(Received for publication, July 19, 1995; and in revised form, December 14, 1995)
From the
The mouse Fas/APO-1 antigen represents a 45-kilodalton
transmembrane receptor that initiates apoptosis by a poorly defined
signaling mechanism. The cytoplasmic domain of Fas does not display any
known enzymatic activities but is capable of interacting with a number
of proteins that were identified recently using the yeast interactive
cloning method. To investigate direct biochemical interactions from
cellular lysates prepared from Fas-responsive cells, a series of
recombinant glutathione S-transferase-mouse Fas fusion
proteins representing different regions of the mouse Fas cytoplasmic
domain was used. Polypeptides of 25, 50, and 70 kilodaltons were found
to associate with the Fas intracellular domain, and this binding was
stable in the presence of 1 M NaCl. These interactions were
also detected using a mouse Fas fusion protein containing an Ile to Asn
mutation, which is responsible for a lymphoproliferative disorder in
certain strains of mice (lpr).
Furthermore, the binding of cellular proteins to Fas could be blocked
upon incubation with a polyclonal antibody directed against the
cytoplasmic domain of Fas. The strong association of cellular proteins
with the cytoplasmic region implies that constitutive interactions may
exist to regulate apoptotic signaling through the Fas antigen.
The activation of the Fas/APO-1 antigen by the Fas ligand or
specific agonist antibodies results in a rapid induction of
apoptosis(1, 2, 3) . The resulting
condensation and fragmentation of nuclei, loss of plasma membrane, and
cell shrinkage are common morphological features of the apoptotic
process. Analysis of a mouse lymphoproliferation mutation, lpr, provides genetic evidence that the
Fas molecule is critically involved in T-cell regulation(4) .
The lpr
mutation has been mapped to a
point mutation in the cytoplasmic domain of the mouse Fas antigen,
which abrogates its ability to initiate
apoptosis(4, 5) .
The Fas antigen is a member of a
large family of cell surface molecules, which includes the p55 and p75
TNF ()receptors, CD27, CD30, CD40, the lymphotoxin-
receptor, and the p75 neurotrophin receptor(6, 7) .
While the homology among these receptors has been dictated primarily by
similar extracellular cysteine-rich repeats, functional analysis of the
p55 TNF receptor and the Fas antigen in transducing apoptosis has
defined a cytoplasmic region of 89 amino acids, which has been
designated the ``cell death'' domain(5, 8) .
A similar sequence has also been discovered in the cytoskeletal protein
ankyrin and in a number of proteins that bind to Fas and the p55 TNF
receptor death domains. These proteins,
MORT1/FADD(9, 10) , RIP(11) , and
TRADD(12) , were identified using the yeast interactive cloning
technique. Each protein can independently initiate cell death when
transiently overexpressed in heterologous cells. The ubiquitous
expression patterns of these proteins, the finding of a similar motif
in ankyrin, and in vitro analysis (9, 10, 11) suggest that the death domain
serves as a protein association motif.
The biochemical mechanisms
responsible for Fas action are incompletely understood. An acidic
sphingomyelinase activity is induced upon cross-linking of Fas with
monoclonal antibodies in U937 cells (13) that gives rise to
higher intracellular ceramide levels. A similar activity has also been
detected upon TNF- binding to the p55 TNF receptor(14) ;
however, recent evidence suggests an important role for a neutral
sphingomyelinase in Fas and p55 TNF receptor signal transduction. In
addition, cellular tyrosine phosphorylation is increased within minutes
after engagement of the Fas antigen in Jurkat and U937
cells(15) . The potential involvement of tyrosine
phosphorylation in Fas signal transduction is supported by the
identification of FAP-1/BAS, a protein tyrosine phosphatase, which
negates Fas-mediated apoptosis(16) . The effects of FAP-1 upon
anti-Fas antibody-induced cell death have been mapped to an interaction
of FAP-1 with the C terminus of the Fas antigen, a region known to
attenuate Fas-mediated apoptosis(5) .
Since the cytoplasmic
domain is clearly necessary for Fas to function as a receptor (4, 5) and numerous binding interactions have been
defined for Fas(17) , we have investigated the binding
properties of the intracellular domain of Fas using recombinant fusion
proteins. Our results indicate that strong protein-protein associations
exist that map to distinct regions of the Fas molecule. The protein
binding interactions are specific, because an anti-Fas antibody can
effectively block the binding. Surprisingly, the predominant binding
interactions are still observed in a cytoplasmic Fas mutation (lpr), suggesting the Fas receptor can
participate in constitutive protein-protein interactions that regulate
its apoptotic function.
