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Proximity and orientation underlie signaling by the non-receptor tyrosine kinase ZAP70

1997; Springer Nature; Volume: 16; Issue: 18 Linguagem: Inglês

10.1093/emboj/16.18.5618

1460-2075

Isabella A. Graef, Leslie J. Holsinger, Steve Diver, Stuart L. Schreiber, Gerald R. Crabtree,

Cell Adhesion Molecules Research

Article15 September 1997free access Proximity and orientation underlie signaling by the non-receptor tyrosine kinase ZAP70 Isabella A. Graef Isabella A. Graef Department of Developmental Biology, Howard Hughes Institute at Stanford University, 300 Pasteur Drive, Beckman Center Room B211, Stanford University Medical School, Stanford, CA, 94305 USA Search for more papers by this author Leslie J. Holsinger Leslie J. Holsinger Department of Developmental Biology, Howard Hughes Institute at Stanford University, 300 Pasteur Drive, Beckman Center Room B211, Stanford University Medical School, Stanford, CA, 94305 USA Search for more papers by this author Steve Diver Steve Diver The Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology and The Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Stuart L. Schreiber Stuart L. Schreiber The Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology and The Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Gerald R. Crabtree Corresponding Author Gerald R. Crabtree Department of Developmental Biology, Howard Hughes Institute at Stanford University, 300 Pasteur Drive, Beckman Center Room B211, Stanford University Medical School, Stanford, CA, 94305 USA Search for more papers by this author Isabella A. Graef Isabella A. Graef Department of Developmental Biology, Howard Hughes Institute at Stanford University, 300 Pasteur Drive, Beckman Center Room B211, Stanford University Medical School, Stanford, CA, 94305 USA Search for more papers by this author Leslie J. Holsinger Leslie J. Holsinger Department of Developmental Biology, Howard Hughes Institute at Stanford University, 300 Pasteur Drive, Beckman Center Room B211, Stanford University Medical School, Stanford, CA, 94305 USA Search for more papers by this author Steve Diver Steve Diver The Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology and The Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Stuart L. Schreiber Stuart L. Schreiber The Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology and The Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Gerald R. Crabtree Corresponding Author Gerald R. Crabtree Department of Developmental Biology, Howard Hughes Institute at Stanford University, 300 Pasteur Drive, Beckman Center Room B211, Stanford University Medical School, Stanford, CA, 94305 USA Search for more papers by this author Author Information Isabella A. Graef1, Leslie J. Holsinger1, Steve Diver2, Stuart L. Schreiber2 and Gerald R. Crabtree 1 1Department of Developmental Biology, Howard Hughes Institute at Stanford University, 300 Pasteur Drive, Beckman Center Room B211, Stanford University Medical School, Stanford, CA, 94305 USA 2The Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology and The Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA The EMBO Journal (1997)16:5618-5628https://doi.org/10.1093/emboj/16.18.5618 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signaling by the antigen receptor of T lymphocytes initiates different developmental transitions, each of which require the tyrosine kinase ZAP70. Previous studies with agonist and antagonist peptides have indicated that ZAP70 might respond differently to different structures of the TCR–CD3 complex induced by bound peptides. The roles of membrane proximity and orientation in activation of ZAP70 signaling were explored using synthetic ligands and their binding proteins designed to produce different architectures of membrane-bound complexes composed of ZAP70 fusion proteins. Transient membrane recruitment of physiological levels of ZAP70 with the membrane-permeable synthetic ligand FK1012A leads to rapid phosphorylation of ZAP70 and activation of the ras/MAPK and Ca2+/calcineurin signaling pathways. ZAP70 SH2 domains are not required for signaling when the kinase is artifically recruited to the