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INTRODUCTION |
Tumor necrosis factor receptor-1 mediates both the proinflammatory
and pro-apoptotic effects of the pleiotropic cytokine TNF (1,
2).1 The proinflammatory
effects are mediated by activation of the transcription factor NF-
B.
Recently it has been shown that NF-
B activation results in both the
transcriptional activation of proinflammatory genes, including IL-8 and
E-selectin (3-5), and in activation of a cell survival pathway
mediated at least in part by induction of anti-apoptotic IAP family
members (6-8). Therefore, quite paradoxically, two diametrically
opposed pathways emanate from TNFR-1: a cell death pathway and a cell
survival pathway mediated by activation of NF-
B.
The intracellular segment of TNFR-1 contains a 70-amino acid homophilic
interaction domain, dubbed the "death domain," which is required
for both signaling and NF-
B activation. Upon activation of TNFR-1, a
multi-component signaling complex is assembled by a series of
homophilic interactions (1). Initially, the death domain-containing
platform adapter molecule TRADD is recruited to TNFR-1 by virtue of a
homophilic death domain interaction (9). TRADD in turn binds the death
adapter molecule FADD (10, 11), which interacts with the zymogen form
of the initiator death protease caspase-8 (12). Subsequent activation
of caspase-8 by an induced proximity mechanism leads to amplification
of the death signal through proteolytic activation of downstream
caspase zymogens (13). Studies done with FADD-deficient embryonic
fibroblasts suggest that this is the major but not the only
pro-apoptotic pathway engaged by TNFR-1, because instead of being
completely resistant to TNF-induced apoptosis, 30% of FADD-null cells
are still sensitive (14, 15). Taken together, these studies suggest that there exists a subsidiary FADD-independent TNFR-1-initiated death
pathway. Earlier biochemical studies indicated that the TNFR-1-associated adapter molecule RAIDD might fulfill this function by
recruiting caspase-2 to the receptor signaling complex (16). However,
caspase-2 null cells do not show any loss of sensitivity to TNF-induced
cytotoxicity (17); therefore, the physiological significance of this
interaction remains unclear. Initial biochemical studies also suggested
a role for the TRADD-binding, RING finger containing adapter molecule
TRAF2 as the major conduit for the flow of NF-
B activating signals
from TNFR-1 (18, 19). Here again, targeted gene deletion showed that
contrary to expectations TRAF2 was not involved in mediating NF-
B
activation but rather engaged the JNK (c-Jun N-terminal kinase) pathway
(20, 21). The TRADD-associated adapter responsible for engaging NF-
B
was shown to be the death domain containing Ser/Thr kinase RIP (22). Overexpression of RIP is sufficient to engage NF-
B, and
RIP-deficient cells fail to activate NF-
B in response to TNF (23,
24). RIP possesses kinase activity as it autophosphorylates itself on
Ser/Thr residues. Surprisingly, the kinase domain is not required for
mediating NF-
B activation; rather, it is the intermediate domain
that resides between the kinase and death domain that mediates this
activity (25). It now appears that RIP is the prototypical member of an
emerging family of related molecules. RIP2 (also known as CARDIAK/RICK)
is a similar molecule possessing a remarkably conserved kinase domain
(26-28). However, instead of having a C-terminal death domain, it
contains a CARD motif, which, like the death domain, is a homophilic
interaction domain found within the prodomains of caspase-1, caspase-2,
and caspase-9 and a variety of adapter molecules including Apaf-1
(which uses its CARD segment to associate with caspase-9) and RAIDD
(which similarly associates with caspase-2) (29). The CARD motif in
RIP2 specifically binds to the prodomain of caspase-1 (27). RIP2
promotes the processing of caspase-1 zymogen to generate the protease
that cleaves and activates the proinflammatory cytokine pro-IL-1
.
Additionally, because of its ability to interact with TRAF adapter
molecules, RIP2 is recruited to TRAF-binding receptors, especially
CD-40 (26). It is therefore tempting to speculate that RIP2 may be part
of a link that connects certain proinflammatory receptors to the
generation of the proinflammatory cytokine IL-1
.