The GST fusion bacterial expression construct
containing the Ile to Asn point mutation at residue 225 as found in lpr mice was generated with two sets of PCR
primers: set 1 (5` primer, 5`-CGGGATCCATGCCTCAAATCTTAGCT-3`; 3` primer,
5`-ATTTTCTCGAGCAAATTT-3`); set 2 (5` primer,
5`-TTTGCTCGAGAAAATAACAACAAGA-3`; 3` primer,
5`-GCTCTAGATCACTCCAGACATTGTCCTTCATT-3`); a point mutation (A) found in
the lpr
form of mouse Fas is indicated. PCR using
each set of primers was carried out independently as described above
with the exception that for primer set 2 Taq polymerase
(Boehringer Mannheim) was used in order to introduce the point mutation
in the PCR product. The PCR products were isolated and analyzed as
described above. The PCR product from primer set 1 was digested with BamHI and XhoI; the PCR product from primer set 2 was
digested with XbaI and XhoI. After isolation by gel
purification, the PCR products from primer set 1 and set 2 were
incubated with pGSTag (BamHI- and XbaI-digested),
ligated using T4 ligase, and used for transformation into SG1117 E.
coli. The clones were verified by restriction analysis, DNA
sequencing, and IPTG-inducible protein expression.
A mFas expression construct encoding amino acids 221-306 fused to GST (GST-mFas-(221-306)) was generated as follows. The pCMV5-mFas vector was first digested with XhoI and the resulting fragment isolated by agarose gel electrophoresis. The single-stranded ends were filled using Klenow DNA polymerase and the fragment recovered by phenol/chloroform extraction and ethanol precipitation. The fragment was then cut with SalI, isolated by agarose gel electrophoresis. Similarly, pGSTag was digested with XhoI, gel-purified, incubated with Klenow fragment to polish the ends, phenol/chloroform-extracted and ethanol-precipitated, digested with SalI, and gel-purified. The digested mFas fragment and pGSTag were then incubated with T4 ligase. The ligation reaction was then used to transform SG1117 E. coli and plated onto LB-ampicillin agar plates. Clones were confirmed by restriction analysis, DNA sequencing, and IPTG-inducible protein expression.
After labeling, the S-containing medium was removed,
and the cells were washed 4 times with 10 ml of phosphate-buffered
saline. HeLa and L929 cells were isolated by scraping. Cells were
harvested by centrifugation at 900
g and lysed for 10
min on ice in Lysis Buffer (0.5% Nonidet P-40, 20 mM Tris, pH
8.0, 200 mM NaCl, 1 mM EDTA, 0.1% 2-mercaptoethanol,
2 µg/ml aprotinin, 1 µg/ml leupeptin, 0.7 µg/ml pepstatin,
and 25 µg/ml PMSF) and vortexed briefly. The lysed cells were then
centrifuged at 900
g for 3 min to remove insoluble
debris. Supernatants were removed and stored at -80 °C until
use. In experiments where anti-GST-mFas or anti-GST IgG were
preincubated with the GST-fusion proteins prior to the addition of
lysates, 2-mercaptoethanol was not present in the Lysis Buffer used to
lyse or wash the protein complexes.
The cytoplasmic domain of the Fas antigen contains an 89-amino acid motif (residues 204-292) required for antibody-induced apoptosis in murine L929 cells(5) . To characterize potential binding interactions of the intracellular sequence of mFas, a series of glutathione S-transferase proteins were prepared that contained different sequences from the cytoplasmic domain of the mouse Fas antigen. Fig. 1shows the FD mutants, which are based upon the work of Itoh and Nagata (5) who assessed the cytotoxic activities of the human Fas deletion mutants after transfection in murine L929 cells. The GST-mFas FD mutants share a common N terminus fused in frame to GST but differ by sequential deletions at the C terminus of the mFas intracellular domain. The longest construct, GST-mFas-(194-306), contained part of the juxtamembrane domain (amino acids 194-203), the entire death domain (amino acids 204-292), and the C-terminal region (amino acids 293-306). The last 14 amino acids define an inhibitory domain by analogy to studies performed with the human Fas antigen(5) .
