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Redirecting Tyrosine Kinase Signaling to an Apoptotic Caspase Pathway Through Chimeric Adaptor Proteins

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Redirecting Tyrosine Kinase Signaling to an Apoptotic Caspase Pathway Through Chimeric Adaptor Proteins 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– 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 death domain (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 death effector domain 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, receptor tyrosine kinase; 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
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