Redirecting tyrosine kinase signaling to an apoptotic
caspase pathway through chimeric adaptor proteins
Perry L. Howard*, Marie C. Chia†, Suzanne Del Rizzo*, Fei-Fei Liu†, and Tony Pawson*‡
*Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, Canada M5G 1X5; and†Department of Medical
Biophysics, University of Toronto, and Department of Radiation Oncology, Ontario Cancer Institute, Princess Margaret Hospital, University
Health Network, 610 University Avenue, Toronto, ON, Canada M6G 1X5
Communicated by Louis Siminovitch, Mount Sinai Hospital, Toronto, ON, Canada, July 25, 2003 (received for review January 15, 2003)
Signal transduction pathways are typically controlled by protein–
protein interactions, which are mediated by specific modular
domains. One hypothetical use of such interaction domains is to
generate new signaling pathways and networks during eukaryotic
evolution, through the joining of distinct binding modules in novel
combinations. In this manner, new polypeptides may be formed
that make innovative connections among preexisting proteins.
Adaptor proteins are specialized signaling molecules composed
exclusively of interaction domains, that frequently link activated
cell surface receptors to their intracellular targets. Receptor ty-
rosine kinases (RTKs) recruit adaptors, such as Grb2 and ShcA, that
activate signaling pathways involved in growth and survival,
whereas death receptors bind adaptors, such as Fadd, that pro-
mote apoptosis. To test the ability of interaction domains to create
new signaling pathways, we have fused the phosphotyrosine
recognition domains of Grb2 (Scr homology 2 domain) or ShcA
Fadd. We find that these chimeric adaptors can reroute mitogenic
or transforming RTK signals to induce caspase activation and cell
death. These hybrid adaptors can be used to selectively kill onco-
genic cells in which RTK activity is deregulated.
tively activated by mutations that induce malignant cell trans-
formation (1). Specific autophosphorylated tyrosine motifs on
activated RTKs serve as docking sites for the Src homology 2
(SH2) and phosphotyrosine-binding (PTB) domains of cytoplas-
mic adaptors, such as Grb2 and ShcA, and these modules can
therefore target specific complexes to activated RTKs (2, 3). In
signaling from normal or oncogenic tyrosine kinases, the nature
of the signal activated by these phosphotyrosine (pTyr) recog-
nition domains depends on the sequences to which they are
linked. The SH2 domain of the Grb2 adaptor, for example, is
flanked by two Src homology 3 (SH3) domains that bind
proteins, such as the Sos guanine nucleotide exchange factor and
the Gab1 docking protein, which are involved in activation of the
Ras and phosphatidylinositol 3? kinase pathways, respectively (4,
5). Grb2 therefore couples pTyr-X-Asn motifs, recognized se-
lectively by the SH2 domain, to signaling pathways that are
recruited by the SH3 domains, and promote cell proliferation,
growth, and survival. A variation on this theme is provided by
mammalian docking proteins, such as Shc, FRS2, and IRS-1
family members. These proteins all possess a PTB domain that
binds phosphorylated NPXY motifs on activated RTKs, and are
phosphorylated on tyrosine on recruitment to the receptor.
Their phosphorylation creates binding sites for the SH2 domains
of cytoplasmic signaling proteins, including Grb2, and thereby
potentiates the activation of specific biochemical pathways that
stimulate growth and survival (6).
The activation of signaling pathways through adaptor proteins
comprised of modular interaction domains is not limited to RTK
signaling, but is a common mechanism used by diverse cell-
surface receptors. For example, members of the tumor necrosis
factor-receptor superfamily contain cytoplasmic domains and
eceptor tyrosine kinases (RTKs) typically transmit signals
that promote cell growth and survival and can be constitu-
motifs that interact with corresponding domains on adaptor
proteins. This occurrence is typified by the Fas receptor, which
contains a death domain (DD) in its C-terminal tail (7, 8).
Trimerization of the receptor leads to binding of the Fas DD to
the DD of an adaptor, Fadd, which also possesses a death
effector domain (DED) (9). The DED of Fadd associates with
the DEDs of procaspase 8?10 (10). Fadd therefore bridges the
Fas receptor to procaspases. The assembly of this multiprotein
complex leads to caspase dimerization and activation, followed
by caspase autocleavage and the stimulation of pathways that
elicit apoptosis (11–13). Thus, both RTKs and death receptors
are activated by oligomerization, and recruit modular adaptors
to couple activated receptors to cytoplasmic signaling pathways.
the joining of interaction domains in nonphysiological combi-
nations might be sufficient to reengineer cellular behavior. We
therefore sought to rewire RTK signaling from proliferation and
survival pathways to apoptosis by exploiting the modular nature
of the pathway components downstream of tyrosine kinase and
composed of interaction domains from both RTK and Fas
signaling pathways could potentially convert a mitogenic ty-
rosine kinase signal into an apoptotic response by recruiting
apical components of the caspase pathway to activated RTKs.
