 h after transfection,
the cells were stimulated with TNF and cycloheximide
(CHX) (50 ng/ml and 10 µg/ml, respectively, for 4 h),
subjected to the TUNEL assay, and stained for CD120a. Nucleic acids
were stained with the Hoechst 33342 dye. The percentage of apoptotic
cells among CD120a-transfected cells was quantified using a confocal
fluorescence microscope, on the basis of characteristic changes in the
morphology of the nucleus (a) or by the TUNEL assay
(b). c, transfected cells were lysed, and equal
amounts of proteins were subjected to Western blotting for uncleaved
procaspase-8, or
-tubulin as a control. Signals were quantified by
scanning densitometry.
B using an
NF-
B-dependent luciferase reporter gene assay. Three
approaches were taken. First, HEK293 cells were transfected with
expression constructs for wild type CD120a and CD120a.4D/E under
conditions that led to constitutive activation of NF-
B as a result
of receptor overexpression. As can be seen in Fig.
2a, transfection of equal
amounts of both wild type CD120a and the phosphorylation mimic
CD120a.4D/E led to approximately equivalent levels of
NF-
B-dependent luciferase reporter gene expression.
Second, we expressed low levels of both receptors in HEK293 and
specifically activated the transfected receptors by cross-linking with
an agonistic hamster anti-mouse CD120a monoclonal antibody. As can be
seen in Fig. 2b, cross-linking of the transfected receptors
also resulted in approximately equivalent increases in luciferase
reporter gene activity, whereas a control hamster monoclonal antibody
(anti-mouse CD120b) was without effect. Third, HEK293 cells were
co-transfected with the NF-
B-luciferase reporter construct and
equivalent amounts of either wild type CD120a, CD120a.4D/E, or empty
vector (2.5 ng/ml) for 18 h prior to stimulation with mouse TNF
(10 ng/ml) for 6 h. As can be seen in Fig. 2c, although
transfection with the reporter gene and empty vector led to a low level
of luciferase expression, transfection with wild type CD120a or
CD120a.4D/E led to a similar increase in basal luciferase expression.
However, stimulation with mouse TNF
resulted in a
ligand-dependent increase in reporter gene expression that
was equivalent for both receptors. Thus, although the induction of
apoptotic activity was lost as a consequence of CD120a phosphorylation,
the ability to activate NF-
B was preserved. Furthermore, specific
ligation of the transfected receptors using both agonistic antibodies
and TNF
resulted in the activation of NF-
B thus indicating that
both the wild type and the phosphorylated receptors are available for
signaling this response.
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Fig. 2.
Activation of NF- B
is unaffected by the state of phosphorylation of CD120a.
a, HEK293 cells were co-transfected with either wild type
CD120a or CD120 4D/E (200 ng of each construct) and the NF-
B
luciferase reporter gene construct. 18 h after transfection, the
cells were lysed and assayed for luciferase activity. b,
wild type CD120a or CD120a.4D/E were expressed at low level in HEK293
cells by co-transfection with 2.5 ng of each construct with the NF-
B
luciferase reporter gene construct. 18 h after transfection, the
cells were stimulated with 0.5 µg of either agonistic hamster
anti-mouse CD120a, hamster anti-CD120b as a control, or medium alone.
Luciferase activity was quantified in cell lysates after 6 h of
stimulation with the indicated antibodies. c, HEK293 cells
were transfected as in b but were stimulated with mouse
TNF
(10 ng/ml for 6 h) before determining luciferase
activity.
to CD120a, the
adapter molecule TRADD binds through its death domain to the death
domain of CD120a (2) thereby forming a platform for the assembly of
other signaling molecules of the apoptosis cascade, including FADD (3) and caspase-8 (3). Other molecules with pro-survival activity such as
TRAF2 and Casper/FLIP are also recruited to the receptor complex. Thus,
protection from apoptosis by phosphorylated CD120a may result from
sequestration of the pro-apoptotic molecules TRADD and FADD, thereby
removing them from their defined sites of action, or from the
recruitment of pro-survival molecules to phosphorylated CD120a-associated tubules. To address this question, HeLa cells were
transfected with the CD120a.4D/E mutant receptor and were co-stained
for CD120a, and TRADD or FADD. As shown in Fig.
3, both endogenous TRADD and FADD showed
a homogenous intracellular staining pattern, which was not affected by
expression of the CD120a.4D/E mutant receptor (Fig. 3, rows
a and b). TRAF2, was also absent from the tubular
structures containing CD120a.4D/E (Fig. 3, row c), rendering
the involvement of cIAP proteins in the survival effect of the
phosphorylated receptor unlikely. Similarly, co-expression of
myc-tagged Casper/FLIP and CD120a.4D/E resulted in a lack of
co-localization (Fig. 3, row d), consistent with the failure
of FADD to be recruited by CD120a.4D/E and with the conclusion that
Casper/FLIP is unlikely to be involved in the pro-survival effect of
CD120a.4D/E. Thus, the adapters TRADD, FADD, and TRAF2, and the
pro-survival molecule Casper/FLIP, are not recruited to the tubular
structures formed by phosphorylated CD120a.
