Redirecting 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) pathways are typically controlled by protein– motifs that interact with corresponding domains on adaptor protein interactions, which are mediated by specific modular proteins. This occurrence is typified by the Fas receptor, which domains. One hypothetical use of such interaction domains is to contains a (DD) in its C-terminal tail (7, 8). generate new signaling pathways and networks during eukaryotic Trimerization of the receptor leads to binding of the Fas DD to evolution, through the joining of distinct binding modules in novel the DD of an adaptor, Fadd, which also possesses a death combinations. In this manner, new polypeptides may be formed effector domain (DED) (9). The DED of Fadd associates with that make innovative connections among preexisting proteins. the DEDs of procaspase 8͞10 (10). Fadd therefore bridges the Adaptor proteins are specialized signaling molecules composed Fas receptor to procaspases. The assembly of this multiprotein exclusively of interaction domains, that frequently link activated complex leads to caspase dimerization and activation, followed cell surface receptors to their intracellular targets. Receptor ty- by caspase autocleavage and the stimulation of pathways that rosine kinases (RTKs) recruit adaptors, such as Grb2 and ShcA, that elicit apoptosis (11–13). Thus, both RTKs and death receptors activate signaling pathways involved in growth and survival, are activated by oligomerization, and recruit modular adaptors whereas death receptors bind adaptors, such as Fadd, that pro- to couple activated receptors to cytoplasmic signaling pathways. mote apoptosis. To test the ability of interaction domains to create Based on these observations, we considered the possibility that new signaling pathways, we have fused the phosphotyrosine the joining of interaction domains in nonphysiological combi- recognition domains of Grb2 (Scr homology 2 domain) or ShcA nations might be sufficient to reengineer cellular behavior. We (phosphotyrosine-binding domain) to the of therefore sought to rewire RTK signaling from proliferation and Fadd. We find that these chimeric adaptors can reroute mitogenic survival pathways to apoptosis by exploiting the modular nature or transforming RTK signals to induce caspase activation and cell of the pathway components downstream of tyrosine kinase and death. These hybrid adaptors can be used to selectively kill onco- death receptors. We hypothesized that chimeric adaptor proteins genic cells in which RTK activity is deregulated. composed of interaction domains from both RTK and Fas signaling pathways could potentially convert a mitogenic ty- eceptor tyrosine kinases (RTKs) typically transmit signals rosine kinase signal into an apoptotic response by recruiting Rthat promote cell growth and survival and can be constitu- apical components of the caspase pathway to activated RTKs. tively activated by mutations that induce malignant cell trans- formation (1). Specific autophosphorylated tyrosine motifs on Materials and Methods activated RTKs serve as docking sites for the Src homology 2 Recombinant Adenoviruses. Plasmids containing SH2, PTB, DED- (SH2) and phosphotyrosine-binding (PTB) domains of cytoplas- R86K, DED-R175Q, DED-SH2, and DED-PTB were cloned mic adaptors, such as Grb2 and ShcA, and these modules can into the KpnI͞NotI sites of the AdTrack cytomegalovirus (CMV) therefore target specific complexes to activated RTKs (2, 3). In vector (14). This vector is bicistronic, containing both the signaling from normal or oncogenic tyrosine kinases, the nature construct of interest, and a GFP reporter, under control of the of the signal activated by these