Materials and Methods
Recombinant Adenoviruses.Plasmids containing SH2, PTB, DED-
R86K, DED-R175Q, DED-SH2, and DED-PTB were cloned
vector (14). This vector is bicistronic, containing both the
construct of interest, and a GFP reporter, under control of the
CMV promoter. These plasmids were used to make recombi-
nant, replication-deficient adenoviruses, according to the
method of He et al. (14). Adenoviruses were amplified and
purified as described (15). Adenoviral titer was determined by
monitoring cytopathic effect in an endpoint dilution assay
(Q-Biogene, Carlsbad, CA). Titers were confirmed by Western
blotting to ensure similar protein expression levels of chimeric
adaptors, and by GFP fluorescence of the different viruses.
Cell Culture. RAT-2 fibroblasts and TWO3 nasopharyngeal car-
cinoma cells were propagated at 37°C in DMEM, supplemented
with 10% FBS, 100 units?ml?1penicillin, and 100 ?g?ml?1
streptomycin. TWO3 cells have lost the Epstein–Barr virus
episome through serial passaging, and are no longer overtly
transformed (16). To derive NTR2 cells, RAT-2 cells were
transfected with ErbB2?NeuNT, and cells were maintained in
media (DMEM plus 5% FBS) until foci developed (2–3 weeks).
Abbreviations: RTK, receptor tyrosine kinase; SH2, Src homology 2; pTyr, phosphotyrosine;
PTB, pTyr binding; DED, death effector domain; moi, multiplicity of infection; EGF, epider-
mal growth factor.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2003 by The National Academy of Sciences of the USA
September 30, 2003 ?
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Foci were picked and expanded. Expression of ErbB2?NeuNT
was confirmed by Western blot analysis.
Survival and Caspase Assays. To determine cell survival, 5 ? 105
cells were plated onto 24-well dishes and grown overnight. The
next day, cells were infected with adenoviruses bearing GFP,
SH2, PTB, DED-R86K, DED-R175Q, DED-SH2, or DED-PTB
at the multiplicities of infection (mois) indicated. Caspase 8
inhibitor was used at a concentration of 20 ?M where indicated.
To determine the effect of epidermal growth factor (EGF)
stimulation, infected TWO3 cells were serum-starved overnight
and stimulated with EGF (0–100 ng?ml). The next day, cell
survival relative to GFP was determined according to the
method of Serrano et al. (17). The experiments were performed
in triplicate and were repeated at least three times.
Colony and Tumorigenicity Assays. NTR2 cells were plated onto
10-cm plates and grown overnight. The next day, cells were
in soft agar was assessed by plating 5 ? 105infected NTR2 cells
(in triplicate) in 0.25% agarose, supplemented with DMEM
(10% FBS), on top of 0.5% agarose in DMEM (10% FBS) on
60-mm plates. Cells were maintained for 21 days (at 37°C in 5%
CO2), and then subsequently stained overnight with MTT
mide). Colonies were counted and averaged.
For xenograft experiments, NTR2 cells were plated on 10-cm
plates and grown overnight. The next day, cells were infected at
an moi of 200 and incubated for 4.5 h. Cells were counted and
resuspended in PBS at a density of 106cells per ml. Female
severe combined immunodeficient (SCID) mice (6–8 weeks old)
were anesthetized, and 105cells (100 ?l) were injected into the
left gastrocnemius muscle. Three mice were injected per con-
struct. Each experiment was performed three times. Tumor
progression was monitored by measuring leg plus tumor diam-
eter. The mice were killed once this diameter reached 15 mm, or
when the tumor was ?1.7 g. All experiments were conducted in
accordance with the guidelines of the Animal Care Committee,
Ontario Cancer Institute.
Focus Assay. RAT-2 cells were transfected with 0.5 ?g ErbB2?
NeuNT cDNA and maintained in culture until foci developed.
Cells were then incubated with adenoviruses bearing SH2,
DED-R86K, DED-SH2, or DED-PTB, and individual foci were
marked and monitored over a 48-h period.
Caspase 8 Activity. Caspase 8 activity was measured by using a
caspase 8 assay kit according to the manufacturer’s instructions
(Calbiochem). Briefly, 2 ? 106NTR2 cells were infected with
adenoviruses bearing (at an moi of 200) SH2, DED-R86K,
DED-R175Q DED-SH2, or DED-PTB, and grown at 37°C. At
2-h intervals, the cells were scraped, pelleted, and washed with
PBS. The cell lysates were incubated with synthetic substrate
(IETD-pNA) at 37°C and monitored for absorbance at 405 nm
every 30 min. For TWO3, the cells were infected (at an moi of
100; DED-PTB, at an moi of 100 or 10 as indicated) and grown
day, the media was replaced with fresh low-serum media, with or
without EGF (100 ng?ml), and then incubated at 37°C. Cells
were restimulated with EGF at 6 h incubation. Caspase activity
was determined as above.
