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  • C07K14/4702—Regulators; Modulating activity
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  • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
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  • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
  • C07K2319/715—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16 containing a domain for ligand dependent transcriptional activation, e.g. containing a steroid receptor domain
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  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/72—Fusion polypeptide containing domain for protein-protein interaction containing SH2 domain

Definitions

• This invention concerns materials, methods and applications relating to the mult- erizing of chimeric proteins with a dimeric or multimeric, preferably non-peptidic, ligand. Aspects of the invention are exemplified by ligand-mediated transformation of cells permitting cell growth in a growth-factor independent manner.
• Described herein are methods and materials which can be used, inter alia, to maintain, via such chemically induced dimerization of proteins, the proliferation of cells, such as stem cells, in culture for the adjuvant treatment of malignancy. These approaches can be used to induce the conditional growth of a variety of cell types and to render the progeny of hematopoietic stem cells, T cells and macrophages in particular, resistant to HIV by targeting essential viral proteins to the cellular degradative pathway.
• Illustrative publications disclosing further background information of interest are provided in PCT/US93/01617, especially on pages 1-4. However, as will be clear from this disclosure, none of the foregoing authors describe or suggest the present invention.
• This invention provides materials and methods for the genetic engineering of host cells to render the cells and their progeny susceptible to conditional transformation.
• the cells are of mammalian origin, more preferably of human origin, and are not terminally differentiated, e.g. stem cells such as hematopoietic stem cells or skin cells.
• stem cells such as hematopoietic stem cells or skin cells.
• Such genetic engineering and the process of conditional transformation are useful, e.g., for expanding a population of such cells.
• the engineered cells, as well as organisms containing them, are useful in clinical applications and as biological reagents for a variety of research and production purposes.
• the invention involves the adaptation of methods and materials for using homo- and hetero-mulrimerization of chimeric "responder" proteins to trigger gene transcription or other biological events in living cells.
• the terms multimer, multimerize and multimerization encompass dimers, trimers and higher order multimers and their formation.
• the chimeric responder proteins are intracellularly expressed fusion proteins which contain one or more specific receptor domains, e.g., F 506 binding protein (FKBP) domains, capable of binding to a corresponding multimerizing agent (e.g. an FK1012 molecule in the case of FKBP domains).
• the multimerizing agent is a multivalent ligand which is capable of binding to receptor domains on more than one of the chimeric protein molecules.
• the multimerizable chimeras contain one or more additional domains, in addition to the receptor domain(s).
• the additional domain(s) may comprise a DNA binding domain, a transcriptional activating domain, a membrane targeting domain (e.g. a myristoylation signal), a cellular destruction domain, a domain such as a single chain antibody (ScFV) or other domain.
• the chimeric proteins are designed such that ligand-mediated multimerization triggers a biological event such as transcription of a transforming gene under the transcriptional control of a DNA element responsive to such multimerization, destruction of a tumor suppressor or viral protein, or direction of a trarisforrning gene product to the cellular site where it assumes transforming activity such as the nucleus or cell membrane.
• FIG. 1 (A) Construction of chimeric soluble and membrane-bound Sos molecules.
• the plasmid coding sequence for the full length hSos, containing amino acid residues 2 to 1333 was cloned immediately downstream of the v-Src myristoylation targeting domain (residues 1-14) in the Xho 1-Sal 1 site of a derivative of the eukaryotic expression vector pBJ5 (Spencer et al. (1993) Science 262, 1019-1024; Pruschy et al (1994) Chemistry & Biology 1, 163-172) to yield MSosE.
• the soluble construct SSosE was constructed by placing hSos in an identical pBJ5-derived vector lacking the myristoylation sequence.
• the FKBP12 module derived from hFKBP was amplified by PCR as described (Spencer et al.,1993, supra) and cloned in three tandem copies into the Xho 1 site of SSosE to yield SF3SosE.
• This FKBP12 module was cloned in three tandem copies downstream of the myristoylation targeting domain in the vector described above to yield MF3E. All constructs contained a C-terminal influenza HA epitope tag to facilitate protein detection. Integrity of the constructs were verified by dideoxy sequencing. S, Sal 1; X, Xho 1; Sc, Sac II; E, Eco Rl.