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Fig. 3.
TRADD, FADD, TRAF2, Casper, and
caspase-8 are not recruited by CD120a.4D/E. HeLa cells were
transfected with CD120a.4D/E (a-c, e),
cotransfected with CD120a.4D/E and myc-Casper (d), or
transfected with myc-TRADD (f) or FLAG-FADD (g).
The cells were fixed, permeabilized, stained as indicated, and observed
by confocal fluorescence microscopy. Text in italics refers
to staining; roman text refers to transfection.
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Fig. 4.
CD120a.4D/E and ERK-phosphorylated
CD120a but not CD120a.wt recruit Bcl-2. HeLa cells were
transfected with CD120a.wt (e-h), CD120a.4D/E
(c, d, j-l), or cotransfected with
CD120a.wt, p42mapk/erk2, constitutively active MEK1 and Bcl-2
(m-p). The cells were stained for CD120a and Bcl-2, and
observed by confocal fluorescence microscopy. Text in
italics refers to staining; roman text refers to
transfection. q, Bcl-2 interacts with ERK-phosphorylated
CD120a in intact cells. Bcl-2 was coexpressed with empty vector
(lane 1), CD120a.wt (lanes 2-5, 8,
9), CD120a.4D/E (lane 6), CD120a.L351N
(lane 7), or CD120a.wt together with p42mapk/erk2
and constitutively active MEK1 (lane 10) in human HEK293
cells. CD120a was immunoprecipitated as indicated under "Experimental
Procedures" using hamster non-immune IgG (lanes 1,
3, 8), no antibody (lane 2), hamster
monoclonal anti-CD120b antibody as control (lane 4), or
hamster monoclonal anti-CD120a antibody (lanes 5-7,
9, 10). Lysates and immunoprecipitates were
separated by SDS-PAGE and subjected to Western blotting with
anti-CD120a and anti-Bcl-2 antibodies. The upper arrow
indicates the gel shift of P-CD120a on SDS-PAGE.
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Fig. 5.
Bcl-xL is not recruited by
phosphorylated CD120a. HeLa cells were cotransfected with Bcl-xL
and CD120a.4D/E, or CD120a.wt, p42mapk/erk2, constitutively
active MEK1 and Bcl-xL. The cells were stained for CD120a and Bcl-xL
and observed by confocal fluorescence microscopy. Text in
italics refers to staining; roman text refers to
transfection.
-tubulin were unchanged, indicating that the effect of the antisense
oligonucleotide was specific for Bcl-2 (Fig. 6b). To examine
whether Bcl-2 accounts for some of the pro-survival effect of
phosphorylated CD120a, HeLa cells were cotransfected with the
CD120a.4D/E mutant receptor together with the Bcl-2 antisense or
control oligonucleotides. HeLa cells were also transfected with wild
type CD120a as a control. The conditions for both receptors were
carefully selected to yield a low level of apoptosis in the absence of
antisense Bcl-2 oligonucleotide thereby ensuring that we would be able
to detect any increase in the degree of apoptosis in the presence of
the oligonucleotide. It was also for this reason that we elected to use
modest receptor overexpression to induce the necessary low level of
apoptosis in the controls as opposed to inducing apoptosis with
ligand exposure. 24 h after transfection, the cells were fixed,
subjected to TUNEL assay, and stained for CD120a. In cells transfected
with the antisense Bcl-2 oligonucleotide, the level of expression
of CD120a was greatly reduced in apoptotic cells and the
localization of CD120a.4D/E to cytoplasmic tubules was lost. Instead,
the receptor exhibited a dim intracytoplasmic punctate staining (Fig.
6c), suggesting that Bcl-2 may be required for the
localization of the phosphorylated receptor to the tubular structures
associated with the endoplasmic reticulum. Importantly, treatment with
the Bcl-2 antisense oligonucleotide (300-450 nM) also
induced a significant increase in the level of apoptosis induced by
CD120a.4D/E, as shown in Fig. 6d. The Bcl-2 antisense
oligonucleotide also potentiated apoptosis in cells transfected with
the wild type receptor, although to a lesser extent than that seen with
CD120a.4D/E. The apoptosis observed was CD120a-specific, because the
antisense oligonucleotide did not promote cell death in cells
transfected with the empty vector. In contrast, treatment with the
control oligonucleotide induced little increase in apoptosis,
indicating that most of the effect of the Bcl-2 antisense
oligonucleotide was sequence specific. However, some
sequence-independent effects of the oligonucleotides may also have
occurred to a much lower extent, as reported (44). Taken together,
these data demonstrate that Bcl-2 forms a complex with phosphorylated
CD120a and participates in the protection against apoptosis conferred
by these events.