Chimeric Adaptors Stimulate Apoptosis in ErbB2?NeuNT-Transformed
Cells. To link RTK signaling to an apoptotic pathway, we
constructed hybrid adaptor proteins containing the N-terminal
DED from Fadd, attached to either the Grb2 SH2 domain
(DED-SH2) or the ShcA PTB domain at the C terminus
(DED-PTB). These chimeric adaptors have the potential to bind
procaspase 8 through their DED, and to target the resulting
complex to pTyr motifs on activated RTKs through their SH2 or
PTB domains. The constructs were C-terminally tagged with an
Myc-His epitope and incorporated into recombinant adenovi-
the PNAS web site, www.pnas.org). We also tested the effects of
domains can spontaneously oligomerize and initiate apoptosis
when overexpressed in fibroblast cell lines (9, 18). As a control
for apoptosis induced by DED overexpression, we constructed a
DED-SH2 adaptor in which Arg 86 of the Grb2 SH2 domain,
which is critical for pTyr binding, was mutated to Lys (DED-
R86K). We also constructed a DED-PTB adaptor in which Arg
175 of the ShcA PTB domain is mutated to glutamine (DED-
R175Q). This substitution abrogates the ability of ShcA PTB
domain to bind pTyr, but not phospholipid (19).
To test the rewiring strategy, we derived a cell line (NTR2) of
RAT-2 fibroblasts transformed by a constitutively active variant
of the ErbB2 RTK (NeuNT), which has binding sites for both the
Grb2 SH2 and ShcA PTB domains (20). Immunoblotting of
lysates of NTR2 cells, which had been incubated overnight with
DED-SH2, DED-PTB, DED-R86K, or SH2-bearing adenovi-
ruses (at an moi of 200), detected equivalent levels of the
different adenovirus-encoded adaptor proteins (Fig. 1A). To
determine the effect of the chimeric adaptors on cell survival,
NTR2 cells were incubated with an increasing amount of ad-
enoviruses, and cell viability was quantified after 24 h (Fig. 1B).
The expression of DED-PTB led to a rapid decrease in cell
survival, even at an moi as low as 10. DED-SH2 also diminished
cell survival, but required mois ?100 to reduce cell survival to
?20%. DED-R86K and DED-R175Q had only a modest effect
on cell survival, and required very high expression levels (at an
moi of 200; Fig. 1B). The SH2 and PTB (data not shown) domains
alone did not affect cell survival. Expression of the DED-PTB and
DED-SH2 chimeric adaptors induced DNA laddering and Poly
(ADP-ribosyl) polymerase cleavage, which is characteristic of apo-
ptotic cell death (Fig. 1 C and D). Thus, expression of chimeric
DED-SH2 and DED-PTB adaptors in ErbB2?NeuNT-trans-
formed fibroblast appears sufficient to initiate apoptosis.
Chimeric Adaptors Stimulate Caspase Activity and Form a Disc-Like
Complex in ErbB2-Transformed Cells. To investigate whether apo-
adaptors depends on caspase 8 activity, we measured cell
survival in the presence or absence of the caspase 8 inhibitor
Z-IETD-FMK. Cotreatment of NTR2 cells with caspase 8
inhibitor restored cell viability to DED-SH2 cells (at an moi of
200), and partially restored viability to DED-PTB-expressing
cells. At low levels of DED-PTB (i.e., at an moi of 10), cell
viability was fully restored with treatment of Z-IETD-FMK,
arguing that DED-SH2?DED-PTB-induced cell death depends
on caspase 8 activation (Fig. 2A). To test whether the expression
of DED-SH2 and DED-PTB leads to caspase 8 activation,
caspase 8 activity in lysates from NTR2 cells was measured. As
shown in Fig. 2B, caspase 8 activity was markedly elevated in
DED-PTB-expressing cells and reached a maximum level within
10 h after infection. Similarly, caspase 8 activity in DED-SH2
lysates increased, peaking at 12 h. Caspase 8 activity in DED-
R86K-expressing cells was not detectable until 16–18 h postin-
fection, 6 h after DED-PTB and DED-SH2 lysates had reached
maximum activity. The activity of DED-R86K is therefore likely
due to the spontaneous oligomerization of the DED, which
depends on accumulation of DED-R86K protein over time. In
contrast, caspase 8 activation by DED-PTB?DED-SH2 occurs
within a few hours of viral addition, even before GFP expression
is detectable, which is most likely due to the recruitment of the
www.pnas.org?cgi?doi?10.1073?pnas.1934711100Howard et al.
wild-type PTB or SH2 domains in the chimeric adaptors to the
activated ErbB2 RTK.