phosphotyrosine (pTyr) recog- CMV promoter. These plasmids were used to make recombi- nition domains depends on the sequences to which they are nant, replication-deficient adenoviruses, according to the linked. The SH2 domain of the Grb2 adaptor, for example, is method of He et al. (14). Adenoviruses were amplified and flanked by two Src homology 3 (SH3) domains that bind purified as described (15). Adenoviral titer was determined by proteins, such as the Sos guanine nucleotide exchange factor and monitoring cytopathic effect in an endpoint dilution assay the Gab1 docking protein, which are involved in activation of the Ј (Q-Biogene, Carlsbad, CA). Titers were confirmed by Western Ras and phosphatidylinositol 3 kinase pathways, respectively (4, blotting to ensure similar protein expression levels of chimeric 5). Grb2 therefore couples pTyr-X-Asn motifs, recognized se- adaptors, and by GFP fluorescence of the different viruses. lectively by the SH2 domain, to signaling pathways that are recruited by the SH3 domains, and promote cell proliferation, Cell Culture. RAT-2 fibroblasts and TWO3 nasopharyngeal car- growth, and survival. A variation on this theme is provided by cinoma cells were propagated at 37°C in DMEM, supplemented mammalian docking proteins, such as Shc, FRS2, and IRS-1 with 10% FBS, 100 units͞mlϪ1 penicillin, and 100 ␮g͞mlϪ1 family members. These proteins all possess a PTB domain that streptomycin. TWO3 cells have lost the Epstein–Barr virus binds phosphorylated NPXY motifs on activated RTKs, and are episome through serial passaging, and are no longer overtly phosphorylated on tyrosine on recruitment to the receptor. transformed (16). To derive NTR2 cells, RAT-2 cells were Their phosphorylation creates binding sites for the SH2 domains transfected with ErbB2͞NeuNT, and cells were maintained in of cytoplasmic signaling proteins, including Grb2, and thereby media (DMEM plus 5% FBS) until foci developed (2–3 weeks). potentiates the activation of specific biochemical pathways that stimulate growth and survival (6). The activation of signaling pathways through adaptor proteins Abbreviations: RTK, ; SH2, Src homology 2; pTyr, phosphotyrosine; comprised of modular interaction domains is not limited to RTK PTB, pTyr binding; DED, death effector domain; moi, multiplicity of infection; EGF, epider- signaling, but is a common mechanism used by diverse cell- mal growth factor.

surface receptors. For example, members of the tumor necrosis ‡To whom correspondence should be addressed. E-mail: [email protected]. BIOCHEMISTRY factor-receptor superfamily contain cytoplasmic domains and © 2003 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.1934711100 PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 ͉ 11267–11272 Downloaded by guest on September 28, 2021 Foci were picked and expanded. Expression of ErbB2͞NeuNT (DED-PTB). These chimeric adaptors have the potential to bind was confirmed by Western blot analysis. procaspase 8 through their DED, and to target the resulting complex to pTyr motifs on activated RTKs through their SH2 or Survival and Caspase Assays. To determine cell survival, 5 ϫ 105 PTB domains. The constructs were C-terminally tagged with an cells were plated onto 24-well dishes and grown overnight. The Myc-His epitope and incorporated into recombinant adenovi- next day, cells were infected with adenoviruses bearing GFP, ruses (see Fig. 7, which is published as supporting information on SH2, PTB, DED-R86K, DED-R175Q, DED-SH2, or DED-PTB the PNAS web site, www.pnas.org). We also tested the effects of at the multiplicities of infection (mois) indicated. Caspase 8 SH2 and PTB domains expressed on their own. In addition, DED inhibitor was used at a concentration of 