Herein, we report the identification and characterization of a third
RIP-related molecule designated RIP3. Like RIP2, its kinase domain is
similar to that of RIP. However, unlike either RIP or RIP2, RIP3
possesses neither a death domain nor a CARD motif at its unique C
terminus. RIP3 overexpression is a potent inducer of apoptosis, an
activity readily localizable to the unique C terminus. Additionally,
RIP3 binds RIP within the TNFR-1 signaling complex and attenuates its
ability to engage the NF-
B pathway. Inhibition of this survival
pathway and binding to initiator caspases may contribute to the
pro-apoptotic activity of RIP3. It is therefore possible that RIP3
represents the alternate death pathway engaged by TNFR-1, especially
because a dominant negative version of RIP3 partially blocks
TNFR-1-mediated cell death.
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MATERIALS AND METHODS |
Cloning of Human RIP3--
A proprietary incyte lifeseq® data
base was screened for sequences that encode homologues of RIP and RIP2.
One expressed sequence tag (incyte accession number 1626570) was found,
and a full-length cDNA encoding RIP3 was cloned from both a human
fetal brain cDNA library and a human aortic endothelial cDNA
library using standard PCR and hybridization protocols. Additional
matching human expressed sequence tag clones (including
GenBankTM accession numbers AI394293, AI082857, and
AA227673) were also identified.
Northern Blot Analysis--
Human multiple tissue
poly(A)+ RNA blots (CLONTECH)
containing 2 µg/lane poly(A)+ RNA were hybridized
according to the manufacturer's instructions using a
32P-labeled RIP3 probe encompassing amino acids 1-223 of
the RIP3 open reading frame.
Expression Vectors--
All eukaryotic expression vectors were
constructed in pFLAG-CMV-2 (N-terminal FLAG tag) or pcDNA3.1/
Myc-His (C-terminal Myc tag) using standard PCR techniques employing
custom-designed primers containing appropriate restriction sites.
Expression constructs encoding RIP, RIP2, TNFR-1, TRADD, CrmA, p35, and
various caspases have been described previously (26). GD tag is an
epitope generated at Genentech that has been described previously (30).
Mutation of the catalytic lysine to an alanine in RIP3 (K50A) was
accomplished by site-directed mutagenesis using the
QuickchangeTM kit from Stratagene. The presence of the
introduced mutation and fidelity of PCR replication were confirmed
by sequence analysis.
Cell Death Assays--
Human MCF7 breast carcinoma cells were
transiently transfected using the Geneporter transfection reagent (Gene
Therapy System). pCMV
-galactosidase was used as reporter plasmid
for marking transfected cells. 16-24 h following transfection, cells
were fixed with 0.5% glutaraldehyde and stained with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside. Approximately 300
-galactosidase-positive cells were assessed by
phase contrast microscopy from each transfection (n = 3) from three randomly selected fields. Viable or apoptotic cells were distinguished based on morphological alterations typical of adherent cells undergoing apoptosis including becoming rounded, condensed, and
detached from the dish. Expression of transfected pro-apoptotic gene
products was confirmed by immunoblotting.
Co-immunoprecipitation and Western Blot Analysis--
293-EBNA
(293E) cells were transiently transfected with the indicated
constructs. Where necessary, a CrmA expression construct was included
to suppress apoptosis. Cells were harvested 24-26 h post-transfection
and lysed in 1 ml of lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40).
Resulting lysates were subjected to immunoprecipitation with antibodies
directed to the epitope tag as described previously (26).
Immunoprecipitates were washed in lysis buffer, resolved by
SDS-polyacrylamide gel electrophoresis, and subsequently analyzed by
protein immunoblotting.
NF-
B Luciferase Assay--
293E cells (1.5 × 105 cells/well) were seeded onto 12-well plates and
transfected with 0.25 µg of a dual luciferase reporter gene plasmid
(10:1 ratio) and indicated amounts of each expression construct.
Additionally, p35 was included in each transfection assay to suppress
apoptosis. The total DNA concentration was kept constant by
supplementation with empty vector to 1 µg. Cells were harvested
24 h post-transfection, and reporter gene activity was determined
using the dual luciferase assay system (Promega).