We determined whether the observed cell death was corre-
lated with the recruitment of caspase 8 to ErbB2?NeuNT by
DED-SH2 and DED-PTB. Expression of either DED-SH2 or
DED-PTB, but not DED-R86K, resulted in a decrease in
ErbB2?NeuNT expression (Fig. 3A). The rapid down-regulation
of ErbB2 induced by the chimeric adaptors suggests that recruit-
ment of caspase 8 to activated ErbB2 by DED-SH2 and DED-
PTB may lead to ErbB2 proteolytic cleavage by caspase 8.
Indeed ErbB2 contains potential caspase 8 sites (amino acids
1013–1016 and 1127–1130, Rattus norvegicus XP?218925.1) that
may be cleaved when caspase 8 is artificially tethered to ErbB2?
NeuNT by the chimeric adaptors. This loss of the receptor
complicated the detection of any complex between ErbB2,
DED-SH2?DED-PTB, and caspase 8. To circumvent this prob-
lem, we cultured the cells with caspase 8 inhibitor (Z-IETD-
FMK), immunoprecipitated adaptor molecules from cell lysates
with anti-Myc antibody, and blotted the resulting immune com-
plexes with ErbB2 antibody (Fig. 3B). Under these circum-
stances, the ErbB2?NeuNT RTK was stabilized, and was copre-
cipitated with the SH2, DED-SH2, PTB, and DED-PTB
proteins; in contrast, the DED-R86K mutant with an inactive
SH2 domain was not significantly associated with ErbB2?
NeuNT. These results argue that wild-type SH2 and PTB
domains retain their ability to bind the activated ErbB2 RTK
when they are coupled to the Fadd DED.
To explore the ability of the DED of the chimeric adaptors to
recruit caspase 8, anti-Myc immunoprecipitates of the various
adaptors were blotted for endogenous caspase 8 in the presence
or absence of caspase 8 inhibitor. Procaspase 8 is cleaved on
activation to produce 43- and 10-kDa products, of which the
43-kDa protein is further processed into 26- and 18-kDa
polypeptides (21). In the presence of caspase 8 inhibitor, full-
length procaspase 8 was coimmunoprecipitated with the DED-
SH2, DED-R86K, and DED-PTB proteins. In the absence of
caspase 8 inhibitor, the 26-kDa (DED-containing) cleavage
product was detected in immunoprecipitates of DED-SH2 and
DED-PTB, but was poorly associated with DED-R86K. These
results indicate that the DED-SH2?DED-PTB adaptors interact
with both ErbB2?NeuNT and caspase 8, stimulate caspase 8
autocleavage, and induce apoptosis. We also tested whether, in
the presence of the DED-SH2 and DED-PTB adaptors (plus
inhibitor), caspase 8 could coprecipitate with ErbB2?NeuNT,
and vice versa. Immunoprecipitation of ErbB2?NeuNT from
5637 cells transfected with ErbB2?NeuNT and Flag-caspase 8,
and blotting with anti-Flag showed that Flag-caspase 8 coim-
munoprecipitated with ErbB2?NeuNT only in the presence of
the chimeric adaptors (Fig. 3D) Similarly, ErbB2?NeuNT co-
with ErbB2?NeuNT) cells infected with SH2-, DED-R86K-, DED-SH2-, and DED-
PTB-bearing adenoviruses. (B) Survival of NTR2 cells (24 h) after treatment
with increasing mois of adenoviruses. (C) Agarose gel electrophoresis of DNA
isolated from NTR2 cells after treatment with the chimeric adaptors.
(D) Western blot analysis [anti-Poly(ADP-ribose) polymerase (PARP)] after
treatment with the chimeric adenoviruses.
(A) Western blot analysis (?-Myc) of NTR2 (RAT-2 cells transformed
presence or absence of caspase 8 inhibitor (Z-IETD-FMK) was measured by
(B) Caspase 8 activity in lysates from SH2-, DED-R86K-, DED-SH2-, or DED-PTB-
expressing NTR2 cells was determined by measuring cleavage of a synthetic
IETD-pNA caspase 8 substrate over time.
(A) NTR2 cells were incubated with DED-PTB, DED-SH2, DED-R86K,
Howard et al.
September 30, 2003 ?
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precipitated with Flag-caspase 8 only in the presence of DED-
SH2 or DED-PTB, suggesting the formation of a ternary com-
plex between the chimeric ErbB2?NeuNT, the chimeric
adaptors, and caspase 8.