membrane, indicating that the SH2 domains function solely in recruitment and not in kinase activation. Using additional synthetic ligands and their binding proteins that recruit ZAP70 equally well but orient it at the cell membrane in different ways, we define a requirement for a specific presentation of ZAP70 to its downstream targets. These results provide a mechanism by which ZAP70, bound to the phosphorylated receptor, could discriminate between conformational changes induced by the binding of different MHC–peptide complexes to the antigen receptor and introduce an approach to exploring the role of spatial orientation of signaling complexes in living cells. Introduction A role for proximity in intracellular regulatory mechanisms was initially raised by the observation that the src-like tyrosine kinases require myristoylation for transformation (Sefton et al., 1982; Cross et al., 1984; Kaplan et al., 1988). Since myristoylation mediates membrane attachment (Resh, 1994), this observation indicated that the role of myristoylation was to bring the src-like tyrosine kinases into proximity with associated signaling molecules or substrates confined to the cell membrane. Similarly, farnesylation is essential for the function of GTPases such as ras (Willumsen et al., 1984; Buss et al., 1989), presumably to allow the ordered propagation of signaling on the inner membrane surface. A broader role for proximity in signaling was suggested by the identification of scaffolding proteins and anchoring proteins that are required for the function of specific signaling pathways (Mochly-Rosen, 1995; Faux and Scott, 1996). Although these proteins may have roles other than mediating proximity, they are postulated to assemble signaling molecules into an ordered spatial array. Testing the role of proximity in signaling has been difficult. The sequence motifs such as the SH2 domain (Koch et al., 1991; Pawson and Gish, 1992) or covalent modifications such as myristoylation that induce proximity might also activate enzymatic activity or other functions through conformational changes. Intuitively one would think that proximity alone could never mediate a qualitative response, since diffusion would allow random interactions. One molecule that may be subject to regulation by membrane proximity is the non-receptor tyrosine kinase ZAP70, which has diverse roles in signaling by the antigen receptor. ZAP70 was originally discovered by Weiss and colleagues (Chan et al., 1992) and shown to be related to the Syk family of tyrosine kinases (Chan et al., 1994a). As predicted by the original studies in cell lines, ablation of the ZAP70 gene in mice results in a profound defect of activation and also defects in positive and negative selection during thymic development (Negishi et al., 1995). In addition, mutations in the human ZAP70 gene are associated with a failure of normal signaling by T cells in peripheral lymphoid organs and skewed development of subpopulations of T cells (Arpaia et al., 1994; Chan et al., 1994b; Elder et al., 1994). Previous work suggests that ZAP70 might be responsive to distinct structural features of the MHC–peptide complex bound to the antigen receptor of T cells. In murine lymphocytes ZAP70 becomes associated with the tyrosine-phosphorylated ITAM motifs (Reth, 1989) of the ζ or ϵ chains of the antigen receptor (Chan et al., 1991, 1992; Wange et al., 1992; Straus and Weiss, 1993). These chains are probably phosphorylated by the src-like tyrosine kinases lck and/or fyn when the antigen receptor binds MHC–antigen complexes on the surface of antigen-presenting cells (Samelson et al., 1985; Reth, 1989; Iwashima et al., 1994). Studies using agonist and antagonist peptides demonstrated that while both were able to induce phosphorylation of TCRζ and recruitment of ZAP70, only the agonist peptides led to signaling and transcriptional activation of the IL-2 gene (Sloan-Lancaster et al., 1994; Madrenas et al., 1995) and presumably other early immune response genes of T cells. While these observations could be explained by the necessity for signaling mechanisms not dependent on ZAP70, the critical role of ZAP70 in signaling in T lymphocytes raises the possibility that ZAP70 