20 ␮M where indicated. domains can spontaneously oligomerize and initiate apoptosis To determine the effect of epidermal growth factor (EGF) when overexpressed in fibroblast cell lines (9, 18). As a control stimulation, infected TWO3 cells were serum-starved overnight for apoptosis induced by DED overexpression, we constructed a and stimulated with EGF (0–100 ng͞ml). The next day, cell DED-SH2 adaptor in which Arg 86 of the Grb2 SH2 domain, survival relative to GFP was determined according to the which is critical for pTyr binding, was mutated to Lys (DED- method of Serrano et al. (17). The experiments were performed R86K). We also constructed a DED-PTB adaptor in which Arg in triplicate and were repeated at least three times. 175 of the ShcA PTB domain is mutated to glutamine (DED- R175Q). This substitution abrogates the ability of ShcA PTB Colony and Tumorigenicity Assays. NTR2 cells were plated onto domain to bind pTyr, but not phospholipid (19). 10-cm plates and grown overnight. The next day, cells were To test the rewiring strategy, we derived a cell line (NTR2) of counted, infected at an moi of 200, and incubated for 1 h. Growth RAT-2 fibroblasts transformed by a constitutively active variant ϫ 5 in soft agar was assessed by plating 5 10 infected NTR2 cells of the ErbB2 RTK (NeuNT), which has binding sites for both the (in triplicate) in 0.25% agarose, supplemented with DMEM Grb2 SH2 and ShcA PTB domains (20). Immunoblotting of (10% FBS), on top of 0.5% agarose in DMEM (10% FBS) on lysates of NTR2 cells, which had been incubated overnight with 60-mm plates. Cells were maintained for 21 days (at 37°Cin5% DED-SH2, DED-PTB, DED-R86K, or SH2-bearing adenovi- CO2), and then subsequently stained overnight with MTT ruses (at an moi of 200), detected equivalent levels of the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro- different adenovirus-encoded adaptor proteins (Fig. 1A). To mide). Colonies were counted and averaged. determine the effect of the chimeric adaptors on cell survival, For xenograft experiments, NTR2 cells were plated on 10-cm NTR2 cells were incubated with an increasing amount of ad- plates and grown overnight. The next day, cells were infected at enoviruses, and cell viability was quantified after 24 h (Fig. 1B). an moi of 200 and incubated for 4.5 h. Cells were counted and The expression of DED-PTB led to a rapid decrease in cell resuspended in PBS at a density of 106 cells per ml. Female survival, even at an moi as low as 10. DED-SH2 also diminished severe combined immunodeficient (SCID) mice (6–8 weeks old) cell survival, but required mois Ͼ100 to reduce cell survival to were anesthetized, and 105 cells (100 ␮l) were injected into the Ͻ20%. DED-R86K and DED-R175Q had only a modest effect left gastrocnemius muscle. Three mice were injected per con- on cell survival, and required very high expression levels (at an struct. Each experiment was performed three times. Tumor moi of 200; Fig. 1B). The SH2 and PTB (data not shown) domains progression was monitored by measuring leg plus tumor diam- alone did not affect cell survival. Expression of the DED-PTB and eter. The mice were killed once this diameter reached 15 mm, or when the tumor was Ϸ1.7 g. All experiments were conducted in DED-SH2 chimeric adaptors induced DNA laddering and Poly accordance with the guidelines of the Care Committee, (ADP-ribosyl) polymerase cleavage, which is characteristic of apo- Ontario Cancer Institute. ptotic cell death (Fig. 1 C and D). Thus, expression of chimeric DED-SH2 and DED-PTB adaptors in ErbB2͞NeuNT-trans- Focus Assay. RAT-2 cells were transfected with 0.5 ␮g ErbB2͞ formed fibroblast appears sufficient to initiate apoptosis. NeuNT cDNA and maintained in culture