Cells in Soft Agar and SCID Mice. To pursue the effects of chimeric
adaptors on mitogenesis, we tested the ability of NTR2 cells
treated with adenoviruses encoding DED-SH2, DED-PTB, and
control proteins to grow in semisolid agar. Infection of NTR2
cells with DED-SH2 or DED-PTB adenoviruses reduced the
ability of these cells to form colonies (Fig. 4A). There was a 50-
to 100-fold reduction in the ability of DED-SH2 or DED-PTB
cells to form colonies when compared with cells expressing
DED-R86K, or the SH2 or PTB domains alone (data not
shown). We also tested the effects of the DED-SH2 and DED-
PTB adaptors on tumor formation in an in vivo xenograft
transplant model. NTR2 cells infected with DED-PTB, DED-
SH2, SH2, or DED-R86K adenoviruses (at an moi of 200), or
uninfected cells, were injected into the gastrocnemius muscle of
SCID mice (Fig. 4B). Muscle plus tumor diameter was deter-
mined over a 3-week period. In control mice, tumors were
detected within 7 days postinjection and progressed very rapidly.
However, treatment of NTR2 cells with DED-PTB, and, to a
lesser extent, DED-SH2, delayed tumor formation by ?4 days
(DED-SH2), or 12 days (DED-PTB), respectively. Eventually,
tumors did arise in both DED-SH2- and DED-PTB-treated
mice, possibly because of cells that had acquired resistance to
apoptosis induced by this mechanism, or to cells that escaped
Receptor Tyrosine Kinase Activation Stimulates Apoptosis in Cells
Expressing Chimeric Adaptors. We investigated whether the chi-
meric adaptors selectively kill cells in which basal tyrosine kinase
constitutively active, or through growth factor stimulation. We
first determined whether the DED-SH2?DED-PTB adaptors
specifically kill ErbB2-transformed cells, as compared with
normal RAT-2 cells. To this end, RAT-2 cells were transfected
with ErbB2?NeuNT to generate foci of transformed cells in a
background of normal, contact-inhibited cells. The cells were
then incubated with equal amounts of adenoviruses encoding
DED-SH2, DED-PTB, or DED-R86K (SH2 and PTB not
shown), and individual foci were examined by phase contrast
microscopy. NeuNT-transformed foci infected with DED-SH2
and DED-PTB displayed significant apoptosis 24–48 h after
infection, and often detached from the substrate (Fig. 5). In
contrast, foci from plates infected with DED-R86K were not
obviously affected. Significantly, the surrounding monolayer of
nontransformed RAT-2 cells survived treatment with DED-
SH2, and with DED-PTB, indicating that the nontransformed
cells are less sensitive than their ErbB2?NeuNT-transformed
fluorescence, which provides a marker for adenovirus infection,
was detected in both the monolayer and foci (data not shown).
These data suggest that the apoptosis induced by the DED-
SH2?DED-PTB adaptors depends on the deregulated pTyr
SH2 polypeptide is not shown because it migrated off the bottom of the gel.
(B) NTR2 cells cultured with the Z-IETD-FMK caspase 8 inhibitor, immunopre-
cipitated with Myc (9E11) antibody, and blotted with ErbB2?Neu (AB3) anti-
body. (C) NTR2 cells were cultured with (?) or without (?) the caspase 8
inhibitor. Myc immunoprecipitates were probed with anti-caspase 8. Full-
length procaspase 8 (? Z-IETD-FMK), or the p26 cleavage product, coimmu-
caspase 8 (Left) or ErbB2?NeuNT immunoprecipitation (Right) from trans-
fected 5637 cells (with caspase 8 inhibitor) probed with either ?-Flag or
?-ErbB2?NeuNT. The ? indicates whether or not a construct was transfected?
infected. Arrows indicate position of caspase 8 and ErbB2?NeuNT.*, a non-
specific band in Flag Ip.
(A) Lysates of NTR2 cells expressing Myc-tagged SH2, DED-R86K,
DED-R86K, DED-SH2, or DED-PTB (at an moi of 200). (B) NTR2 cells were either
or DED-PTB (at an moi of 200) adenoviruses, and then injected into the leg
muscle of SCID mice. Leg plus tumor diameter was measured over a 3-week
period. The plotted data represent the mean ? SE for the experiments.