• Jurkat-TAg cells were cotransfected with 2 ⁇ g of NFAT-SX reporter plasmid and 8 ⁇ g the indicated expression plasmids MDblE, McVavE, and ScVavE, stimulated, and analyzed for SEAP activity as described in (B).
• MDblE contains amino acid residues 2 to 498 from oncogenic Dbl and McVavE and ScVavE contain residues 2 to 845 of the full length Vav proto-oncogene, cloned into the myristoylated and soluble vectors as described in Figure 1A.
• Activation is again presented as percent following ionomycin stimulation alone relative to cells stimulated with ionomycin + PMA.
• Inset Expression of epitope-tagged constructs verified as in (B). Asterisk denotes a nonspecific band recognized by the 12CA5 antibody.
• Cos-1 cells were transfected on coverslips with SSosE (A,B) or MSosE (C,O) expression plasmids, fixed and stained with 12CA5 mAb and FITC-conjugated rabbit anti-mouse secondary antibody, and analyzed by confocal microscopy.
• MSosE localizes predominantly to the plasma membrane while SSosE remains cytosolic.
• FIG. 3 (A) Construction of Sos molecules containing mutations in the C-terminal proline-rich regions. Using PCR-mediated site-specific mutagenesis, one or both of the proline-rich sequences containing amino acids shown were changed to alanines as illustrated to abolish the consensus for SH3 binding. The location of these mutations in reference to the catalytic domain and pleckstrin homology domain (PH) is shown. The altered versions of Sos were cloned into the vector described in Figure 1 to generate MSosPlE and MSosPl,2E. (B) Membrane-bound Sos molecules with mutant C-terminal proline-rich domains no longer bind to Grb-2.
• FIG. 4 (A) Using FK1012 to mimic the role of Grb-2-induced localization of Sos. Upper panel, the proposed physiologic role of Grb-2 to localize Sos to the cell membrane. Lower panel, FK1012 induces the localization of a chimeric Sos/FKBP to a myristoylated FKBP at the cell membrane. (B) Activation of Ras by inducible membrane localization of Sos. Jurkat-TAg cells cotransfected with 2 ⁇ g NFAT-SX, 9 ⁇ g SF3SosE or 4.5 ⁇ g (molar equivalent) SF3E, and 1 ⁇ g MF3E.
• Fig 5. Construction of chimeric intracellular signaling molecules. Schematic of Src-family kinase-FKBP12 chimeras used for inducible membrane targeting. Src-family kinases are deregulated by mutation of the C-terminal tyrosine residue as shown and inactivated by truncation of the N-terminal myristoylation targeting peptide (residues 1-10). SFl ⁇ SH3Fyn lacks residues 1 - 144, SFl ⁇ SH3,SH2Fyn lacks residues 1 - 254 and SFl ⁇ KFyn has substitution K296E.
• Membrane targeting is achieved by subcloning these modified kinases into the FKBP12- tagging vector MF1E (myristoylated) or SF1E (cytosolic).
• M myristoylation targeting sequence from v-src (residues 1-14) (Spencer et al.,1993, supra; Cross, 1984, MCB); S, soluble, nonmyristoylated; U, unique domain; E, influenza hemagglutinin epitope tag (Field et al, 1988, MCB); TAIL, C-terminal regulatory peptide.
• B Model of the regulation of Lck/Fyn by the CD45 protein-tyrosine phosphatase and Csk PTK.
• Fig 6. Inducible signal transduction using synthetic dimers by membrane targeting of Src-family kinases.
• A The ability of dimeric ligand FK1012 to recruit SFlFyn to the plasma membrane docking protein MFIE is assayed by the induction of an NF-AT-responsive reporter plasmid NF ⁇ AT-SX in Jurkat-TAg cells (Clipstone and Crabtree, Nature, 1992; Northrup et al, 1993, Nature). This is compared to the FK1012-dependent recruitment of wild-type Fyn (SFlFynwt) or cytosolic FKBP12 (SF1E).
• ⁇ SH3,2 or kinase ( ⁇ K) domain is compared to the parent constructs SFlFyn (C) or SFlLck (D) by membrane targeting with FK1012.
• C SFlFyn
• D SFlLck
• MlLck myristoylated FKBP-Lck
• All of the constructs were similarly expressed as assayed by Western blot using the 12CA5 mAb against the influenza hemagglutinin epitope (Cross, 1984, MCB).