somehow senses the nature of the bound peptide and assumes a configuration that is either effective or ineffective in signaling. Much of our understanding of signaling pathways has grown out of genetic modifications in the germlines of mice or transfection studies of cells overexpressing constitutively active or dominant negative proteins. While these studies have contributed immensely to our understanding of signaling, such stable genetic modifications set in motion a ricocheting series of actions, reactions and compensations at both the molecular and cellular levels, leading to a new steady-state. Since the outcome of the introduction of these modified signaling molecules is commonly examined many hours or days following their introduction or germline modification, while most signaling molecules are normally only active for seconds or minutes, compensation and indirect effects are inevitable and sometimes impossible to distinguish from direct effects. Such compensatory mechanisms and indirect effects are likely to be consistent with one another, and independent approaches may lead to a coherent steady-state yet not reflect the physiological activity of the protein under study. In an effort to avoid these difficulties and to mimic the evanescent nature of signaling, we have developed an approach to allow real-time analysis of the function of isolated signaling molecules in living cells (Spencer et al., 1993). In this approach we have made use of the importance of induced proximity in activating biological functions (Mochly-Rosen, 1995; Crabtree and Schreiber, 1996; Faux and Scott, 1996). In the following studies we have extended this approach to analyze the roles of configuration or architecture in signaling and applied it to the study of the mechanism of action of the non-receptor tyrosine kinase ZAP70. Results Membrane recruitment can activate the signaling potential of ZAP70 We devised an approach to analyze the role of orientation of signaling molecules, such as ZAP70, using cell-permeable synthetic ligands or chemical inducers of dimerization (CIDs) (Figure 1A and B). This approach makes use of the fact that different linker elements in the CIDs differ in their length, rigidity and preferred orientation and hence allow bound molecules to sample subsets of all possible membrane-associated configurations. In addition, we used ligand-binding domains in fusion proteins that generate different geometries of the associated signaling complex based on the relative positions of the C- and N-termini (Figure 1C). The chimeric molecule SF1ZAPwt was constructed by fusion of the full-length, wild-type murine and/or human ZAP70 cDNA (Chan et al., 1992; Gauen et al., 1994) to one copy of the cDNA encoding the FK506-binding protein FKBP12 (Figure 1C). The membrane-docking construct MF3E consisted of three tandem copies of FKBP12 fused to the myristoylation domain of v-src (Holsinger et al., 1995; Spencer et al., 1995). SF1ZAPwt and MF3E were transiently co-expressed in the human leukemic T cell line TAg Jurkat, along with a construct in which tandem binding sites for the NF-AT transcription factor direct expression of secreted alkaline phosphatase (NFAT SEAP). This plasmid has been shown to initiate transcription at the correct site both in Jurkat cells and in transgenic murine lymphocytes (Durand et al., 1988; Verweij et al., 1990) and, like activation of many early immune response genes in T lymphocytes, requires signals from both the Ras/MAPK pathways and calcium/calcineurin pathways (Clipstone and Crabtree, 1992). Hence it serves as a monitor for both the ras and calcium pathways. Furthermore, since this plasmid replicates in the TAg Jurkat cell line, it assembles into chromatin that is likely to be similar to chromosomal genes (Stillman, 1996). Addition of FK1012 to TAg Jurkat cells that had been co-transfected with MF3E and SF1ZAPwt resulted in dose-dependent induction of NF-AT-SEAP activity to levels comparable with those achieved with either PMA and ionomycin or stimulation through the antigen receptor (Figure 2A). Western blot analysis using an antibody specific for ZAP70 showed that chimeric human and murine SF1ZAPwt were expressed at levels near those