In Vitro Kinase Assay--
Immunoprecipitated RIP3 and RIP3
(K50A) were obtained from transfected 293E cells and subjected to an
auto-kinase assay exactly as described previously for RIP2 (26).
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RESULTS AND DISCUSSION |
Cloning and Structure of RIP3--
Data base searching revealed a
partial cDNA that possessed substantial homology to the previously
characterized Ser/Thr kinase domain of RIP and RIP2 (RICK/CARDIAK). A
human fetal brain cDNA library was screened to obtain a full-length
cDNA. It contained a 1554-base pair open reading frame encoding a
novel 518-residue protein with a predicted molecular mass of 57 kDa
(Fig. 1A). An in-frame stop
codon was present upstream of the initiator methionine. Because it
shared extensive homology to the kinase domain of RIP (34% identity,
60% similarity) and RIP2 (31% identity, 58% similarity) (Fig.
1B), it was termed RIP3. However, the C terminus of RIP3 contained neither a death domain (like RIP) nor a CARD domain (like
RIP2) and had no discernible homology to any known protein.

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Fig. 1.
RIP3 sequence analysis, expression and kinase
activity. A, predicted amino acid sequence of RIP3. The
kinase domain is underlined, and the catalytic lysine is
indicated by an asterisk. B, the N-terminal
kinase domain of RIP3 shares substantial similarity to the
corresponding domain in RIP and RIP2. Darker shading
indicates identical residues, whereas lighter shading
indicates conserved substitutions. C, multiple human adult
tissue mRNA blot (CLONTECH) was probed with a
32P-labeled cDNA probe specific for RIP3. D,
in vitro kinase assay. 293E cells were transiently
transfected with empty vector (lane 1) or expression vectors
encoding Myc-tagged native RIP3 (lane 2) or Myc-tagged RIP3
(K50A) point mutant (lane 3). Cell lysates were
immunoprecipitated with Myc antibody 24 h following transfection.
The immunoprecipitates were subjected to in vitro kinase
assay (upper panel) or immunoblot analysis with anti-Myc
monoclonal antibody (lower panel).
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Tissue Distribution of RIP3--
Human tissue RNA blots were
hybridized with a 32P-labeled cDNA probe specific for
the RIP3 kinase domain. RIP3 transcript was present in a variety of
human tissues, being especially prominent in the pancreas where two
species of approximately 2.1 and 2.6 kb were clearly discernible (Fig.
1C). The 2.1-kb transcript corresponds in size to the cloned
full-length cDNA.
RIP3 Is a Protein Kinase--
To determine whether RIP3 was indeed
a protein kinase, 293E cells were transfected with Myc epitope-tagged
RIP3 or RIP3 (K50A) in which the invariant lysine conserved in all
protein kinases was mutated (31). Immunoprecipitated RIP3 was subjected
to an in vitro kinase assay followed by SDS-polyacrylamide
gel electrophoresis and visualized by autoradiography. An approximately
60-kDa 32P-labeled band corresponding to RIP3 was observed
only when the native sequence was expressed (Fig. 1D). As
expected, mutant RIP3 (K50A), in which the lysine essential for
enzymatic activity and ATP binding was altered to alanine, did not
possess kinase activity. Taken together, these observations are
consistent with RIP3 being a bona fide autophosphorylating protein kinase.
RIP3 Induces Apoptosis--
During the course of transfection
studies, it became evident that RIP3 induced dramatic apoptosis that
required cotransfection with a caspase inhibitor to allow for cell
survival and detectable RIP3 expression. This was surprising because as
noted earlier RIP3 possesses none of the pro-apoptotic homophilic
interaction domains involved in activation of the death pathway. RIP3
induced apoptosis in both 293E renal epithelial cells and MCF7 breast carcinoma cells. The transfectants rapidly developed morphological characteristics typical of adherent cells undergoing apoptosis, including membrane blebbing, becoming rounded and shrunken and detaching from the culture dish. To identify the pro-apoptotic region
within RIP3, truncated versions were analyzed for their ability to
induce cell death (Fig. 2B).