• Fig 7. Conditional activation of Src-family kinases mimics TCR signaling. Comparison of the induction of a panel of transcription factors (Spencer et al.,1993) by CID-induced Fyn (A) or Lck (B). (C) The ability of dominant-negative Ras (RSV-N17Ras, 2 ⁇ g) or FK506 (2 ng/ml) to block NF-AT activation by membrane recruitment of Fyn or Lck by 300 nM FK1012 (D) An examination of the kinetics of activation by Fyn and Lck using 1 ⁇ M FK1012 relative to that by mitogen or direct TCR crosslinking (see Materials and Methods).
• the cells are engineered to contain and be capable of expressing one or more transf orrning genes under the expression control of a transcriptional control element responsive to the presence of a predetermined ligand, e.g. by adaptation of the regulated transcription technology disclosed in International Patent Applications PCT/US93/01617 and PCT/US94/08008 and in Spencer et al, Science, 1993.
• Transforming genes may be of two types. The first are DNA sequences encoding transforming proteins such as myc, fos, myb, etc. whose transforming activity is attributed to their overexpression. These transforming genes may be linked to a desired transcriptional regulatory element for regulatable expression as discussed below.
• the second type of transforming genes encode proteins such as ras, raf, sos or src-like tyrosine kinases, the transforming activity of which is attributed to their localization to the cell membrane and /or allosteric changes that can be induced by physical proximity of an activated protein.
• the DNA sequence comprising the transforming gene encodes a protein containing at least the activated form of the kinase portion of the src-family member.
• Transforming genes of the second type e.g. ras, raf, sos, activated kinase, etc., are regulatably expressed as chimeric genes linked to a DNA sequence encoding a cellular targeting domain.
• the transforming gene product in such cases comprises a fusion protein containing the peptide sequence encoded by the transforming gene fused to a targeting domain such as a nuclear localization sequence or a myristoylation sequence which targets the fusion protein to the cell membrane.
• Regulatable expression involves recombinant DNA constructs ("target gene constructs") containing a first DNA sequence encoding a transforming gene (e.g. myc or a fusion protein of sos linked to a myristoylation sequence), and a second DNA sequence comprising a transcriptional regulatory element, such as a promoter or enhancer sequence, which is responsive to the multimerization of chimeric responder proteins.
• Target genes of these embodiments comprise transforming genes, as discussed below.
• DNA sequences for the desired transforming genes may be readily obtained by conventional means.
• primers may be designed based on the published sequence of a desired target cDNA, synthesized by conventional procedures and used in obtaining target gene DNA through standard PCR techniques.
• DNA sequence information and other information relevant to the cloning and use of transforming gene sequences are readily available.
• chimeric responder proteins contain at least one ligand-binding (or "receptor") domain and an action domain capable, upon multimerization of the chimeric responder molecules, of initiating transcription of the transforming gene within a cell.
• the chimeric proteins may further contain additional domains.
• chimeric responder proteins and the responder constructs which encode them are recombinant in the sense that their various components are derived from different sources, and as such, are not found together in nature (i.e., are mutually heterologous).
• the transcriptional control element is responsive in the sense that transcription of the transforming gene is activated by the presence of the multimerized responder chimeras in cells containing these constructs.
• the constructs of this invention may contain one or more selectable markers such as a neomycin resistance gene (neor) and herpes simples virus- thymidine kinase (HSV-tk).
• neomycin resistance gene neor
• HSV-tk herpes simples virus- thymidine kinase
• the modified cells into which one or more constructs have been successfully introduced may then be selected, separated from other cells and cultured, again by conventional methods.
• the multimerizing ligands useful for triggering the expression of the transforming gene in the practice of this invention are capable of binding to two (or more) of the receptor domains, i.e. to two or more chimeric responder proteins containing such receptor domains.
• the multimerizing ligand may bind to the chimeras in either order or simultaneously, preferably with a Kd value below about 10-6, more preferably below about 10-7, even more preferably below about 10-8, and in some embodiments below about 10-9 M.
• the ligand preferably is a non-protein and has a molecular weight of less than about 5 kDa. Even more preferably, the multimerizing ligand has a molecular weight of less than about 2 kDa, and even more preferably, less than 1500 Da.