Figure 1:
GST-mFas fusion proteins. A series of
GST-fusion proteins were purified from E. coli as described
under ``Materials and Methods.'' The full-length mouse Fas
antigen is shown for reference and is a 306-amino acid transmembrane
protein. The extracellular domain (ECD) spans from amino acids
1 to 148, the transmembrane domain (TM) from 149 to 165, and
the intracellular domain from 166 to 306. The death domain (solid
region) spans from amino acids 204 to 292 and the inhibitory
domain from 293 to 306(5) . GST-mFas-(194-306) represents
the parent fusion protein in this study, as it encodes 10 residues in
the juxtamembrane region, the entire death domain, and the inhibitory
domain fused to GST. The GST-mFas FD mutants are based on truncations
made by Itoh and Nagata (5) in the human Fas antigen and
contain mouse Fas sequence from amino acid 194 to 292 (FD5),
283 (FD8), 276 (FD7), 268 (FD4), and 221 (FD2); GST-mFas lpr contains
residues 194-306 from mouse Fas with a mutation at residue 225
where an asparagine is substituted for an isoleucine; and
GST-mFas-(221-306) lacks the N-terminal 17 amino acids of the
death domain and the 10 residues of the juxtamembrane domain found in
GST-mFas-(194-306).
Two other constructs, GST-mFas-(221-306)
and GST-mFas lpr, were also generated. The fusion
protein, GST-mFas-(221-306), differs from
GST-mFas-(194-306) by lacking 27 amino acids at the N terminus of
the mFas coding region that includes 17 amino acids of the death domain (Fig. 1). The GST-mFas lpr
construct
represents a protein with identical primary structure to
GST-mFas-(194-306) except for a single amino acid change at
residue 225, where an isoleucine was replaced by an asparagine residue (Fig. 1). Certain strains of mice carrying this mutation in the
Fas antigen develop lymphadenopathy and an autoimmune syndrome. This
point mutation has been shown to interfere with the ability of Fas to
transmit an apoptotic signal(4) .
Several cellular proteins were observed to bind nonspecifically to GST and GST-mFas-(194-306). However, three polypeptides of relative molecular masses 25, 50, and 70 kilodaltons from HeLa, L929, and Jurkat cell lines (referred to as p25, p50, and p70) were observed to bind to the GST-mFas-(194-306) protein but not to GST (Fig. 2). In Jurkat cells, p25 is clearly present but is obscured by a closely migrating protein found in the GST and GST-fusion protein lanes. Similar protein associations were observed in Hut78 cells (data not shown).
Figure 2:
GST-mFas-associated polypeptides from
metabolically labeled HeLa, L929, and Jurkat cell lysates. S-Labeled cell lysates were prepared as described under
``Materials and Methods,'' then precleared with 25 µg of
GST on 50 µl of Sepharose-GSH, and incubated with either 10 µg
of GST or GST-mFas-(194-306) on 50 µl of Sepharose-GSH at 4
°C for 2 h. After repeated washes with Lysis Buffer and
equilibration with 50 mM Tris, pH 8.0, the GST or
GST-mFas-(194-306) and any associated polypeptides were eluted
twice with 50 µl of 20 mM GSH in 50 mM Tris, pH
8.0. Eluates were analyzed by 12% SDS-PAGE and fluorography. aa, amino acids.
Figure 3: GST-mFas-associated polypeptides are stable to high salt concentrations. Metabolically labeled HeLa cell lysates were prepared and screened with either GST or GST-mFas-(194-306) as described in Fig. 2except that the Sepharose-protein complexes were washed with Lysis Buffer containing different salt concentrations (as indicated). The eluted material was subjected to 12% SDS-PAGE and fluorography. aa, amino acids.
The specificity of these interactions was tested using an anti-GSTmFas antibody. A polyclonal antibody was generated to the recombinant GST-mFas-(221-306) protein. The antibody recognized the Fas intracellular domain by Western blot analysis (Fig. 4A). The antibody was affinity-purified and then used to assess the nature of the binding interactions between the GST-mFas-(194-306) recombinant protein and p25, p50, and p70. The GST-mFas-(194-306) protein was preincubated with increasing amounts of the anti-GST-mFas or anti-GST antibody and used to screen HeLa cell metabolically labeled lysates as described under ``Materials and Methods.'' Incubation with the anti-GST-mFas IgG blocked binding of p25 and p70 to GST-mFas (Fig. 4B). A longer exposure indicated that the p50 protein was similarly disrupted. Control experiments indicated that the anti-GST antibody had no effect up to 100 µg of IgG. These findings indicate that the three polypeptides associate specifically with the mFas intracellular domain containing amino acid residues 194-306.