Expression of a version encoding the first 431 residues, RIP3 (1-431),
failed to induce apoptosis, suggesting that the missing 87 C-terminal
residues were critical for pro-apoptotic function. Additionally, the
kinase domain of RIP3 was not required for killing activity because
expression of a C-terminal segment of RIP3 (224-518), which lacks the
kinase domain, induced apoptosis at levels comparable with that of the intact molecule (Fig. 2B). As anticipated, RIP3-induced
apoptosis was blocked by caspase inhibitors including CrmA, a cowpox
virus encoded serpin and by p35, a baculovirus gene product that
equipotently inhibits multiple caspases (Fig. 2C).
RIP3-induced death was not inhibited by a dominant negative version of
FADD, implying that it engages a pathway independent or downstream of
this receptor-associated pro-apoptotic adapter molecule (data not
shown).

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Fig. 2.
RIP3 induces apoptosis and selectively binds
large prodomain caspases. A, 293E cells were
transiently transfected with the indicated FLAG epitope-tagged
expression constructs. Expression of RIP3 and its truncated versions
was confirmed by immunoblotting cell lysates from transiently
transfected cells with FLAG antibody (lower panel).
B, MCF7 breast carcinoma cells were transiently transfected
with the indicated constructs and the -galactosidase reporter gene
that was used as a marker for transfection. Cells were fixed, and the
morphology of
5-bromo-4-chloro-3-indolyl- -D-galactopyranoside-stained
cells was examined by light microscopy. Data (mean ± S.E.) shown
are the percentages of apoptotic cells among the total number of cells
counted (n = ~300). C, MCF7 cells were transiently
transfected with either empty vector or RIP3 expression construct and a
4-fold molar excess of the apoptosis inhibitory genes CrmA or p35.
D, RIP3 interacts with large prodomain caspases. 293E cells
were cotransfected with FLAG-RIP3 and indicated GD epitope-tagged
caspases. 24 h after transfection, extracts were prepared and
immunoprecipitated (IP) with FLAG antibody and immunoblotted
with GD-Biotin antibody to detect coprecipitating caspases. The
lower panel is an immunoblot of 10% of the lysate used in
the immunoprecipitation showing relative expression of the transfected
constructs.
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An adapter-independent mechanism by which RIP3 may induce apoptosis
would be binding and activating caspases. In keeping with this notion,
co-precipitation studies revealed an association between transfected
RIP3 and the large prodomain caspases 2, 8, 9, and 10, but no
detectable binding to downstream small prodomain caspases, such as
caspase-3 (Fig. 2D). A number of important caveats with
regard to this experiment should be noted, including that the
interaction is observed in an overexpression system and could still be
indirect (for example, mediated by an endogenous intervening adapter
molecule). Additional studies will be required to confirm the exact
mechanism by which RIP3 engages the caspase death machinery.
RIP3 Does Not Activate NF-
B--
Given the sequence homology
between RIP3 and RIP, a known activator of NF-
B, we asked if it,
too, could induce the NF-
B pathway (23, 25).
NF-
B-dependent reporter constructs were co-transfected
into 293E cells with RIP, TNFR-1, or RIP3 expression constructs.
Despite being expressed to an extent no less than RIP or TNFR-1, RIP3
activated the NF-
B reporter gene to a negligible level (4.5-fold)
when compared with RIP (460-fold) and TNFR-1 (124-fold) (Fig.
3A). Given this, we asked the
converse question, namely, whether RIP3 could inhibit RIP- or
TNFR-1-induced NF-
B activation. As shown in Fig. 3B,
cotransfection studies revealed that RIP3 inhibited RIP and TNFR-1
induced NF-
B activation in a dose-dependent manner. It
did not influence activation of this pathway by TRAF6 (32), an
unrelated inducer that mediates IL-1-induced NF-
B activation (data
not shown).

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Fig. 3.
RIP3 inhibits NF- B
activation, binds RIP, and is recruited to the TNFR-1 signaling
complex. A, Overexpression of RIP3 fails to
significantly activate NF- B. 293E cells were transiently
cotransfected with an NF- B-driven luciferase construct and the
indicated expression constructs. Data (mean ± S.E.) represent
luciferase activities normalized for transfection efficiency.
