(Received for publication, June 29, 1995; and in revised form, January 19, 1996)
From the
The Fas antigen (Apo1/CD95) is a transmembrane protein belonging
to the nerve growth factor receptor family. It is expressed on a
variety of cells, including activated T lymphocytes. Ligation of Fas
with its natural ligand or with anti-Fas antibodies often results in
the apoptotic death of the cell, making Fas an important mediator of
down-regulating immune responses. The signal transduction pathways
utilized by Fas are currently unknown, although tyrosine kinase
activity has recently been strongly implicated. Here, we report that
the tyrosine kinase p59 physically associates
with Fas in Fas-sensitive cells. In addition, we show that activated T
lymphocytes from fyn knockout mice exhibit elevated lifespans
and reduced apoptosis in vitro compared to their normal
counterparts. Furthermore, activated T lymphocytes from the fyn- deficient mice are less sensitive to killing by both
anti-Fas antibody and Fas-ligand cytotoxic T cells. These results
suggest that p59
plays an important role in Fas
signal transduction.
Deletion of activated peripheral T cells is important in the down-regulation of immune responses and controlling T cell numbers. The mechanisms by which this is accomplished are unclear, but the expression and action of a transmembrane death protein known as the Fas antigen has recently been implicated(1, 2, 3, 4, 5) . It is now believed that activated T cells eventually co-express Fas and Fas ligand, leading to the apoptotic death of the cells upon interaction with each other (see (6, 7, 8) for recent reviews). The nature of the Fas death signal is currently unknown and has become the focus of intense research.
A cytoplasmic region in Fas that is essential for
killing has been identified and termed the ``death
domain''(9) . A similar motif exists in the cytotoxic TNF ()receptor (TNFR1), a member of the nerve growth factor
receptor family, to which Fas also belongs(10) . This death
domain also shares some homology with part of the Drosophila protein reaper, which is an important mediator in
apoptosis(11) . Both Fas and TNFR appear to be coupled to a
sphingomyelinase, ceramide-producing pathway, which could explain, at
least in part, the apoptotic outcome of ligating these receptors (12, 13, 14, 15) . Whether this
coupling is direct or not is unknown.
Fas contains no previously known signaling motifs or catalytic activities. In an effort to identify molecules that might interact with Fas and link it to signal transduction pathways, a number of groups have employed the yeast 2-hybrid system, with the Fas cytoplasmic region as ``bait.'' This approach has identified three different proteins that can interact with Fas: FADD (MORT1)(16, 17) , RIP(18) , and FAP-1 (PTP-BAS)(19) , although evidence that these proteins interact in a physiologically relevant system is lacking. FADD and RIP both contain death domain-like motifs, and interaction with Fas may occur via dimerization of this region. The functions of FADD and RIP are not known, although RIP may be a protein kinase. FAP-1 is a tyrosine phosphatase and, interestingly, interacts with the Fas carboxyl terminus, which has been shown to be involved in negatively regulating Fas killing(9) . The level of endogenous FAP-1 seems to correlate with the Fas sensitivity of a cell, and transfection of cells with FAP-1 encoding DNA also leads to a reduction in Fas sensitivity. This suggests that one or more tyrosine kinases may play a role in Fas killing. Indeed, it has previously been demonstrated that Fas activity is inhibited by tyrosine kinase inhibitors, and ligation of Fas leads to the tyrosine phosphorylation of a number of cellular proteins(20) . Therefore, there is now strong evidence from two different sources that tyrosine kinase activity is involved in Fas signal transduction.