of the endogenous protein (Figure 2A and B). In contrast, signaling as manifested by activation of NF-AT-induced transcription was not detected when ZAP70 was homodimerized in the absence of the membrane-docking molecule MF3E (Figure 2A). Since previous studies had found that ZAP70 could not be fully activated by stable membrane association with the transmembrane molecule CD16 (Kolanus et al., 1993), we tested the effects of inducing relatively stable membrane association by directly fusing the c-src myristoylation site to the N-terminus of ZAP70. Transfection of membrane-associated myristoylated ZAP70 resulted in a small but reproducible signal, usually from 20 to 35% of that obtained by transient recruitment with FK1012 (data not shown) or PMA and ionomycin. To determine if the myristoylated ZAP70 or the docking construct could be concentrated in caveoli and perhaps signal as a consequence of this, we examined the localization of the docking construct by confocal microscopy and found that it was uniformly distributed over the inner cell membrane (data not shown). Caveoli in contrast have a focal distribution (Conrad et al., 1995). Figure 1.(A) Model of the conformations sampled by ZAP70 upon membrane recruitment with synthetic ligands. (B) Molecular structure of synthetic ligands. Me, methyl. (C) Schematic representation of ligand-binding domains. FKBP, FK506-binding protein; FRB, FK506-binding region; CpH, cyclophilin A. Download figure Download PowerPoint Figure 2.Activation of T cell signal transduction by recruitment of ZAP70 to a myristilated docking molecule at the cell membrane. (A) TAg Jurkat cells were co-transfected with 2 μg murine SF1ZAPwt together with 1 μg MF3E (Spencer et al., 1995) and 2 μg NFAT-SEAP (Bram et al., 1993) or 2 μg murine SF1ZAPwt alone and 2 μg NFAT-SEAP. Twenty-four hours following electroporation, cells were divided equally and duplicates were treated for 18 h with medium alone, 25 ng/ml PMA and 1 μM ionomycin or titrations of FK1012A, and SEAP activity was measured. Data are presented as the percentage of maximal activation obtained by stimulation with 1 μM ionomycin plus 25 ng/ml PMA. Data represent the mean of four independent transfections ± SEM. Protein expression levels of human and murine SF1ZAPwt chimeras were verified by immunoblotting with the 12CA5 mAb specific for the HA epitope Tag (insert). (B) Near physiological levels of chimeric ZAP70 are recruited to the membrane fraction. TAg Jurkat cells were co-transfected with 2 μg human SF1ZAPwt and 1 μg MF3E. Twenty four hours post-transfection cells were stimulated with 200 nM FK1012A and membrane and cytosolic fractions were prepared. Cytoplasmic (C) and membrane (M) fractions were analyzed by Western blot with an antibody specific for ZAP70 (UBI). (C) Monomeric synthetic ligand inhibits signaling by membrane-recruited ZAP70. TAg Jurkat cells were co-transfected with 2 μg SF1ZAPwt, 1 μg MF3E and 2 μg NFAT SEAP and after 24 h cells were stimulated with 200 nM FK1012A. At the indicated time points a 10-fold molar excess of FK506M monomer (2 μM) (Spencer et al., 1995) or 2 ng/ml of the immunosuppressant FK506 were added. Data are presented as the percentage of maximal activation obtained by stimulation with FK1012A alone for 18 h. Download figure Download PowerPoint To determine the amount of SF1ZAPwt recruited to the membrane-docking molecule MF3E following addition of FK1012, we used fractionated membrane and cytosol preparations from TAg Jurkat cells that had been transiently transfected with human SF1ZAPwt and MF3E. Membrane and cytosolic fractions prepared from stimulated and unstimulated cells were subjected to SDS–PAGE, followed by immunoblotting with an antibody specific for ZAP70. These results demonstrate that SF1ZAPwt, but not endogenous ZAP70, was translocated to the membrane fraction upon addition of FK1012 (Figure 2B). The amount of SF1ZAPwt recruited to the membrane fraction was ∼10% of the level of total endogenous ZAP70 (Figure 2B). This degree of membrane recruitment is similar to the level of endogenous ZAP70 found in membrane fractions following TCR stimulation (van Oers et al., 1994; I.Graef and G.R.Crabtree, unpublished results). When