until foci developed. Cells were then incubated with adenoviruses bearing SH2, Chimeric Adaptors Stimulate Caspase Activity and Form a Disc-Like DED-R86K, DED-SH2, or DED-PTB, and individual foci were Complex in ErbB2-Transformed Cells. To investigate whether apo- marked and monitored over a 48-h period. ptosis of ErbB2-transformed NTR2 cells induced by the chimeric adaptors depends on caspase 8 activity, we measured cell Caspase 8 Activity. Caspase 8 activity was measured by using a survival in the presence or absence of the caspase 8 inhibitor caspase 8 assay kit according to the manufacturer’s instructions Z-IETD-FMK. Cotreatment of NTR2 cells with caspase 8 (Calbiochem). Briefly, 2 ϫ 106 NTR2 cells were infected with inhibitor restored cell viability to DED-SH2 cells (at an moi of adenoviruses bearing (at an moi of 200) SH2, DED-R86K, 200), and partially restored viability to DED-PTB-expressing DED-R175Q DED-SH2, or DED-PTB, and grown at 37°C. At cells. At low levels of DED-PTB (i.e., at an moi of 10), cell 2-h intervals, the cells were scraped, pelleted, and washed with viability was fully restored with treatment of Z-IETD-FMK, ͞ PBS. The cell lysates were incubated with synthetic substrate arguing that DED-SH2 DED-PTB-induced cell death depends (IETD-pNA) at 37°C and monitored for absorbance at 405 nm on caspase 8 activation (Fig. 2A). To test whether the expression every 30 min. For TWO3, the cells were infected (at an moi of of DED-SH2 and DED-PTB leads to caspase 8 activation, 100; DED-PTB, at an moi of 100 or 10 as indicated) and grown caspase 8 activity in lysates from NTR2 cells was measured. As overnight in low serum media (DMEM plus 0.5% FBS). The next shown in Fig. 2B, caspase 8 activity was markedly elevated in day, the media was replaced with fresh low-serum media, with or DED-PTB-expressing cells and reached a maximum level within without EGF (100 ng͞ml), and then incubated at 37°C. Cells 10 h after infection. Similarly, caspase 8 activity in DED-SH2 were restimulated with EGF at 6 h incubation. Caspase activity lysates increased, peaking at 12 h. Caspase 8 activity in DED- was determined as above. R86K-expressing cells was not detectable until 16–18 h postin- fection,6hafterDED-PTB and DED-SH2 lysates had reached Results maximum activity. The activity of DED-R86K is therefore likely Chimeric Adaptors Stimulate Apoptosis in ErbB2͞NeuNT-Transformed due to the spontaneous oligomerization of the DED, which Cells. To link RTK signaling to an apoptotic pathway, we depends on accumulation of DED-R86K protein over time. In constructed hybrid adaptor proteins containing the N-terminal contrast, caspase 8 activation by DED-PTB͞DED-SH2 occurs DED from Fadd, attached to either the Grb2 SH2 domain within a few hours of viral addition, even before GFP expression (DED-SH2) or the ShcA PTB domain at the C terminus is detectable, which is most likely due to the recruitment of the

11268 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1934711100 Howard et al. Downloaded by guest on September 28, 2021 Fig. 2. (A) NTR2 cells were incubated with DED-PTB, DED-SH2, DED-R86K, PTB, or SH2 encoding adenoviruses (at an moi of 200), and survival (24 h) in the presence or absence of caspase 8 inhibitor (Z-IETD-FMK) was measured by crystal violet staining. The data represent the average Ϯ SE for the experiment. (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.