Although it has not been shown that Fas itself
is tyrosine-phosphorylated, we examined the context of the tyrosine
residues in the cytoplasmic domains of both human and mouse Fas for any
similarities to those in other signaling molecules that are known to
interact with tyrosine kinases. Interestingly, one of the tyrosines is
found in the death domain within a conserved YXXL motif. This
sequence is reminiscent of half of an immunoreceptor tyrosine-based
activation motif (ITAM) found in the T cell receptor CD3 complex chains
and elsewhere (see (21) for a review) and is also suggestive
of a binding site for proteins with SH2 domains(22) . ZAP-70
and p59 are two tyrosine kinases known to
interact with similar sequences, although p59
is
much more flexible in its binding sequence requirements than
ZAP-70(23) . Also, since disruption of the fyn gene
has been correlated with lack of programmed cell death in certain
regions of the mouse brain (24) (which, along with T
lymphocytes, expresses the highest level of
p59
), we decided to investigate the possibility
that p59
might interact with Fas and contribute
to Fas signal transduction. Here we report that there is indeed a
specific physical interaction between p59
and
Fas in Fas-sensitive cell lines. The biological relevance of this
interaction was confirmed by the observation of an increased survival
and decreased rate of apoptosis in alloantigen-stimulated splenocytes
from fyn knockout mice. Furthermore, fyn-deficient
activated T cells are resistant to killing when their surface Fas is
ligated by either anti-Fas antibody or Fas ligand presented by
Fas-dependent cytotoxic T cells. This provides the first report of a
protein with known catalytic activity capable of linking Fas to a
signal transduction pathway that has been shown to occur in a
physiologically relevant system.
Immunoprecipitates were washed
extensively in lysis buffer without EDTA and either dissociated in SDS
sample buffer, separated by SDS-polyacrylamide gel electrophoresis
(8%), and electroblotted onto nitrocellulose, or subjected to in
vitro kinase assays. Western blotting was performed using either
rabbit-anti-fyn antibody (FYN3, Santa Cruz Biotechnology) or
rabbit anti-Fas peptide Fab fragment (kindly provided by
Dr. K. Elkon, (28) ) and horseradish peroxidase-conjugated
donkey anti-rabbit (Amersham) and visualized using the ECL method
(Amersham).
Standard cytotoxicity assays were carried out
with PMM-1 effector cells stimulated with PMA (10 ng/ml) and ionomycin
(3 µg/ml). Effector cells were stimulated for 3 h at 37 °C and
then washed in RPMI media. Target cells, C57BL/6 or
Fyn splenocytes were labeled with chromium
sulfate for 60 min at 37 °C before being washed in RPMI and PBS.
Assays were set up in 200-µl volumes containing a maximum of 5
10
effector cells and 1
10
target cells. To examine the impact of effector to target cell
ratios, the concentration of the PMM-1 cells was serially diluted from
5
10
cells per well to 0.625
10
cells per well. The assays were incubated for 4 h at 37 °C
after which the cells were pelleted and the amount of
Cr
in the supernatants was measured in a LKB Rack Gamma Counter.
Cell-specific lysis was calculated to determine the relative amount of
cell death.
To determine if p59 associates with the Fas
antigen, co-immunoprecipitation experiments were performed. We chose to
study YAC-1 cells which express relatively high levels of both Fas and
p59
(
)and are very sensitive to killing by
PMM-1, a killer cell that has been shown to kill via a Fas-dependent
pathway(25) . Either Fas or p59
were
immunoprecipitated from extracts of YAC-1 target cells. Western blot
analysis revealed that p59
was co-immunoprecipitated with
anti-Fas antibody, but not with an isotype-matched control or
antibodies to another cell surface molecule CD45 (Fig. 1).
Interestingly, ligation of Fas on YAC-1 cells does not appear to be
required for the association of p59
, as we obtained the
same results from YAC-1 cells preincubated in the presence or absence
of PMM-1 Fas-dependent killer cells (data not shown).
Figure 1:
Antibodies to the
Fas-antigen immunoprecipitate fyn. Western blot showing that
p59 specifically associates with Fas. Various
antibodies were used to precipitate material from YAC cell lysates.
Material bound to protein A/G-agarose was fractionated on an 8%
polyacrylamide gel, and blotted with anti-fyn antibody.
Anti-Fas and anti-fyn antibodies immunoprecipitate detectable
levels of p59
from cell lysates, but an
isotype-matched control and anti-CD45 antibodies do not. Even extreme
overexposure of the filter did not reveal any fyn in the CD45
immunoprecipitates.
In order to establish the reciprocity of this interaction, extracts were first immunoprecipitated with either anti-Fas or anti-fyn antibodies. The resulting precipitates were then probed with both antibodies. As can be seen in Fig. 2, fyn is present in the Fas precipitates, and Fas can be detected in the anti-fyn precipitated sample. Together, these experiments establish an association between Fas and fyn.