Figure 4: Association of GST-mFas with cellular polypeptides is blocked by preincubation with a polyclonal antibody against GST-mFas. A, detection of Fas intracellular domain with anti-GST-Fas antibody. Each lane contains 1 µg of purified GST or GST-mFas-(221-306) fusion protein. GST-mFas-(221-306) was either untreated(-) or incubated with 0.01 unit/µl thrombin (+) as indicated under ``Materials and Methods.'' Samples were run out on 12% SDS-PAGE and immunoblotted using anti-GST-Fas serum at a dilution of 1:1000. The indicated band represents the mFas fragment cleaved from the GST carrier after thrombin treatment. B, HeLa cell lysates from metabolically labeled cells were prepared and screened for GST or GST-mFas binding proteins as in Fig. 2. GST-mFas-(194-306) was incubated for 30 min at 4 °C with different amounts of anti-GST-mFas IgG or anti-GST IgG prior to incubation with HeLa lysates. The eluted material was subjected to 12% SDS-PAGE and fluorography. aa, amino acids.
Binding
experiments with HeLa and L929 cell extracts indicated that while both
p25 and p70 interacted strongly with both GST-mFas lpr and GST-mFas-(194-306), only p25 was found to be strongly
associated with GST-mFas-(221-306) (Fig. 5). These
findings imply that p25 and p70 interact with mFas via distinct
domains. Binding of p70 to Fas appears to require a region between
residues 194 and 221, while p25 binds to more C-terminal sequences.
Furthermore, the fact that both p25 and p70 bind to GST-mFas lpr
is intriguing as the lpr
mutant is believed to represent an inactive form of Fas.
Figure 5:
p25 and p70 differentially associate with
mutant forms of GST-mFas. Metabolically labeled HeLa (A) and
L929 (B) cell lysates were prepared and screened with
GST-fusion proteins as described in Fig. 2. In the above
experiment, lysates were screened in duplicate with GST, GST-mFas lpr, GST-mFas-(194-306), or
GST-mFas-(221-306). The eluted material was subjected to 12%
SDS-PAGE and fluorography. aa, amino
acids.
To define further the binding of p50 to the cytoplasmic domain of wild-type Fas, a series of C-terminal mutations (FD) were assayed, which corresponded to deletions made in the human Fas antigen(5) . Metabolically labeled HeLa cell lysates were assayed for p50 binding using these GST-fusion proteins (Fig. 1). The p50 protein was found to strongly associate with all the FD mutants except GST-mFas FD2, which contains residues 194-221 of mouse Fas (Fig. 6A). Since p50 binds tightly to the FD4 mutant (mFas-(194-267)), the binding domain for p50 spans residues 222 to 268 of mFas.
Figure 6: p50 maps to residues 222-268 and p70 maps to residues 194-221 of the mFas intracellular domain. Metabolically labeled lysates were prepared and screened with GST-fusion proteins as in Fig. 2. In this experiment, lysates were screened in duplicate with GST, GST-mFas-(194-306), GST-mFas FD5, GST-mFas FD8, GST-mFas FD7, GST-mFas FD4, or GST-mFas FD2. The eluted material was subjected to 12% SDS-PAGE and fluorography. aa, amino acids.
In addition, all the FD mutant proteins used in this analysis were found to bind equally well to labeled p70 from HeLa cells (Fig. 6B). These results are consistent with the mapping of p70 binding to a region of Fas between amino acids 194 and 221 (Fig. 5). Attempts to visualize binding of p25 to the FD mutants did not provide conclusive mapping information; however, the binding of p25 to GST-mFas-(221-306) (Fig. 5) indicated that p25 interacts with the C-terminal 85 amino acids of Fas. Taken together, the GST-fusion protein data demonstrate that each cellular protein displays a distinctive binding interaction with the cytoplasmic domain of mouse Fas. Binding of p70 has been localized to residues 194-221; p50 binds to residues 222-268; and p25 binds a region between residues 221 and 306. The mouse Fas intracellular domain is therefore capable of multiple protein-protein interactions.
The Fas antigen belongs to a group of cell surface receptors, the TNF receptors, CD40, CD30, and the p75 neurotrophin receptor, for which intracellular receptor signaling pathways are not well defined. A major question regarding the mechanism of Fas-mediated cell killing is how activation of the receptor leads to the biochemical reactions responsible for apoptosis. A number of activities, including sphingomyelin turnover(13) , tyrosine phosphorylation and dephosphorylation(15, 16) , and activation of ras(21) have been implicated. However, none of these enzymatic activities have been physically linked to the Fas receptor.