signaling was initiated by FK1012 in cells transfected with the myristoylated docking construct and chimeric ZAP70wt, the addition of monomeric competing ligand FK506M (Spencer et al., 1993) rapidly blocked signaling with a time course similar to that of blocking signaling with FK506. FK506 inhibits calcineurin (Liu et al., 1991; Clipstone and Crabtree, 1992) and blocks NF-AT nuclear translocation and therefore NF-AT-dependent transcription (Flanagan et al., 1991). FK506M is modified at C21 and hence does not inhibit calcineurin (Spencer et al., 1993). It blocks signaling with essentially identical kinetics as blocking signaling with FK506 (Figure 2C), further indicating that signaling induced by FK1012 is related to recruitment of ZAP70 to the membrane. Activation of signaling by ZAP70 recruitment parallels activation by the antigen receptor To determine if FK1012-mediated recruitment of SF1ZAPwt mimicked physiological activation by antigen receptor stimulation, we compared TCR crosslinking with activation of SF1ZAPwt by FK1012. The time course of activation of signaling was similar in cells treated with FK1012 or stimulated by crosslinking of the T cell receptor with an antibody specific for the Jurkat TCR (C305, kindly provided by Dr A.Weiss) (Figure 3A). ZAP70 becomes phosphorylated on tyrosine residues that are essential for its in vitro kinase activity after antigen receptor engagement (Chan et al., 1995; Wange et al., 1995). FK1012 addition to Jurkat cells co-expressing the myristoylated docking construct MF3E and the chimeric ZAP70, SF1ZAPwt, led to phosphorylation of SF1ZAPwt within 10 min of addition of FK1012 (Figure 3B). This is similar to the time course of ZAP70 phosphorylation induced by anti-TCR antibodies. Antigen receptor stimulation also induces a significant reduction in mobility of the transcription factor NF-ATc on SDS–PAGE, which appears to be related to phosphorylation within the N-terminus of NF-ATc (I.Graef, N.Clipstone and G.R.Crabtree, unpublished results). TCR stimulation as well as membrane recruitment of SF1ZAPwt resulted in a rapid shift in the mobility of co-transfected NF-ATc, which was detectable 15 min after stimulation (Figure 3C). Figure 3.Activation of signaling by FK1012 parallels activation by antigen receptor signaling. (A) TCR stimulation and membrane recruitment of ZAP70 activate NFAT-dependent transcription with similar kinetics. TAg Jurkat cells that had been transfected with 2 μg SF1ZAPwt, 1 μg MF3E and 2 μg NFAT-SEAP were stimulated in duplicate with either 200 nM FK1012A or 1:1000 anti-TCR antibody (C305). The immunosupressant FK506 (2 ng/ml) was added after signaling had proceeded for the indicated times. Data are presented as the percentage of maximal stimulation obtained with either FK1012 alone or anti-TCR alone for 18 h. (B) Membrane recruitment of ZAP70 results in tyrosine phosphorylation of ZAP70. TAg Jurkat cells were transiently transfected with 2 μg SF1ZAPwt and 1 μg MF3E and treated with either medium alone or 200 nM FK1012A for the indicated times. NP-40 lysates were first immunoprecipitated with the anti-HA antibody 12CA5 and then subjected to Western Blot analysis with 12CA5 or an mAb specific for phosphotyrosine (4G10; UBI). (C) Membrane recruitment of ZAP70 and anti-TCR treatment induce a change in NF–ATc1 mobility. TAg Jurkat cells were co-transfected with 2 μg SF1ZAPwt, 1 μg MF3E and 3 μg Flag epitope-tagged NF–ATc1 (pSH160c). Twenty-four hours following transfection, cells were treated with either medium alone or anti-TCR (C305) or 200 nM FK1012A for the indicated times. Changes in NF-ATc1 mobility were analyzed by SDS–PAGE and immunoblotting with an mAb specific for the Flag epitope tag (M2; Eastman Kodak). The change in NF–ATc1 mobility upon activation is indicated by an arrow. Download figure Download PowerPoint Previous studies had shown that ZAP70 stably associated with the membrane by covalent fusion to a transmembrane protein failed to signal (Kolanus et al., 1993). Since we also found minimal stimulation upon transfecting cells with myristoylated ZAP70 we determined whether prolonged stimulation via the antigen receptor in Jurkat cells would lead to accommodation of the