(A) Colony formation in soft agar of NTR2 cells incubated with SH2,
www.pnas.org?cgi?doi?10.1073?pnas.1934711100Howard et al.
signal of the oncogenic ErbB2?NeuNT RTK, and that residual
tyrosine kinase signaling in quiescent cells (contact inhibited for
2 weeks) is inefficient at inducing apoptosis. We have confirmed
these results by using fusion proteins of DED-SH2, DED-R86K,
and SH2, in which the adaptors are fused to a membrane
permeable sequence from the HIV TAT protein, which allows
results were similar to adenovirus-mediated delivery and rule
out spurious contribution of adenoviral proteins to DED-SH2-
mediated apoptosis (data not shown). To quantify the sensitiv-
ities of ErbB2?NeuNT-transformed and parental cells to DED-
SH2 or DED-PTB treatment, we treated serum-starved parental
RAT-2 and NTR2 cells with identical amounts of DED-SH2 and
DED-PTB adenoviruses. Whereas DED-SH2 or DED-PTB (at
an moi of 200) reduced the viability of NTR2 cells to ?20% within
24 h, parental RAT-2 cells remained viable (70–80%) at the same
dose. Thus, we conclude that expression of activated ErbB2 leads
to an ?4-fold increase in killing by chimeric adaptors.
We also investigated the effects of growth factor stimulation
on cells expressing the chimeric adaptors. For this purpose, we
used TWO3 human epithelial cells, which express the EGF
receptor (EGFR) and respond to EGF (16). As shown in Fig. 6A,
expression of DED-SH2 and DED-PTB in TWO3 cells stimu-
lated with EGF (100 ng?ml) produced DNA laddering, which is
characteristic of apoptosis. Unstimulated cells displayed reduced
levels of DNA laddering (data not shown). To test the effect of
EGF stimulation on the survival of TWO3 cells, cells were
treated with SH2, PTB, DED-R86K, DED-SH2, or DED-PTB
adenoviruses, serum-starved overnight, and then stimulated
with serum free medium (?EGF) or medium containing EGF
(100 ng?ml) for 24 h. EGFR activation decreased the survival of
DED-SH2-expressing cells, whereas cells expressing the control
DED-R86K, SH2, or PTB proteins were not obviously affected
by EGFR stimulation (Fig. 6B). At high mois (?100), DED-PTB
However, at reduced mois (an moi of 10), we observed that EGF
induced apoptosis in DED-PTB-expressing cells, indicating that
this adaptor is more potent in nucleating an apoptotic response.
Consistent with these observations, EGF stimulation of TWO3
cells expressing DED-PTB or DED-SH2 caused a rapid rise in
caspase 8 activity that reached a maximum at ?4 h after
stimulation (see Fig. 8, which is published as supporting infor-
mation on the PNAS web site). Control DED-R86K and DED-
an important mechanism for controlling signal transduction
pathways (6, 23). One possible reason for the prevalent use of
interaction domains is to facilitate the formation of new con-
nections between existing proteins during the course of evolu-
tion, and thus to create new signaling pathways. Conversely,
aberrant protein interactions can disturb cellular phenotype, and
thereby contribute to disease. Proteins encoded by pathogenic
bacteria and viruses, or chimeric cellular oncoproteins, fre-
quently exert their effects by usurping normal protein–protein
interactions, and thus reorganizing cellular behavior. For exam-
the actin cytoskeleton of the host cell by binding the SH2?SH3
adaptor protein Nck (24), whereas the Bcr-Abl oncoprotein is
phosphorylated within its Bcr region at a YVNV motif that binds
the Grb2 adaptor (25, 26). The modular nature of signaling
pathways thus lends itself to rewiring, in which proteins are
assembled into novel complexes with new biological properties.
Previous work (27–29) has suggested that signaling proteins
can be experimentally modified to form novel interactions. For
example, the oncogenic potential of Bcr-Abl can be attenuated
by chimeric adaptors comprising the catalytic domain of the
tyrosine phosphatase Shp1 fused to the Abl-binding domain of
Rin1 (27). Similarly, caspase 8 can be artificially activated
through chemically inducible dimerization (CID) domains fused
in tandem to Fadd or caspase 8 (30), likely resulting in higher-
order oligomerization of the fusion proteins in response to
chemical dimerizer. Chimeric scaffold proteins have also been
used to reroute signaling between distinct MAP kinase pathways
in yeast (28). In the example discussed here, we have linked two
entirely different signaling pathways by using chimeric adaptors.
The ErbB2 RTK has multiple Grb2 SH2-binding sites (at least
one direct and two through ShcA) and one ShcA PTB-binding
recruit several caspase 8 molecules through the DED-SH2
adaptor, and two caspase 8 molecules through DED-PTB.
Although the activated Fas receptor itself forms a trimer, recent
data suggest that dimerization is the crucial step in caspase 8
activation (11, 13). These observations suggest a possible mech-
R86K, DED-SH2, or DED-PTB proteins, and individual foci were examined over
is less sensitive to apoptosis induced by DED-SH2?DED-PTB. (B) The survival of
adenoviral delivery (at an moi of 200).