NeuNT by the chimeric adaptors. This loss of the receptor complicated the detection of any complex between ErbB2, DED-SH2͞DED-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 ErbB2͞NeuNT 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 ErbB2͞ 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 ␣ Fig. 1. (A) Western blot analysis ( -Myc) of NTR2 (RAT-2 cells transformed recruit caspase 8, anti-Myc immunoprecipitates of the various with ErbB2͞NeuNT) cells infected with SH2-, DED-R86K-, DED-SH2-, and DED- PTB-bearing adenoviruses. (B) Survival of NTR2 cells (24 h) after treatment adaptors were blotted for endogenous caspase 8 in the presence with increasing mois of adenoviruses. (C) Agarose gel electrophoresis of DNA or absence of caspase 8 inhibitor. Procaspase 8 is cleaved on isolated from NTR2 cells after treatment with the chimeric adaptors. activation to produce 43- and 10-kDa products, of which the (D) Western blot analysis [anti-Poly(ADP-ribose) polymerase (PARP)] after 43-kDa protein is further processed into 26- and 18-kDa treatment with the chimeric adenoviruses. 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 wild-type PTB or SH2 domains in the chimeric adaptors to the caspase 8 inhibitor, the 26-kDa (DED-containing) cleavage activated ErbB2 RTK. product was detected in immunoprecipitates of DED-SH2 and We determined whether the observed cell death was corre- DED-PTB, but was poorly associated with DED-R86K. These ͞ lated with the recruitment of caspase 8 to ErbB2 NeuNT by results indicate that the DED-SH2͞DED-PTB adaptors interact DED-SH2 and DED-PTB. Expression of either DED-SH2 or with both ErbB2͞NeuNT and caspase 8, stimulate caspase 8 DED-PTB, but not DED-R86K, resulted in a decrease in autocleavage, and induce apoptosis. We also tested whether, in ErbB2͞NeuNT expression (Fig. 3A). The rapid down-regulation the presence of the DED-SH2 and DED-PTB adaptors (plus of ErbB2 induced by the chimeric adaptors suggests that recruit- inhibitor), caspase 8 could coprecipitate with ErbB2͞NeuNT, ment of caspase 8 to activated ErbB2 by DED-SH2 and DED- and vice versa. Immunoprecipitation of ErbB2͞NeuNT from PTB may lead to ErbB2 proteolytic cleavage by caspase 8. 5637 cells transfected with ErbB2͞NeuNT and Flag-caspase 8, Indeed ErbB2 contains potential caspase 8 sites (amino acids and blotting with anti-Flag showed that Flag-caspase 8 coim-

1013–1016 and 1127–1130, Rattus norvegicus XP࿝218925.1) that munoprecipitated with ErbB2͞NeuNT only in the presence of BIOCHEMISTRY may be cleaved when caspase 8 is artificially tethered to ErbB2͞ the chimeric adaptors (Fig. 3D) Similarly, ErbB2͞NeuNT co-

Howard et al. PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 ͉ 11269 Downloaded by guest on September 28, 2021 Fig. 4. (A) Colony formation in soft agar of NTR2 cells incubated with SH2, DED-R86K, DED-SH2, or DED-PTB (at an moi of 200). (B) NTR2 cells were either left untreated (uninfected control) or infected with SH2, DED-R86K, DED-SH2, 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 Ϯ SE for the experiments.

mined over a 3-week period. In control mice, tumors were Fig. 3. (A) Lysates of NTR2 cells expressing Myc-tagged SH2, DED-R86K, detected within 7 days postinjection and progressed very rapidly. DED-SH2, or DED-PTB were immunoblotted with the indicated antibodies. The However, treatment of NTR2 cells with DED-PTB, and, to a SH2 polypeptide is not shown because it migrated off the bottom of the gel. Ϸ (B) NTR2 cells cultured with the Z-IETD-FMK caspase 8 inhibitor, immunopre- lesser extent, DED-SH2, delayed tumor formation by 4 days cipitated with Myc (9E11) antibody, and blotted with ErbB2͞Neu (AB3) anti- (DED-SH2), or 12 days (DED-PTB), respectively. Eventually, body. (C) NTR2 cells were cultured with (ϩ) or without (Ϫ) the caspase 8 tumors did arise in both DED-SH2- and DED-PTB-treated inhibitor. Myc immunoprecipitates were probed with anti-caspase 8. Full- mice, possibly because of cells that had acquired resistance to ϩ length procaspase 8 ( Z-IETD-FMK), or