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Fig. 6.
Bcl-2 participates in the survival activity
of phosphorylated CD120a. HeLa cells were cotransfected for
24 h with CD120a mutants as indicated, together with 300-450
nM 3'-biotinylated Bcl-2 antisense phosphorothioate
oligonucleotides, or a control oligonucleotide of the same nucleotide
content but scrambled sequence. a, staining with
Cy5-conjugated streptavidin showed the efficient nuclear delivery of
the oligonucleotides. b, equal amounts of proteins from
cells lysates were subjected to Western blot analysis for Bcl-2,
Bcl-xL, and -tubulin. Concentration of antisense (lanes
1-4) or scrambled (lanes 5-8) oligonucleotides was 0 (lanes 1 and 5), 150 nM (lanes
2 and 6), 300 nM (lanes 3 and
7), 450 nM (lanes 4 and
8). c, 24 h after the transfection, cells
were subjected to the TUNEL assay and stained for CD120a. The
upper cell was transfected with the scrambled
oligonucleotide, whereas the lower cell was transfected with
the antisense oligonucleotide. d, cotransfection of the
antisense oligonucleotide restores the ability of P-CD120a to induce
apoptosis. The percentage of TUNEL-positive cells among
CD120a-transfected cells was quantitated using a confocal fluorescent
microscope.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. In the present
study we show that phosphorylation of CD120a by p42mapk/erk2
differentially regulates its function as a death receptor. When phosphorylated, CD120a loses its ability to induce apoptosis and thus
confers a survival advantage to cells expressing the phosphorylated receptor. However, the receptor retains its ability to stimulate the
activation of NF-
B either by high level overexpression or by agonist
antibody- or TNF
-mediated ligation of the transfected receptor
following low level expression. In addition, we show that Bcl-2 is
specifically recruited by phosphorylated CD120a to tubular structures
associated with elements of the endoplasmic reticulum and contributes
to the survival activity of the phosphorylated receptor. Thus, the
phosphorylated CD120a·Bcl-2 complex localized within endoplasmic
reticulum tubules may represent an alternative signaling complex that
is protective against apoptosis in contrast to the previously
described pro-apoptotic CD120a·TRADD complex.
B activation.
B, a
transcription factor that regulates the expression of both pro-survival
molecules, including c-IAPs as well as pro-inflammatory cytokines (59).
Whether similar events regulate the apoptotic pathway induced by other
death receptors such as Fas, or whether this mechanism is specific for
CD120a signaling, remains to be elucidated. However, these findings
suggest that post-translational modification of the cytoplasmic domain
of CD120a may play an important role in the regulation of receptor
function and may thus have significant implications in cell survival
and function in cancer and in chronic inflammatory diseases, which are
associated with persistent activation of the
Ras-Raf-MEK-p42mapk/erk2 pathway by activated oncogenes and
cytokines, respectively.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Linda Remigio and Cheryl Leu for outstanding technical assistance. We also wish to thank Dr. Gary L. Johnson and Nancy Johnson for their generous help with the confocal microscopy; Dr. Hong-Bing Shu for helpful discussions and for advice for co-immunoprecipitation experiments; and Drs. Lynn Heasley, Gary L. Johnson, Gabriel Nunez, Hong-Bing Shu, and Hong Zhou for providing us with expression vectors.
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States Public Health Services Grants HL55549 and SCOR HL 56556 from the National Institutes of Health.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 Traveling Fellowship grants from the Société de Pneumologie de Langue Française, the Association pour la Recherche contre le Cancer, Fondation Alain Philippe, Fondation Lavoisier du Ministère des Affaires Etrangères, and a Michael and Eleanore Stobin 1999 Pediatric Fellowship from National Jewish Medical and Research Center, Denver, CO.
§§ To whom correspondence should be addressed: Dept. of Pediatrics, National Jewish Medical and Research Center, Neustadt Rm. D405, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1188; Fax: 303-398-1381; E-mail: richesd@njc.org.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M010681200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor
necrosis factor-
;
TRADD, TNF receptor-associated death domain
protein;
FADD, Fas-associated death domain proteins;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated
kinase;
JNK, c-Jun NH2-terminal kinase;
MEK, MAPK/ERK
kinase;
MEK1.ca, constitutively active MEK1;
FLIP, FLICE-inhibitory
protein;
IAP, inhibitor of apoptosis family of proteins;
Fas-L, Fas
ligand;
DISC, death-inducing signaling complex;
PBS, phosphate-buffered
saline;
PAGE, polyacrylamide gel electrophoresis;
TUNEL, terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling;
TRAF2, TNF receptor-associated factor-2.
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