signaling pathways or silencing of signaling. Jurkat cells stimulated with an antibody to the TCR complex for up to 1 week showed a pronounced defect in their ability to activate NF-AT- and AP-1-dependent transcription (Figure 4A). In addition, phosphorylation of the mitogen-activated protein (MAP) kinases ERK-1 and ERK-2 was also defective in long-term TCR-stimulated cells compared with non-treated cells (Figure 4B). These results parallel previous studies in T and B cells (Wilde and Fitch, 1984; Goodnow et al., 1988; Fields et al., 1996; Li et al., 1996) and indicate that prolonged stimulation through the antigen receptor leads to a refractory state and reduced signaling, possibly by MAP kinase inactivation. This refractory state probably explains why previous studies either failed to detect signaling or found reduced signaling when ZAP70 was stably associated with the membrane. Figure 4.Accommodation of TCR signaling to prolonged stimulation. (A) TAg Jurkat cells were treated for 5 days with an antibody to CD3 (OKT-3; ATCC) or left untreated. Following the long-term stimulation cells were transfected with 2 μg NFAT-SEAP or 2 μg AP-1-SEAP (Spencer et al., 1993). Stimulation and assays for reporter gene activity were performed as described above. (B) TAg Jurkat cells were treated for 5 days with an antibody to CD3 or left untreated. After the prolonged stimulation the cells were stimulated with either PMA and ionomycin, anti-CD3 antibody or PHA for 15 min. The cell lysates were analyzed for activation of MAPK, identified by the change in MAPK electrophoretic mobility indicating phosphorylated forms of MAPK. The change in MAPK mobility upon activation is indicated by an arrow. Download figure Download PowerPoint The role of specific orientations of the membrane-recruited ZAP70 in signaling We examined the requirement for a precise conformation of the recruited ZAP70 using synthetic ligands that orient the signaling domains of fusion proteins differently relative to their signaling partners (Figure 1A and B). These were used in combination with different membrane-docking molecules (Figure 1C) to vary the geometry of the membrane-associated complex. A heterodimeric CID was synthesized made up of FK506 chemically linked to CsA, which we termed FKCsA (Belshaw et al., 1996) and used to recruit chimeric FKBP–ZAP70 to the membrane with a single myristoylated cyclophilin A. Although FKCsA is predicted to sample very different conformations than FK1012A, it was ∼30–40% as effective as FK1012A in inducing signaling (Figure 5A). An additional CID was synthesized, FK1012H2 (Figure 1B), in which the distance between the twisted amide surrogate structures (Rosen et al., 1990) that bind FKBP was reduced by several Ångstroms (S.Diver and S.L.Schreiber, unpublished results). FK1012H2 was able to induce signaling by recruitment of FKBP–ZAP70 that was quantitatively and qualitatively similar to FK1012A (Figure 5B). To explore the role of rotational freedom around the twisted amide surrogate structures, a fourth synthetic ligand FK1012Z was synthesized. This molecule is constrained by the Z-cis double bond in the linker between the two FK506 structures and hence samples fewer and different configurations than FK1012A and the above molecules. Despite this more constrained geometry, FK1012Z induced signaling nearly as well as FK1012A (Figure 5B). As an additional means of exploring the issue of presentation or geometry of the recruited ZAP70, we used the heterodimeric CID rapamycin to recruit ZAP70. Rapamycin binds FKBP through its twisted amide surrogate structure on one side and FRB through the opposite side of the molecule (Choi et al., 1996). Furthermore, the N- and C-termini of the 89 amino acid region of FRB which bind rapamycin protrude from the molecule at right angles, resulting in an orientation of fusion proteins different from either cyclophilin A or FKBP (Figure 1C). Hence, rapamycin produces a relatively rigid connection between FKBP12 and FRB, and would be expected to sample only those structures that are allowed by the flexibility within the FRB–membrane chimeric docking construct and within the FKBP–ZAP70 chimeric protein. Although rapamycin was highly