• the action domains of the chimeric proteins may be selected from any of the proteins or protein domains (preferably of the species of the desired host cells or organism) which upon multimerization are capable of activating transcription of a target gene which is under the transcriptional control of a cognate control element.
• the action domain of the chimeric responder protein molecules may comprise a protein domain such as a CD3 zeta subunit which is capable, upon exposure to the ligand and subsequent multimerization, of initiating a detectable intracellular signal leading to transcriptional activation via the IL-2 promoter.
• a DNA-binding protein such as GAL4
• another contains as its action domain a transcriptional activation domain such as VP16.
• Heterodimerization of such responder proteins to form a GAL4-VP16 dimer activates the transcription of genes (in our case, the transforming gene ) under the transcriptional control of elements to which the (hetero)dimerized responder proteins can bind.
• multimerization activates transcription of the transforming gene under the transcriptional control of a transcriptional control element (e.g. enhancer and /or promoter elements and the like) which is responsive to the multimerization event.
• a transcriptional control element e.g. enhancer and /or promoter elements and the like
• DNA constructs for the various embodiments of this invention may be assembled in accordance with the design principles, and using materials and methods, disclosed in the patent documents cited herein, including PCT/US94/01617, with modifications as described herein and as disclosed in the examples which follow
• This invention further involves DNA vectors containing the various constructs described herein (for these and other embodiments), whether for introduction into host cells in tissue culture, for introduction into embryos or for administration to whole organisms for the introduction of the constructs into cells in vivo.
• the construct may be introduced episomally or for chromosomal integration.
• the vector may be a viral vector, including for example an adeno-, adeno associated- or retroviral vector.
• the constructs or vectors containing them may also contain selectable markers permitting selection of transfectants containing the construct.
• This invention further encompasses the genetically engineered cells containing and /or expressing the constructs described herein, including prokaryotic and eucaryotic cells and in particular, yeast, worm, insect, mouse or other rodent, and other mammalian cells, including human cells, of various types and lineages, whether frozen or in active growth, whether in culture or in a whole organism containing them.
• the genetically engineered cells of such embodiments may contain and be capable of regulatably expressing more than one such transforming gene (e.g. myc and a sos-myristoylation sequence fusion), each of which may be under the same or different multimerizer-regulated expression control. Exposure of the engineered cells or their progeny to the multimerizing ligand(s) recognized by the chimeric transcription control proteins results in expression of the transforming gene(s) and in cellular growth characteristic of a transformed phenotype.
• transforming gene e.g. myc and a sos-myristoylation sequence fusion
• the transforming genes may be used in a conditionally transforming manner, i.e. where the peptide sequence encoded by a transforming gene of the second type e.g., ras, raf, sos, activated kinase, etc., is fused to a ligand-binding domain, such as an FKBP domain.
• a ligand-binding domain such as an FKBP domain.
• the ligand-binding domain confers targeting capabilities to the transforming gene product.
• the resultant fusion proteins are capable of ligand-mediated association with a membrane docking protein or other localization protein, and thus constitute a targetable transforming factor.
• Localization proteins are fusion proteins containing a ligand-binding domain and a targeting domain which directs the fusion protein to a particular cellular location, e.g. the cell membrane in the case of a myristoylation sequence or the nucleus in the case of a nuclear localization sequence, for example.
• the targetable transforming factor is directed to the desired cellular location by association with the localization protein.
• the cells are engineered to contain and be capable of expressing recombinant DNA sequences encoding one or more targetable transforming factors and localization protein(s).
• the first DNA sequence encodes a chimeric protein comprising a ligand binding domain fused to a peptide sequence encoded by a transforming gene, which can, upon localization to the appropriate cellular environment (e.g. the nucleus or, in the case of proteins such as raf or sos, the cell membrane), activate a transformation pathway.
• the second DNA sequence encodes a chimeric protein comprising a cellular or subcellular localization domain (e.g.
• the chimeric transforming factor molecules multimerize with the chimeric localization proteins and thus become localized at the cell membrane or other targeted site.
• the DNA sequences encoding the two chimeric proteins may themselves be expressed in a ligand-regulated manner as described above, using the same or different ligand to which the chimeric transforming and localization proteins bind.
• the cells may further contain at least one transforming gene encoding a protein such as myc, fos, myb, etc.