Figure 2:
Reciprocity of Fas-fyn interaction. Western blot showing co-immunoprecipitation of Fas
and fyn from PMM-1 stimulated YAC-1 cell lysates. The right lane of each panel is present solely as a positive
control for the blotting antibodies and to show the mobilities of fyn and Fas, but is not meant to be quantitative. In the left panel, the positive control was a small portion of an
anti-fyn immunoprecipitate from a RIPA buffer lysate of YAC-1
cells previously determined to contain specifically immunoprecipitated
p59 using two separate anti-fyn antibodies. In the right panel, the positive control is
20 µl of a non-immunoprecipitated YAC-1 postnuclear cell
lysate.
Elimination of activated T
cells is believed to occur via Fas/Fas ligand
interactions(1, 2, 3, 4, 5, 6, 7, 8) .
Therefore, in order to determine if p59 plays a role in
Fas function, we studied mixed lymphocyte cultures (MLCs) of
splenocytes obtained from normal and fyn
mice(26) . Throughout the early stages of MLC, the T
cells from normal and fyn
mice
exhibited nearly identical growth characteristics (Fig. 3A). [
H]thymidine uptake
experiments also indicated comparable levels of proliferation within
the early stages of culture (data not shown). However, by day 17 after
stimulation, the cultures of fyn-deficient T cells had
substantially more viable cells than control cultures, indicating an
impairment in the normal (presumably Fas-based) mechanisms of activated
T cell deletion (Fig. 3A). Additionally, analysis of
several MLCs indicated an increase in fyn
lymphocyte lifespan manifested as a greatly elevated cloning
efficiency (Fig. 3B). A representative cloning
experiment is shown. In 3 other cloning experiments, the actual number
of clones obtained varied, but an enhancement in cloning efficiency of
the fyn
T cells of approximately 5:1
was consistently observed. This apparent increase in fyn
T cell lifespan was closely
paralleled by a reduction in the level of apoptosis as assessed by
characteristic DNA fragmentation (Fig. 4). No discernable
differences in the relative amounts of Fas surface expression on fyn
and control splenocytes was
observed upon flow cytometric analysis (data not shown). These data are
all consistent with a role for p59
in Fas signal
transduction.
Figure 3:
fyn-deficient lymphocytes
stimulated with alloantigen exhibit increased lifespans and cloning
efficiency. A, elevation of viable cell numbers at late time
points of fyn mixed lymphocyte
cultures. Viable cell numbers were recorded over an extended time
course after stimulation with alloantigen by eosin exclusion staining.
The cultures were equilibrated and restimulated on day 17 (arrow). B, increased cloning efficiency of fyn
T cells. Cells were removed from
the primary MLCs on day 7, set up at the indicated densities in 96-well
plates, and stimulated with 5
10
irradiated Balb/c
splenocytes per well. Plates were scored for growth 7 days after
plating. The results are typical of 4 separate cloning experiments from
independent MLCs.
Figure 4:
Alloantigen-stimulated fyn splenocytes exhibit reduced
apoptosis in culture. Pictured is a typical DNA fragmentation gel of
Ficoll-purified normal (+/+) and fyn-deficient
(-/-) splenocytes from various days of mixed lymphocyte
culture.
Finally, we sought to directly determine if fyn played a role in Fas-mediated cell death. To do this, we utilized
alloantigen-stimulated splenocytes from fyn and fyn
mice and determined their
susceptibility to anti-Fas antibody. Wells of 96-well plates were
coated with either anti-Fas or bovine serum albumin as a control, and
day 5 alloantigen-activated splenocytes were incubated in them
overnight. The next day, cell viability was assessed by vital dye
exclusion. As seen in Fig. 5A, the fyn
splenocytes were resistant to
the effects of anti-Fas antibody while the control cells were
susceptible. We also determined the relative sensitivities of fyn
and fyn
splenocytes to killing by PMM-1.
Normal and fyn-deficient splenocytes were used as targets in a
Cr-release lytic assay. The fyn
cells were found to be far less
sensitive to PMM-1 than their normal counterparts (Fig. 5B). These results provide strong and direct
evidence that fyn activity is involved in the Fas death
pathway in these cells.