(A) RAT-2 cells were incubated with adenoviruses encoding DED-
was analyzed by agarose gel electrophoresis. (B) TWO3 cells were infected
with SH2, PTB, DED-R86K, DEDSH2, or DED-PTB adenoviruses at an moi of 100
(an moi of 100 or 10 for DED-PTB), and were serum-starved overnight. Cells
were stimulated with serum-free medium (?EGF) or medium containing EGF
(100 ng?ml). Quantification of the survival of TWO3 cells after EGF stimula-
tion, 48 h after infection (24 h after EGF stimulation).
(A) TWO3 cells were treated with SH2, DED-R86K, DED-SH2, or
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September 30, 2003 ?
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anism through which the DED-SH2?DED-PTB chimeric adap- Download full-text
tors induce caspase 8 activation in response to RTK signaling,
namely that the adaptors recruit caspase 8 to the activated
receptor in a fashion that suffices for caspase dimerization and
activation. These data support the view that quite distinct
receptors and signaling pathways operate through the same
general principles of modular protein interactions and proximity
Our results show that chimeric adaptors containing a pTyr-
recognition domain (SH2 or PTB) fused to a DED can nucleate
a complex in which an activated RTK is coupled to caspase 8,
leading to stimulation of caspase activity and consequent cell
death. We have demonstrated this rewiring of tyrosine kinase
and caspase pathways in cells transformed by an oncogenic
variant of ErbB2, and in tumors arising from such cells, as well
as in epithelial cells stimulated with EGF. The ShcA PTB
domain appears more potent than the Grb2 SH2 domain in
stimulating apoptosis when linked to the Fadd DED domain,
and, indeed, when a high level of the DED-PTB adenovirus is
increased ability of DED-PTB to induce apoptosis are unclear,
but they likely reflect innate differences between the binding
properties of the ShcA PTB domain and Grb2 SH2 domain, or
their roles in signaling from ErbB1?ErbB2. The ShcA PTB
domain has a slower off-rate for phosphorylated motifs than do
SH2 domains (31), and this rate may enhance the potential for
caspase 8 dimerization. Furthermore, the PTB domain also
binds PIP2, which promotes ShcA association with the plasma
membrane (19). Although the lipid binding ability of ShcA PTB
is likely not sufficient to induce significant apoptosis, because
this activity should be retained by the mutant PTB domain in the
DED-R175Q adaptor, it may contribute. Another distinguishing
feature of the PTB and SH2 domains is that Grb2 contributes to
the recruitment of the Cbl E3 protein-ubiquitin ligase to ErbB1,
raising the possibility that overexpression of the Grb2 SH2
domain could interfere with this negative regulator of RTK
signaling (32). Regardless, the data illustrate the inherent dif-
ferences between these two classes of pTyr-binding modules.
Our results raise the possibility that cells can be engineered to
alter their responses to external signals, in ways that might be
therapeutically beneficial. With detailed knowledge of pTyr
signaling in individual tumors, for example, through SH2 pro-
filing (33), chimeric adaptors could be tailored to specifically
target the RTK expression profile of the individual tumor.
Alternatively, small molecules that could bridge endogenous
components of RTK signaling pathways to apoptotic pathways
might also reengineer cellular behavior in a manner that could
antagonize the growth of transformed cells. Indeed, drugs such
as cyclosporin and rapamycin exert their effect by nucleating
novel protein–protein interactions (34, 35). In summary, our
data support the idea that new signaling pathways can be
engineered by the joining of interaction domains in novel
We thank Drs. Jerry Gish and Henry Klamut for helpful discussions and
Dr. Ingemar Ernberg for the TWO3 cells. This work was supported by
grants from the National Cancer Institute of Canada (T.P.), the Cana-
dian Institutes of Health Research (T.P.), and the Elia Chair in Head and
Neck Cancer Research (F.-F.L.). P.L.H. is a Postdoctoral Fellow of the
National Cancer Institute of Canada and is supported by funds received
from the Terry Fox Run; M.C. is a Ph.D. student and is supported by a
Canadian Institutes of Health Research student award and a Cancer
Research Society student fellowship; and T.P. is a distinguished scientist
of the Canadian Institutes of Health Research.
1. Blume-Jensen, P. & Hunter, T. (2001) Nature 411, 355–365.
2. Pawson, T. (1995) Nature 373, 573–580.
4. Li, N., Batzer, A., Daly, R., Yajnik, V., Skolnik, E., Chardin, P., Bar-Sagi, D.,
Margolis, B. & Schlessinger, J. (1993) Nature 363, 85–88.
5. Holgado-Madruga, M., Emlet, D. R., Moscatello, D. K., Godwin, A. K. &
Wong, A. J. (1996) Nature 379, 560–564.