• transforming gene may be expressed constitutively or under the control of the ligand-regulated system alluded to above, under the regulation of the same or different ligand to which the chimeric proteins above bind.
• an activation construct encoding a human SOS protein fused to multiple FKBP domains.
• a second construct encoding a membrane docking protein was prepared which encodes the 20 amino acid myrsitoylation signal from the c-src protein fused to multiple FKBP domains.
• constructs were prepared which encode a targetable transforming factor comprising a fusion protein containing a number of FKBP domains and a modified src- family tyrosine kinase such as fyn, lck, lyn, etc.
• the modifications to the src-family tyrosine kinase included incorporation of known transforming mutations, deletion of the myristoylation sequence which is required for transforming activity, and optional deletion of other domains including all non-kinase domains.
• Host cells were then transfected with such a construct and with a construct encoding an FKBP-containing membrane docking protein.
• the targetable transforming factors associate with the membrane docking protein and are thus directed to the cell membrane where they activate the transforming process. This is evidenced by the development of transcriptional activation of transforming proteins such as AP-1 and others.
• the cells are engineered to contain and be capable of expressing recombinant DNA sequences encoding chimeric proteins comprising various combinations of one or more of the following domains: a target binding domain (such as a single chain FV or other antibody moiety), a multimerizing ligand-binding domain, and a domain targeting the chimera for degradation or destruction.
• a target binding domain such as a single chain FV or other antibody moiety
• a multimerizing ligand-binding domain such as a single chain FV or other antibody moiety
• a domain targeting the chimera for degradation or destruction.
• the first DNA sequence encodes a first chimeric protein containing a ligand-binding domain and a tumor suppressor binding domain.
• the second such DNA sequence encodes a second chimeric protein containing a ligand-binding domain (which may be the same or different from the ligand-binding domain of the first chimera) and a domain targeting the chimera for degradation or destruction.
• the chimeras multimerize.
• the tumor suppressor to which the first chimera binds is thus linked in trans to the degradation targeting domain and is thereby targeted for destruction and effectively removed from the engineered cells.
• Tumor suppressors to be targeted in such embodiments of this invention include pl5, pl6, p21, p27, Rb and the like. See e.g. Weinberg, 1991, "Tumor Suppressor Genes" Science 254:1138- 1146.
• Degradation targeting domains include domains such as the cyclin destruction box and the jun degradation signal.
• Tumor suppressor binding domains may be readily prepared in the form of single chain FV fragments (ScFV's) capable of recognizing the relevant tumor suppressor.
• the cells are engineered to contain a DNA sequence encoding a chimeric protein containing a target binding domain such as a ScFV directed to the desired target and a domain targeting the chimera for degradation or destruction, e.g. via a proteolyric pathway. That DNA sequence is linked to and under the expression control of a transcriptional control element responsive to the presence of a predetermined ligand, e.g. by adaptation of the regulated transcription technology disclosed in International Patent Applications PCT/US93/01617 and PCT/US94/08008, as as discussed above.
• the two approaches to regulated destruction described above may be extended from targeting one or more tumor suppressors to targeting one or more viral proteins, and in particular, one or more essential proteins of an HIV virus, for example.
• recombinant DNA sequences encoding chimeras containings ScFV's are used as above, but directed to HIV proteins such as the HIV protease, nef of others.
• Introduction of the recombinant DNA molecules into hematopoietic stem cells provides a route to macrophages and T cells capable of expressing the recombinant DNAs.
• Such cells contain the ligand-regulated system for degradation of the targeted viral proteins, and in that sense, would be characterized by ligand-induced resistance to the virus.
• DNA sequences for incorporation into recombinant DNAs of this invention may be obtained as described in PCT/US94/1617. Those include DNA sequences encoding cellular localization signals (such as myristoylization sites for directing chimeras to the cell membrane) and DNA encoding ligand binding domains (including naturally occurring or genetically engineered FKBPs or cyclophilins).
• cellular localization signals such as myristoylization sites for directing chimeras to the cell membrane
• DNA encoding ligand binding domains including naturally occurring or genetically engineered FKBPs or cyclophilins.