Figure 5:
Reduced sensitivity to Fas-based death of fyn-deficient splenocytes. Normal or fyn-deficient
splenocytes from day 5 MLCs were assessed for their sensitivity to Fas
ligation by either anti-Fas (A) or PMM-1 (B). A, splenocytes were incubated overnight in floppy high
protein-binding 96-well plates previously coated with either anti-Fas
or bovine serum albumin as a control. The next day, viable cell numbers
were determined by trypan blue exclusion staining and light microscope
examination of multiple fields. B, Cr-labeled
splenocytes were mixed with PMM-1 effector cells at a range of effector
to target ratios and incubated for 4 h before being
harvested.
We are currently working on determining how
the fyn tyrosine kinase interacts with Fas. As mentioned
previously, the conserved YXXL motif in the death domain of
Fas is intriguing in that it is very similar to ITAM sequences involved
in signal transduction via the T cell receptor-associated CD3 chains.
We have so far not been able to demonstrate tyrosine phosphorylation of
Fas. Furthermore, to our knowledge, no death domain-containing protein
has ever been shown to be phosphorylated on tyrosine, even though many
of these proteins, including TNFR1 and RIP, possess conserved
YXXL motifs in their death domains. ()Clearly, the
importance of these sequences in binding fyn and/or relaying
an apoptotic signal needs further study.
It should be noted that
lack of phosphorylation of the tyrosine residue within the
YXXL motif of Fas would not preclude the possibility that
p59 interacts with Fas at this sequence. These sequences
can bind tyrosine kinases in at least two different ways: via SH2
domain interactions with the phosphorylated tyrosine residues or via a
mechanism independent of both SH2 domains and tyrosine phosphorylation.
An intact tyrosine activation motif consists of 2 YXXL
sequences separated by approximately 10 amino acids. Binding of ZAP-70
to the CD3-
ITAM has recently been shown to be dependent on very
specific sequence criteria(23) . These include the requirement
for an intact ITAM, accurately spaced tyrosine residues, and prior
phosphorylation of the tyrosine residues. Binding of ZAP-70 to the ITAM
is likely achieved via its 2 SH2
domains(23, 30, 31) . Binding of fyn to an ITAM is much more flexible, however. Even mutating the
tyrosine residues in the ITAM or altering their spacing does not affect
the interaction of p59
, which is thought to be initially
mediated by its unique amino-terminal region (23) . Given the
plasticity of the sequence requirements for fyn binding to
YXXL-containing motifs, it is tempting to speculate that this
region of Fas could represent an ``ITAM-equivalent'' for fyn interaction.
It is interesting to note that in some
circumstances, Fas ligation can cooperate with T cell receptor/CD3
stimulation in signaling T cell mitogenesis(32) , and that T
cells from Fas-defective lpr mice exhibit reduced
responsiveness to antigen(2) . Our observation that p59 binds to Fas provides an explanation for both of these phenomena,
by identifying a specific tyrosine kinase that is common to the two
systems. Fas-bound p59
may, under some conditions, be
brought into close proximity with the CD3 chains and may be able to
contribute additional tyrosine phosphorylation of CD3 or other
complexed proteins, leading to enhanced T cell stimulation. It will be
interesting to determine if the lpr mutation disrupts the
association between Fas and p59
.
Although we believe
that p59 plays an important role in Fas signal
transduction, it is clearly not absolutely essential. Apoptosis still
occurs, albeit at a reduced rate, in activated fyn
splenocytes. It is likely that
other tyrosine kinases may be able to partially compensate for
p59
in its absence, as may be the case in CD3 signal
transduction(26) . To our knowledge, fyn-deficient
mice have not been shown to exhibit an lpr-like phenotype,
with large numbers of T lymphocytes accumulating with age. This has not
yet been specifically studied, however, and our data would predict that
in older fyn
mice, there may be some
degree of lymphoproliferative disease apparent. Our observations are
also important in that they predict that some types of human
lymphoproliferative and autoimmune diseases could result in mutations
that affect fyn-Fas interactions or p59
activity.
Clearly, the identification of a physiologically relevant kinase
associating with the apoptosis-inducing Fas antigen opens up numerous
new avenues of investigation for elucidating the entire nature of the
death signal.