the p26 cleavage product, coimmu- apoptosis induced by this mechanism, or to cells that escaped noprecipitate with adaptors containing DED. (D) Flag immunoprecipitation of adenovirus infection. caspase 8 (Left) or ErbB2͞NeuNT immunoprecipitation (Right) from trans- fected 5637 cells (with caspase 8 inhibitor) probed with either ␣-Flag or ␣-ErbB2͞NeuNT. The Ϯ indicates whether or not a construct was transfected͞ Receptor Tyrosine Kinase Activation Stimulates Apoptosis in Cells infected. Arrows indicate position of caspase 8 and ErbB2͞NeuNT. *, a non- Expressing Chimeric Adaptors. We investigated whether the chi- specific band in Flag Ip. meric adaptors selectively kill cells in which basal tyrosine kinase activity is elevated, either through mutation that renders an RTK constitutively active, or through growth factor stimulation. We precipitated with Flag-caspase 8 only in the presence of DED- first determined whether the DED-SH2͞DED-PTB adaptors SH2 or DED-PTB, suggesting the formation of a ternary com- specifically kill ErbB2-transformed cells, as compared with ͞ plex between the chimeric ErbB2 NeuNT, the chimeric normal RAT-2 cells. To this end, RAT-2 cells were transfected adaptors, and caspase 8. with ErbB2͞NeuNT to generate foci of transformed cells in a background of normal, contact-inhibited cells. The cells were ͞ Chimeric Adaptors Suppress the Growth of ErbB2 NeuNT-Transformed then incubated with equal amounts of adenoviruses encoding Cells in Soft Agar and SCID Mice. To pursue the effects of chimeric DED-SH2, DED-PTB, or DED-R86K (SH2 and PTB not adaptors on mitogenesis, we tested the ability of NTR2 cells shown), and individual foci were examined by phase contrast treated with adenoviruses encoding DED-SH2, DED-PTB, and microscopy. NeuNT-transformed foci infected with DED-SH2 control proteins to grow in semisolid agar. Infection of NTR2 and DED-PTB displayed significant apoptosis 24–48 h after cells with DED-SH2 or DED-PTB adenoviruses reduced the infection, and often detached from the substrate (Fig. 5). In ability of these cells to form colonies (Fig. 4A). There was a 50- contrast, foci from plates infected with DED-R86K were not to 100-fold reduction in the ability of DED-SH2 or DED-PTB obviously affected. Significantly, the surrounding monolayer of cells to form colonies when compared with cells expressing nontransformed RAT-2 cells survived treatment with DED- DED-R86K, or the SH2 or PTB domains alone (data not SH2, and with DED-PTB, indicating that the nontransformed shown). We also tested the effects of the DED-SH2 and DED- cells are less sensitive than their ErbB2͞NeuNT-transformed PTB adaptors on tumor formation in an in vivo xenograft counterparts to apoptosis induced by the chimeric adaptors. GFP transplant model. NTR2 cells infected with DED-PTB, DED- fluorescence, which provides a marker for adenovirus infection, SH2, SH2, or DED-R86K adenoviruses (at an moi of 200), or was detected in both the monolayer and foci (data not shown). uninfected cells, were injected into the gastrocnemius muscle of These data suggest that the apoptosis induced by the DED- SCID mice (Fig. 4B). Muscle plus tumor diameter was deter- SH2͞DED-PTB adaptors depends on the deregulated pTyr

11270 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1934711100 Howard et al. Downloaded by guest on September 28, 2021 Fig. 6. (A) TWO3 cells were treated with SH2, DED-R86K, DED-SH2, or DED-PTB adenoviruses in the presence of EGF (100 ng͞ml), and DNA laddering 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 (ϪEGF) or medium containing EGF (100 ng͞ml). Quantification of the survival of TWO3 cells after EGF stimula- tion, 48 h after infection (24 h after EGF stimulation).