• ScFV's may be produced by conventional methods using cloned DNA encoding portions of antibodies against the desired tumor suppressor (or viral protein or other target), which may also be prepared by conventional methods. For instance, using conventional methods one may obtain mAbs which specifically recognize a desired tumor suppressor. Starting with murine hybridoma or spleen cells which produce such antibodies, one may generate phage which contain DNA encoding the desired ScFV using the commercially available Recombinant Phage Antibody System and pCANTAB 5 Gene Rescue and Sequencing Primers (Pharmacie Biotech)(or the equivalent). See Analects 22(l):l-7 (Winter 1993) and references cited therein.
• DNA encoding the desired ScFV may be readily linked at its 3' end to DNA encoding the ligand-binding domain to form a recombinant DNA encoding the chimeric protein mentioned above which comprises a ligand binding domain and a tumor suppressor binding domain.
• Degradation targeting domains such as the jun and cyclin destruction boxes or a ubiquitin conjugating enzymatic domain, for example, may be cloned via PCR or synthesized using automated oligonucleotide synthesis procedures.
• transforming genes are known which may be obtained from the ATCC, by cloning (PCR) or by assembly of overlapping synthetic oligonucleotides. See e.g. McCormick, "ras Oncogenes” ppl25-145; Hunter, “Oncogene Products in the Cytoplasm: The Protein Kinases”, ppl47-173; Eisenman, “Nuclear Oncogenes” pp 175-221, and other chapters in Oncogenes and the Molecular Origins of Cancer (Cold Spring Harbor Press, 1989, Weinberg, ed.).
• DNA sequences encoding the various components may be assembled into recombinant DNA molecules encoding the desired chimeras by analogy to the methodology described in
• the recombinant molecules may be assembled or transferred into vectors for propagation or transfection which may additionally contain transcriptional control elements such as the desired promoter/enhancer elements and conventional genetic elements such as origins of replication and selection markers.
• eukaryotic cells may be engineered in accordance with this invention.
• hematopoietic stem cells of mammalian origin, e.g. murine or preferably primate, and in particular human origin, are preferred.
• Cells are obtained, manipulated and cultured using methods conventional for the respective cell type and origin.
• CTA stem cells of this invention may be expanded by culture in a culture medium containing the multimerizing ligand in an effective amount for growth of the cells.
• Cells so produced may be administered to a patient in need thereof as an adjuvant to cancer chemotherapy, e.g. for leukemia, lymphoma and various solid tumors as an alternative to bone marrow transplantation with donated bone marrow cells.
• Bone marrow transplantation may also be effected using CTA stem cells rendered resistant to HIV as described above.
• Administration to the patient of the multimerizing ligand in an amount effective to cause multimerization of the chimeric proteins present in the engineered cells renders those cells and their progeny resistant to HIV.
• epithelial cells may be engineered to impart conditional transformation characteristics in accordance with this invention. Culture of such cells is then effected in media containing the multimerizing ligand in an amount sufficient to permit cell growth. Cells so produced may then be transplanted with CTA cutaneous stem cells.
• the methods and materials of this invention may also be used for non-clinical purposes. For instance, they may be used to produce expanded populations of stem cells for providing to the research community for the study of asymetric cell division, to study the mechanism of transformation and other research purposes. Examples
• Example 1 Regulatable activation of the Ras pathway via ligand-mediated association of a targetable Sos protein and membrane docking protein.
• COS cells used in this study were grown in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal calf serum and penicillin/ streptomycin.
• Jurkat-TAg cells (16) were maintained in RPMI 1640 supplemented with 10% fetal calf serum, L-glutamine, and penicillin/ streptomycin.
• 10 7 Jurkat-TAg cells were electroporated at 960 ⁇ FD, 250 V, in 0.4 ml media with the indicated amount of expression plasmids.
• HRP horseradish peroxidase
• ECL horseradish peroxidase
• Lysates were immunoprecipitated with 12CA5 as described above and washed extensively in HNTG buffer (50 mM HEPES, 10% glycerol, 0.1% TritonXlOO, 150 mM NaCl). Identical immunoprecipitat.es were analyzed on 10% SDS-PAGE gels and blots probed with 12CA5, or on 12% SDS-PAGE gels and blots probed with anti-Grb-2 mAb (Transduction Laboratories).
• NF-AT is a transcription factor that binds regions within the IL-2 enhancer and is essential for transcription of genes such as IL-2, IL-4, GMCSF, and CD40 ligand that coordinate the actions of cells necessary for an immune response.