To begin to understand Fas receptor function, we have utilized recombinant GST-fusion proteins to assay for proteins that associate with cytoplasmic sequences of Fas. In contrast to the yeast two-hybrid approach, this approach provides direct biochemical evidence for interacting proteins from cell lysates. Three proteins of apparent molecular masses 25, 50, and 70 kilodaltons, from HeLa, Jurkat, L929, and Hut78 cells, display strong interactions with the intracellular domain of mouse Fas. These proteins are not restricted to Fas-expressing cells, since HeLa and L929 cells do not express the Fas antigen(5, 20) ; these two cell lines are rendered Fas-sensitive upon introduction of the Fas cDNA by transfection. The binding interactions are stable even in the presence of 1 M NaCl and could be specifically blocked by polyclonal antibodies against the Fas intracellular domain. Mapping data suggest that each protein binds to a different Fas cytoplasmic sequence (Fig. 7). Since these proteins were found to bind to the Fas intracellular domain in the absence of ligand, it is possible that these interactions are constitutive in nature. However, given that the intracellular domain of Fas has been demonstrated to self-associate(9, 22) , it is also possible that the resultant recombinant fusion protein complexes contain the Fas intracellular domain in dimeric or oligomeric form. This suggests that the recombinant Fas proteins used here may mimic the behavior of the receptor upon agonist binding.
Figure 7: Schematic representation of the mouse Fas antigen and its binding proteins. The p25, p50, and p70 interacting proteins are aligned along the cytoplasmic domains of Fas, deduced from the results presented in Fig. 5and Fig. 6.
Supporting
the idea that these interactions are constitutive in nature is the
behavior of the labeled HeLa cell proteins toward the mFas lpr mutant fusion protein. A comparable level of
binding was observed to the lpr
and wild-type
fusion proteins, suggesting that the lpr
mutation
does not interfere with these associations. These results are of
interest because mouse experiments clearly demonstrate that the Ile to
Asn lpr
change results in a biologically inactive
receptor.
The 25- and 70-kilodalton proteins are similar in size to
two cellular proteins, MORT1/FADD (9, 10) and
RIP(11) , respectively, both of which were identified using a
yeast interaction screening technique with the human Fas cytoplasmic
domain. The MORT1/FADD and RIP proteins were shown to induce apoptosis
when overexpressed in heterologous cells. In contrast to FADD and RIP
proteins, the p25 and p70 proteins were found to interact with the lpr mutant form of Fas. Also, p70 maps to a
different binding domain than the RIP protein(11) . Several
reasons may account for the differential results observed with these
metabolically labeled proteins. First, the p25 and p70 proteins may be
different from MORT1/FADD and RIP proteins. Second, there may be
species-specific differences, because the mouse Fas lpr
mutation (Ile to Asn) is somewhat different from the human Fas lpr
mutation (Val to Asn). Third, the p25 and p70
proteins may bind to the Fas lpr
protein through
different conformational states, which may be exhibited either by Fas
or the interacting proteins. In this regard, a potential disadvantage
with the use of recombinant fusion proteins is that the exact folding
of the cytoplasmic domains may not be reproduced.
For many receptor systems, ligand binding leads to dimerization or aggregation of receptors, which facilitates post-translational modifications such as tyrosine phosphorylation. The action of agonist anti-Fas antibodies indicates that aggregation of Fas may serve as a key step in initiating its apoptotic function. This model of Fas action has been borne out by experiments demonstrating that the intracellular domains of both Fas and the p55 TNF receptor self-associate(22, 23) . The experiments here demonstrate that strong and multiple protein-protein interactions exist in the absence of antibody or Fas ligand. Similar sized proteins to p25 and p50 using Fas cross-linking followed by immunoprecipitation were recently reported(24) . Our results suggest that ligand-independent protein interactions are possible for Fas. If the cell death proteins that have been identified as potential partners for the Fas antigen and the p55 TNF receptor (17) function as protein association domains, the binding of the Fas ligand or agonist antibodies to the Fas receptor may lead to one of several possibilities: (a) additional protein interactions; (b) dissociation and relocalization of Fas-associated proteins to their cellular site of action; or (c) appropriate conformational changes that unmask a cell death activity in the constitutively associated protein complex. Direct assays of receptor proximal events will be required in order to discriminate among these possibilities.