effective in inducing transcription by recruitment of a transcription activation domain (Ho et al., 1996) or by recruiting the src-like tyrosine kinase fyn or the exchange factor Sos to the membrane (Figure 5C and data not shown), it was inactive when used to recruit ZAP70 to the membrane (Figure 5C), despite the fact that ZAP70 was membrane associated in the extracts of these cells (Figure 5C, insert). These observations indicate that while simple proximity is capable of activating the signaling function of ZAP70, the induction of signaling requires a specific configuration of the kinase at the membrane, presumably one suitable for further interaction with downstream targets. These results also demonstrate that the docking construct is not simply taking ZAP70 to a specific membrane compartment, such as caveoli, where signaling is initiated by virtue of localization within a specific membrane domain. Figure 5.The role of configuration and the SH2 domains in the activation of ZAP70 by FK1012-induced membrane recruitment. (A) FKCsA induces signaling by recruitment of ZAP70 to a myristoylated cyclophilin A. TAg Jurkat cells were transfected with 2 μg SF1ZAPwt plus 1 μg MC1E or 2 μg SF1E plus 1 μg MC1E and 2 μg NFAT-SEAP. Stimulation and assays for reporter gene activity were performed as described above. Data are presented as the percentage of maximal activation obtained by stimulation with 1 μM ionomycin plus 25 ng/ml PMA. Data represent the mean of four independent transfections ± SEM. (B) Activation of ZAP70 induced by different CIDs. TAg Jurkat cells were transfected with 2 μg SF1ZAPwt plus 1 μg MF3E and 2 μg NFAT-SEAP. Stimulation and assays for reporter gene activity were performed as described above. Data are presented as the percentage of maximal activation obtained by stimulation with 1 μM ionomycin plus 25 ng/ml PMA. Data represent the mean of four independent transfections ± SEM. (C) Orientation sensitivity of ZAP70 but not Fyn. TAg Jurkat cells were co-transfected with 2 μg SF1ZAPwt, 2 μg SF1ΔNΔCSH2 or 2 μg SF1ΔSH3Fyn (Spencer et al., 1995) plus 1 μg MFRB and 2 μg NFAT-SEAP. Stimulation and assays for reporter gene activity were performed as described above. Data are presented as the percentage of maximal activation obtained by stimulation with 1 μM ionomycin plus 25 ng/ml PMA. Data represent the mean of four independent transfections ± SEM. Cell fractionations after stimulation for 2 h with 10 nM rapamycin were performed as described above. Cytoplasmic (C) and membrane (M) fractions were analyzed by Western blot with the anti-HA 12CA5 antibody. Download figure Download PowerPoint The SH2 domains can be replaced by membrane recruitment with synthetic ligands The SH2 domains and inter-SH2 region of ZAP-70 have been proposed to contribute to the catalytic activity of ZAP70 by inter- or intramolecular interactions (Hatada et al., 1995). The functional significance of the SH2 domains and inter-SH2 region of ZAP70 was addressed by preparing chimeric FKBP–ZAP70 proteins lacking either the N-terminal (SF1ΔNSH2) or both (SF1ΔNΔCSH2) of the SH2 domains. SF1ΔNSH2 and SF1ΔNΔCSH2 were expressed at levels comparable with the wild-type (Figure 6B) and induced signaling comparable with that observed after activation of the full-length construct SF1ZAPwt (Figure 6A). These results indicate that in this context the SH2 domains of ZAP70 solely direct localization of ZAP70 to the membrane and are unnecessary for activation of kinase activity or other functions through conformational changes induced by binding of the SH2 domains to the ITAM motif. Figure 6.The tandem SH2 domains can be fully replaced by membrane recruitment. (A) TAg Jurkat cells were transfected with 2 μg SF1ZAPwt or 2 μg SF1ΔNSH2 or 1.5 μg SF1ΔNΔCSH2 plus 1 μg MF3E and 2 μg NFAT-SEAP. Stimulation and assays for reporter gene activity were performed as described above. Data are presented as the percentage of maximal activation obtained by stimulation with 1 μM ionomycin plus 25 ng/ml PMA. Data represent the mean of two independent transfections ± SEM. (B) Protein expression levels of the SH2 deletion constructs SF1ΔNSH2 and SF1ΔNΔCSH2 in comparison with full-length SF1ZAPwt were verified by immun