• the NF-AT transcription complex responds to signaling through the TCR as well as by pharmacological agents such as phorbol ester and ionomycin, a calcium ionophore, which synergize to activate NF-AT-dependent transcription (18-21).
• pharmacological agents such as phorbol ester and ionomycin, a calcium ionophore, which synergize to activate NF-AT-dependent transcription (18-21).
• Activation of T cell signal rransducrion was assayed by induction of NF-AT-dependent transcription of a secreted alkaline phosphatase reporter gene, and accumulation of alkaline phosphatase in the media.
• Transfection of a constitutively active calcineurin can functionally replace the calcium-dependent events in T cell signaling (17,22,23), while expression of constitutively active Ras (v-Ha-Ras) can bypass the need for phorbol ester stimulation (24).
• Activities of the soluble and myristoylated Sos constructs were assayed for their ability to provide a signal complimentary to the calcium signal induced by ionomycin stimulation.
• Either constitutively active Ras or myristoylated Sos synergize with ionomycin to activate NF-AT- dependent transcription in T cells (Figure IB).
• soluble Sos exhibited only a small activation above the level of vector alone.
• Sos for Ras activation in T cells we compared the activity of Sos to that of the Dbl oncoprotein, thought to be a GEF specific for members of the Rho/Rac subfamily of small GTP binding proteins, including the human CDC42 protein (28-30).
• Sos the activity of the product of the Vav proto-oncogene (31), a hematopoietic-specific protein implicated in a variety of signal transduction pathways, reviewed in (32).
• Vav was of particular interest since it has been shown to be tyrosine phosphorylated following TCR activation (33), and unconfirmed reports suggests it exhibits a Ras-specific GEF activity following phosphorylation by Lck as well as by diacylglycerol binding (34,35).
• myristoylated Dbl was unable to activate the Ras-dependent pathway in T cell activation ( Figure ID), indicating that specificity for Ras by a membrane-targeted GEF is essential.
• Soluble Sos was fused to three domains of FKBP12 (SF3SosE) and coexpressed in Jurkat- TAg cells with membrane-bound myristoylated FKBPs (MF3E). Addition of the CID, FK1012, resulted in the localization of soluble Sos with membrane-bound FKBPs, and the activation of Ras (Figure 4B).
• T cell activation A role for Ras in T cell activation has been clearly established (42), however the mechanism and full consequences of its activation have been the subject of much debate.
• Our studies indicate that at least one mechanism in T cells may be mediated by membrane recruitment of Sos. Membrane proximity is essential for Ras-dependent events as other components of the Ras pathway such as RasGAP (43) and Raf (44,45) can also be activated by membrane localization.
• RasGAP RasGAP
• Raf 44,45
• Grb- 2/ Sos membrane-recruitment by the TCR may be mediated by She, only low levels of tyrosine phosphorylation on She are detected following TCR stimulation (46).
• Grb-2 induces a conformational change in Sos and thereby activates its catalytic activity is now unlikely in light of the ability of MSosE to fully activate the Ras pathway in the absense of a detectable interaction between Grb-2 and SOS with mutated SH3- binding regions. This is consistent with data indicating that Grb-2 binding to Sos in vitro has no measurable effect on guanine nucleotide exchange activity (49). Furthermore, biophysical studies have indicated that Grb-2 SH3 domains exhibit no conformational change following Grb- 2 SH2 binding to phosphotyrosine peptides (50).
• Vav is not a membrane-recruited GEF for Ras in T cells.
• Vav contains a variety of motifs important for protein-protein interactions, including SH2, SH3, pleckstrin homology, and leucine-rich domains. Indeed, Vav may also play a role as a Rho/Rac-specific GEF essential for altering components of the cytoskeleton, cell shape, and motility.
• the precise definition of Vav GEF substrate specificity and the roles of its various domains are needed to define the precise role of Vav in T cell activation.
• linker-mediated dimerization results in more favorable kinetics for signal transduction since the biologic response can be terminated by dissolution of either of the two linkages with Grb-2. If the in vivo dissociation rates of Grb-2 with receptor are similar to those of Grb-2 with Sos, a 2-fold gain in dissociation rate would be realized. At present only solution measurements for individual peptides in vitro are available (56,57), and high asymmetric interactions would reduced the kinetic advantage of this form of mediated dimerization.