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 Ϸ4hafter stimulation (see Fig. 8, which is published as supporting infor- Fig. 5. (A) RAT-2 cells were incubated with adenoviruses encoding DED- mation on the PNAS web site). Control DED-R86K and DED- R86K, DED-SH2, or DED-PTB proteins, and individual foci were examined over R175Q adaptors did not induce this strong spike of EGF-induced a 48-h period. The surrounding monolayer (48 h) of nontransfected RAT-2 cells caspase activity. is less sensitive to apoptosis induced by DED-SH2͞DED-PTB. (B) The survival of parental RAT-2 cells versus NTR2 cells (serum-starved) was compared 24 h after Discussion adenoviral delivery (at an moi of 200). Protein–protein interactions mediated by modular domains form an important mechanism for controlling signal transduction pathways (6, 23). One possible reason for the prevalent use of signal of the oncogenic ErbB2͞NeuNT RTK, and that residual interaction domains is to facilitate the formation of new con- tyrosine kinase signaling in quiescent cells (contact inhibited for nections between existing proteins during the course of evolu- 2 weeks) is inefficient at inducing apoptosis. We have confirmed tion, and thus to create new signaling pathways. Conversely, these results by using fusion proteins of DED-SH2, DED-R86K, aberrant protein interactions can disturb cellular phenotype, and and SH2, in which the adaptors are fused to a membrane thereby contribute to disease. Proteins encoded by pathogenic permeable sequence from the HIV TAT protein, which allows bacteria and viruses, or chimeric cellular oncoproteins, fre- fusion proteins to transfer across biological membranes (22). The results were similar to adenovirus-mediated delivery and rule quently exert their effects by usurping normal protein–protein out spurious contribution of adenoviral proteins to DED-SH2- interactions, and thus reorganizing cellular behavior. For exam- ple, the Tir protein of enteropathogenic Escherichia coli modifies mediated apoptosis (data not shown). To quantify the sensitiv- ͞ ities of ErbB2͞NeuNT-transformed and parental cells to DED- the actin cytoskeleton of the host cell by binding the SH2 SH3 SH2 or DED-PTB treatment, we treated serum-starved parental adaptor protein Nck (24), whereas the Bcr-Abl oncoprotein is RAT-2 and NTR2 cells with identical amounts of DED-SH2 and phosphorylated within its Bcr region at a YVNV motif that binds DED-PTB adenoviruses. Whereas DED-SH2 or DED-PTB (at the Grb2 adaptor (25, 26). The modular nature of signaling an moi of 200) reduced the viability of NTR2 cells to Ͻ20% within pathways thus lends itself to rewiring, in which proteins are 24 h, parental RAT-2 cells remained viable (70–80%) at the same assembled into novel complexes with new biological properties. dose. Thus, we conclude that expression of activated ErbB2 leads Previous work (27–29) has suggested that signaling proteins to an Ϸ4-fold increase in killing by chimeric adaptors. can be experimentally modified to form novel interactions. For We also investigated the effects of growth factor stimulation example, the oncogenic potential of Bcr-Abl can be attenuated on cells expressing the chimeric adaptors. For this purpose, we by chimeric adaptors comprising the catalytic domain of the used TWO3 human epithelial cells, which express the EGF tyrosine phosphatase Shp1 fused to the Abl-binding domain of receptor (EGFR) and respond to EGF (16). As shown in Fig. 6A, Rin1 (27). Similarly, caspase 8 can be artificially activated expression of DED-SH2 and DED-PTB in TWO3 cells stimu- through chemically inducible dimerization (CID) domains fused lated with EGF (100 ng͞ml) produced DNA laddering, which is in tandem to Fadd or caspase 8 (30), likely resulting in higher- characteristic of apoptosis. Unstimulated cells displayed reduced order oligomerization of the fusion proteins in response to levels of DNA laddering (data not shown). To test the effect of chemical dimerizer. Chimeric scaffold proteins have also been EGF stimulation on the survival of TWO3 cells, cells were used to reroute signaling between distinct MAP kinase pathways treated with SH2, PTB, DED-R86K, DED-SH2, or DED-PTB in yeast (28). In the example discussed here, we have linked two adenoviruses, serum-starved overnight, and then stimulated entirely different signaling pathways by using chimeric adaptors. with serum free medium (ϪEGF) or medium containing EGF The ErbB2 RTK has multiple Grb2 SH2-binding sites (at least (100 ng͞ml) for 24 h. EGFR activation decreased the survival of one direct and two through ShcA) and one ShcA PTB-binding DED-SH2-expressing cells, whereas cells expressing the control site. The activated receptor exists as a dimer, and could therefore DED-R86K, SH2, or PTB proteins were not obviously affected recruit several caspase 8 molecules through the DED-SH2 by EGFR stimulation (Fig. 6B). At high mois (Ͼ100), DED-PTB adaptor, and two caspase 8 molecules through DED-PTB. expression was lethal to TWO3 cells, even in the absence of EGF. Although the activated Fas receptor itself forms a trimer, recent