6. Pawson, T. & Nash, P. (2000) Genes Dev. 14, 1027–1047.
7. Itoh, N. & Nagata, S. (1993) J. Biol. Chem. 268, 10932–10937.
8. Tartaglia, L. A., Ayres, T. M., Wong, G. H. & Goeddel, D. V. (1993) Cell 74,
9. Boldin, M. P., Varfolomeev, E. E., Pancer, Z., Mett, I. L., Camonis, J. H. &
Wallach, D. (1995) J. Biol. Chem. 270, 7795–7798.
10. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V. & Wallach, D. (1996) Cell 85,
11. Boatright, K. M., Renatus, M., Scott, F. L., Sperandio, S., Shin, H., Pedersen,
I. M., Ricci, J. E., Edris, W. A., Sutherlin, D. P., Green, D. R., et al. (2003) Mol.
Cell 11, 529–541.
12. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni,
J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., et al. (1996) Cell 85, 817–827.
13. Shiozaki, E. N., Chai, J., Rigotti, D. J., Riedl, S. J., Li, P., Srinivasula, S. M.,
Alnemri, E. S., Fairman, R. & Shi, Y. (2003) Mol. Cell 11, 519–527.
14. He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W. & Vogelstein, B.
(1998) Proc. Natl. Acad. Sci. USA 95, 2509–2514.
15. Graham, F. L. & Prevec, L. (1992) Bio?Technology 20, 363–390.
16. Teramoto, N., Maeda, A., Kobayshi, K., Hayashi, K., Oka, T., Takahashi, K.,
Takada, K., Klein, G. & Akagi, T. (2002) Lab. Invest. 80, 303–312.
17. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W. (1997)
Cell 88, 593–602.
18. Chinnaiyan, A. M., O’Rourke, K., Tewari, M. & Dixit, V. M. (1995) Cell 81,
19. Ravichandran, K. S., Zhou, M. M., Pratt, J. C., Harlan, J. E., Walk, S. F., Fesik,
S. W. & Burakoff, S. J. (1997) Mol. Cell. Biol. 17, 5540–5549.
20. Dankort, D., Jeyabalan, N., Jones, N., Dumont, D. J. & Muller, W. J. (2001)
J. Biol. Chem. 276, 38921–38928.
21. Medema, J. P., Scaffidi, C., Kischkel, F. C., Shevchenko, A., Mann, M.,
Krammer, P. H. & Peter, M. E. (1997) EMBO J. 16, 2794–2804.
22. Schwarze, S. R., Ho, A., Vocero-Akbani, A. & Dowdy, S. F. (1999) Science 285,
23. Pawson, T. & Nash, P. (2003) Science 300, 445–452.
24. Gruenheid, S., DeVinney, R., Bladt, F., Goosney, D., Gelkop, S., Gish, G. D.,
Pawson, T. & Finlay, B. B. (2001) Nat. Cell Biol. 3, 856–859.
25. Pendergast, A. M., Muller, A. J., Havlik, M. H., Maru, Y. & Witte, O. N. (1991)
Cell 66, 161–171.
26. Goga, A., McLaughlin, J., Afar, D. E., Saffran, D. C. & Witte, O. N. (1995) Cell
27. Lim, Y. M., Wong, S., Lau, G., Witte, O. N. & Colicelli, J. (2000) Proc. Natl.
Acad. Sci. USA 97, 12233–12238.
28. Park, S. H., Zarrinpar, A. & Lim, W. A. (2003) Science 299, 1061–1064.
29. Harris, K., Lamson, R. E., Nelson, B., Hughes, T. R., Marton, M. J., Roberts,
C. J., Boone, C. & Pryciak, P. M. (2001) Curr. Biol. 11, 1815–1824.
30. Fan, L., Freeman, K. W., Khan, T., Pham, E. & Spencer, D. M. (1999) Hum.
Gene Ther. 10, 2273–2285.
31. Zhou, M. M., Harlan, J. E., Wade, W. S., Crosby, S., Ravichandran, K. S.,
Burakoff, S. J. & Fesik, S. W. (1995) J. Biol. Chem. 270, 31119–31123.
32. Waterman, H., Katz, M., Rubin, C., Shtiegman, K., Lavi, S., Elson, A., Jovin,
T. & Yarden, Y. (2002) EMBO J. 21, 303–313.
33. Nollau, P. & Mayer, B. J. (2001) Proc. Natl. Acad. Sci. USA 98, 13531–13536.
34. Husi, H., Luyten, M. A. & Zurini, M. G. (1994) J. Biol. Chem. 269, 14199–
35. Woerly, G., Weber, E. & Ryffel, B. (1994) Biochem. Pharmacol. 47, 1435–1443.
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