• Example 2 Targetable, conditional alleles of Src-family tyorosine kinases
• Plasmid Clones The expression plasmids used in this study are described in Fig. 5A or below. AU of the constructs made by PCR were sequenced. Protein expression was verified by Western blot analysis using the influenza hemagglutinin epitope tag (12CA5) (7).
• the murine Lck, Fyn and Lyn templates are from m-lck, pmTF and lynAF, respectively (18, 19).
• Primers were flanked by Xho I (5' primer) or Sal I (3' primer) sites, and the resulting fragments were subcloned into pKS (Stratagene), sequenced, and subcloned into the Sal I site of SF1E (SF1 series) or MFIE (MF1 series) described previously (5).
• RSV-N17Ras (20) is dominant-negative Harvey Ras mutant.
• the reporter plasmids NF-AT-SX, IL-2-SX, AP-l-SX, NF ⁇ B-SX and Oct/OAP-SX have been described (5). Briefly, they contain multiple binding sites for the various transcription factors cloned upstream of a minimal interleukin 2 (IL-2) promoter [-70 to +47 (21)] driving secreted alkaline phosphates (SEAP) expression.
• IL-2 minimal interleukin 2
• SEAP alkaline phosphates
• J.RT- T3.5 is a TCR ⁇ -chain-deficient subclone of Jurkat cells (20). AU cells were grown and electroporated in RPMI 1640 medium. 10% (vol/vol) fetal calf serum, 10 mM Hepes (pH 7.4) and penicillin / streptomycin .
• Jurkat-TAg ceUs were electroporatcd (B ⁇ »-Rad Gene Pulser; 960 ⁇ F and 250 V in a 0.4-cm-wide cuvette) with 2-3 ⁇ g of the reporter plasmid NF-AT-SX or one of its derivatives (5, 23), 1 ⁇ g of the pBJ5 expression vector containing the "docking protein" MFIE or MF3E and 2 ⁇ g of pBJ5 containing one of the Src-fam y kinases or the control construct SF1. Alternatively, 1 ⁇ g of MFlLck was cotransfected with 2 ⁇ g of reporter.
• mAb Anti-TCR monoclonal antibody
• UCHTl (Sigma)-coated microtiter wells were incubated for 1 hr at 37°C with 10 ⁇ g of mAb per ml of phosphate-buffered saline (PBS) and blocked for 1 hr 37°C in PBS containing 1% fetal calf serum.
• CeUs were divided into these wells or untreated wells containing mitogen or FK1012. Ahquots of ceUs were removed from the microtiter wells at each time point and frozen until the last time point. The data are presented as the average of two experiments performed in dupUcate.
• chimeric Lck (SFlLck), Fyn (SFlFyn) and Lyn (SFlLyn) were deregulated by eliminating their regulatory, C-terminal tyrosine residues (Fig 5A).
• Fig 5A the administration of the CID FK1012 (5, 26) should lead to the formation of hetero- and homodimers (Fig 5C).
• NF-AT-SX reporter plasmid
• NF-AT-dependent transcription of this reporter is eUcited by the antigen receptor through a bifurcating signaling pathway (28-30) that requires both calcineurin and Ras and that is inhibited by cyclosporin A and FK506 (31).
• cytosolic FKBP12-Fyn chimera SFlFyn
• MFIE docking protein
• NF-AT activity is undetectable at all concentrations of FK1012 (Fig. 6A).
• the docking protein and the FKBP12-Fyn chimera are cotransfected into Jurkat-TAg cells, FK1012 activates signaling at concentrations as low as 1 nM.
• Membrane recruitment of "wild-type" Fyn, SFlFynwt is insufficient for signaling (Fig. 6A).
• FK1012-mediated membrane recruitment and crosslinking of SF3Fynwt which contains three FKBP12s, signaled effectively.
• SFlFyn or SFlLck plus MF3E were transiently transfected into the TCR- subclone of Jurkat ceUs, J.RT-T3.5 (22). Since these ceUs lack the TCR ⁇ chain, they do not assemble a TCR complex at the plasma membrane, resulting in the enhanced degradation or retention in the endoplasmic reticulum of the unassembled TCR complex subunits (36). Surprisingly, membrane recruitment of Fyn (Fig.8A) or Lck initiated signaling in the absence of a functional TCR.

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