However, at reduced mois (an moi of 10), we observed that EGF data suggest that dimerization is the crucial step in caspase 8 BIOCHEMISTRY induced apoptosis in DED-PTB-expressing cells, indicating that activation (11, 13). These observations suggest a possible mech-

Howard et al. PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 ͉ 11271 Downloaded by guest on September 28, 2021 anism through which the DED-SH2͞DED-PTB chimeric adap- feature of the PTB and SH2 domains is that Grb2 contributes to tors induce caspase 8 activation in response to RTK signaling, the recruitment of the Cbl E3 protein- to ErbB1, namely that the adaptors recruit caspase 8 to the activated raising the possibility that overexpression of the Grb2 SH2 receptor in a fashion that suffices for caspase dimerization and domain could interfere with this negative regulator of RTK activation. These data support the view that quite distinct signaling (32). Regardless, the data illustrate the inherent dif- receptors and signaling pathways operate through the same ferences between these two classes of pTyr-binding modules. general principles of modular protein interactions and proximity Our results raise the possibility that cells can be engineered to effects. alter their responses to external signals, in ways that might be Our results show that chimeric adaptors containing a pTyr- therapeutically beneficial. With detailed knowledge of pTyr recognition domain (SH2 or PTB) fused to a DED can nucleate signaling in individual tumors, for example, through SH2 pro- a complex in which an activated RTK is coupled to caspase 8, filing (33), chimeric adaptors could be tailored to specifically leading to stimulation of caspase activity and consequent cell target the RTK expression profile of the individual tumor. death. We have demonstrated this rewiring of tyrosine kinase Alternatively, small molecules that could bridge endogenous and caspase pathways in cells transformed by an oncogenic components of RTK signaling pathways to apoptotic pathways variant of ErbB2, and in tumors arising from such cells, as well might also reengineer cellular behavior in a manner that could as in epithelial cells stimulated with EGF. The ShcA PTB antagonize the growth of transformed cells. Indeed, drugs such domain appears more potent than the Grb2 SH2 domain in as cyclosporin and rapamycin exert their effect by nucleating stimulating apoptosis when linked to the Fadd DED domain, novel protein–protein interactions (34, 35). In summary, our and, indeed, when a high level of the DED-PTB adenovirus is used, apoptosis is constitutive in TWO3 cells. The reasons for the data support the idea that new signaling pathways can be increased ability of DED-PTB to induce apoptosis are unclear, engineered by the joining of interaction domains in novel but they likely reflect innate differences between the binding combinations. properties of the ShcA PTB domain and Grb2 SH2 domain, or their roles in signaling from ErbB1͞ErbB2. The ShcA PTB We thank Drs. Jerry Gish and Henry Klamut for helpful discussions and domain has a slower off-rate for phosphorylated motifs than do Dr. Ingemar Ernberg for the TWO3 cells. This work was supported by grants from the National Cancer Institute of Canada (T.P.), the Cana- SH2 domains (31), and this rate may enhance the potential for dian Institutes of Health Research (T.P.), and the Elia Chair in Head and caspase 8 dimerization. Furthermore, the PTB domain also Neck Cancer Research (F.-F.L.). P.L.H. is a Postdoctoral Fellow of the binds PIP2, which promotes ShcA association with the plasma National Cancer Institute of Canada and is supported by funds received membrane (19). Although the lipid binding ability of ShcA PTB from the Terry Fox Run; M.C. is a Ph.D. student and is supported by a is likely not sufficient to induce significant apoptosis, because Canadian Institutes of Health Research student award and a Cancer this activity should be retained by the mutant PTB domain in the Research Society student fellowship; and T.P. is a distinguished scientist DED-R175Q adaptor, it may contribute. Another distinguishing of the Canadian Institutes of Health Research.

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