Inset is an immunoblot of 293E extracts showing relative
expression of the transfected constructs. B, RIP3 suppresses
RIP and TNFR-1-induced NF- B activation. 293E cells were
cotransfected with the NF- B luciferase construct together with a
constant amount of RIP or TNFR-1 expression construct and varying
amounts of RIP3 expression vector. The total amount of transfected DNA
was kept constant by adding empty vector. Data (mean ± S.E.,
n = 3) are displayed as fold inhibition of reporter
luciferase activity. C, RIP3 binds RIP. 293E cells were
cotransfected with Myc-RIP and FLAG-RIP3 or truncated versions of
FLAG-RIP3. Cell extracts were immunoprecipitated (IP) with
anti-Myc antibody, and associating RIP3 was detected by immunoblotting
with FLAG antibody. The amount of Myc-RIP and FLAG-RIP3 present in the
extract was assessed by direct immunoblotting of 10% of the extract
with epitope tag antibodies (lower panels). D,
RIP3 is recruited to the TNFR-1 signaling complex. 293E cells were
co-transfected with the indicated epitope-tagged constructs. TNFR-1 was
immunoprecipitated with anti-FLAG antibody, and associating proteins
were detected by immunoblotting. Lower panels are direct
immunoblots of 10% of the extract used in the immunoprecipitation to
confirm expression of the transfected gene product. E, RIP3
(1-431) can partially inhibit TNFR-1-induced cell death. TNFR1 were
transiently transfected to MCF7 cells together with empty vector, CrmA,
or RIP3 (1-431). The molar ratios between TNFR1 and other expression
constructs are indicated.
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This result prompted us to look for a potential physical interaction
between RIP and RIP3. Cotransfection studies deploying differentially
epitope-tagged expression constructs revealed strong binding of RIP3 to
RIP (Fig. 3C). RIP3 also bound RIP2, but to a much lesser
extent, possibly because of overexpression unveiling a weak promiscuous
interaction (data not shown). Truncated versions of RIP3 were
co-expressed to delineate the segment that associated with RIP.
Reminiscent of the studies mapping the pro-apoptotic activity of RIP3,
an intact C terminus was found to be essential, as evidenced by the
inability of RIP3 (1-431) to bind RIP. Additionally, the kinase domain
was dispensable because RIP3 (224-518) still bound. Finer mapping
studies will be needed to delineate the exact, possibly overlapping
domains responsible for mediating apoptosis and RIP association.
RIP3 Is Recruited to the TNFR-1 Signaling Complex in a
RIP-dependent Manner--
Because RIP is recruited to the
TNFR-1 signaling complex through interaction with the
receptor-associated platform adapter molecule TRADD (25), we asked
whether RIP3 could similarly be recruited to the TNFR-1 complex by
virtue of its interaction with RIP. As shown in Fig. 3D,
RIP3 is recruited to the TNFR-1 signaling complex at stoichiometric
amounts in a RIP-dependent manner, consistent with the
ability of these two related molecules to bind each other. Interestingly, exceedingly small, nonstoichiometric amounts of overexpressed RIP and RIP3 can also be recruited to the TNFR-1 signaling complex in the absence of co-transfected TRADD. This is
presumably mediated by the low level of endogenous TRADD present in the
transfected cells. Once recruited to the TNFR-1 signaling complex (or
possibly to other receptor complexes that bind TRADD and RIP such as
the TNFR-1 related receptor, DR3), RIP3 could exert a
pro-apoptotic activity. This may in part be accomplished by
attenuating the NF-
B survival pathway and/or by activating caspases.
Regardless, a dominant negative version of RIP3 should be able to
partially attenuate TNFR-1-induced cell death. The inhibition can only
be partial because the TRADD-FADD-caspase-8 pathway would still
function even in the presence of dominant negative RIP3 (12). In
keeping with this notion, we found that RIP3 (1-431) could act as a
dominant negative and partially inhibit TNFR-1-induced cell death (Fig.
3E). The partial nature of the inhibitory effect was
evident, especially when compared with the potent caspase-8 inhibitor CrmA.
In summary, RIP3 is a pro-apoptotic molecule that binds RIP and
antagonizes its ability and that of TNFR-1 to engage the NF-
B pathway.