ZAP-70 is essential for the T cell antigen receptor-induced plasma membrane targeting of SOS and Vav in T cells.

Translocation of the SOS and Vav GDP/GTP exchange factors proximal to Ras and Rac GTPases localized in the plasma membrane glycolipid-enriched microdomains is a pivotal step required for T cell antigen receptor-induced T cell activation. Here we demonstrate that the T cell antigen receptor zeta-chain-associated ZAP-70 kinase and T cell antigen receptor zeta-chain immunoreceptor tyrosine-based activation motifs are essential for the membrane recruitment of SOS and Vav. Plasma membrane targeting of SOS or Vav begins with the assembly of ZAP-70 with Grb-2 and SOS. The subsequent tyrosine phosphorylation of LAT (linker for activation of T cell) by ZAP-70 leads to a shift in equilibrium from the ZAP-70.Grb-2.SOS(Vav) complex to the (Vav)SOS.Grb-2.LAT complex. This shift results in the targeting of SOS and Vav into glycolipid-enriched microdomains and initiation of the Ras and Rac signaling cascades involved in T cell activation, proliferation, and cytokine production.

involved in Rac GDP/GTP exchange (14,15). Disruption of either the TCR or CD28 signaling pathways in Vav-deficient mice markedly decreases the amount of interleukin-2 secretion, maturation, and TCR-mediated cytoskeletal reorganization in T cells (16,17). These Vav-deficient mice also display defects in TCR-induced intracellular calcium fluxes as well as in the activation of mitogen-activated protein kinase and the NF-B transcription factor (18).
Recruitment of SOS proximal to Ras in the plasma membrane is an essential step for Ras activation in T cells (13,19), and plasma membrane-targeted SOS derivatives activate components of the Ras signaling pathway, particularly the ERK kinase and AP-1 transcription factor (20). Previously, we (7) and others (8,21) reported that a block in Ras activation occurs in anergic T cells. More recently, we showed that this Ras block is mediated by the impaired membrane translocation of SOS (22). This result prompted us to further investigate the mechanisms of recruitment of SOS, as well as Vav, to GTPasecontaining signaling complexes in the plasma membrane of T cells.
SOS and Vav activation and their membrane targeting may be controlled by the adaptor protein Grb-2, which binds to SOS and Vav (13,23). These Grb-2/SOS and Grb-2/Vav interactions involve the association of the Src homology 3 (SH3) domain of Grb-2 with proline-rich regions of SOS and Vav (13,23). Considerably less is known about phosphoproteins that form docking sites for adaptor proteins, such as Grb-2, and mediate the recruitment of "SOS(Vav)⅐adaptor protein" complexes to the plasma membrane in an activated T cell. Upon TCR ligation, Grb-2 forms a complex with a pp36 -38 tyrosine phosphoprotein (13,24) recently identified as LAT (linker for activation of T cells) (25). LAT is localized primarily in plasma membrane glycolipid-enriched microdomains (GEM) of T cells (13,24,26) and is a substrate for the TCR -chain (TCR)-associated ZAP-70 protein-tyrosine kinase (25). Consistent with its membrane localization, LAT may function as a central adaptor that recruits multiple proteins required for downstream signaling (25,(27)(28)(29). Upon TCR stimulation, ZAP-70 is translocated to the plasma membrane, and activated Lck enhances the plasma membrane accumulation of ZAP-70 (30). Immunoreceptor tyrosine-based activation motifs (ITAMs) of TCR-associated CD3 subunit phosphoproteins, which recruit ZAP-70 from the cytoplasm to the membrane-associated TCR⅐CD3 complex (31)(32)(33), may also form docking sites for the binding of secondary signaling proteins containing SH2 domains, including Grb-2 (32,34). Conceivably, Lck-and ZAP-70-dependent phosphorylation of LAT may play a critical role in the assembly of LAT⅐Grb-2containing GEM-associated signaling complexes proximal to TCR and downstream effectors, such as GEM-associated Ras and Rho/Rac.
In this study, we analyzed the roles of ZAP-70 and LAT in the recruitment of SOS, Vav, and Grb-2 from the cytoplasm to * This work was supported by the Vern Bruder grant from the Canadian Diabetes Association, a grant from the Juvenile Diabetes Foundation International, and a Diabetes Interdisciplinary Research Program grant from the Medical Research Council of Canada and Juvenile Diabetes Foundation International. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a postdoctoral fellowship from the Juvenile Diabetes Foundation International. 1 The abbreviations used are: ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signalregulated kinase kinase; GEM, glycolipid-enriched microdomains; GST, glutathione S-transferase; IB, immunoblotting; IP, immunoprecipitation; ITAM, immunoreceptor tyrosine-based activation motif; NF-B, nuclear factor-B; SH2 and SH3, Src homology 2 and 3, respectively; TCR, T cell antigen receptor; TCR, TCR -chain; Tg, transgene; Ab, antibody; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid. the plasma membrane-and GEM-associated signaling complexes in activated T cells. We demonstrate that TCR-associated ZAP-70 and TCR ITAMs are essential for the membrane recruitment of SOS and Vav. ZAP-70 initiates the membrane translocation of SOS and Vav by promoting the assembly of signaling complexes comprising ZAP-70, Grb-2, SOS, and Vav. This process precedes and is independent of the ZAP-70-mediated tyrosine phosphorylation of LAT. LAT amplifies this multistep recruitment process and is essential for the TCR-induced GEM targeting of SOS and Vav, which subsequently enables SOS and Vav to activate Ras and Rac.
T Cell Lines-Lck-negative JCaM1.6 and parental E6.1 Jurkat human leukemic T cell lines (38) were obtained from the American Type Culture Collection (Manassas, VA). The P116 ZAP-70-deficient cell line, a variant of the Jurkat E6.1 T cell line (39), was kindly provided by Dr. R. T. Abraham (Department of Immunology, Mayo Clinic, Rochester, MN). The mutant Jurkat T cell line, J.CaM2, which is defective in pp36 -38/LAT expression (27), was generously supplied by Dr. A. Weiss. Cells were maintained in RPMI 1640 (Life Technologies Inc., Burlington, Canada) medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma). The level of TCR surface expression is very similar in all of the Jurkat T cell lines and stable transfectants studied (data not shown).
Cell Activation and Lysis-Mouse spleen T cells were purified using murine T cell enrichment columns (R & D Systems, Minneapolis, MN) (purity Ն95%). If not otherwise indicated, mouse T cells were stimulated (3 min, 37°C) with 1 g/10 7 cells of the biotin-conjugated anti-TCR or anti-CD3⑀ mAb either alone or together with the biotin-conjugated anti-CD4 mAb. Cross-linking of mAbs was accomplished using streptavidin (Sigma) at a 4:1 (w/w) ratio for the indicated times. The anti-human CD3⑀ mAb was cross-linked using AffiniPure F(abЈ) 2 fragments of a donkey anti-mouse IgG Ab (Jackson Laboratories, West Grove, PA). Herbimycin A treatment of Jurkat T cells was performed for 3 h in complete RPMI plus 10% fetal bovine serum medium supplemented with 5 M herbimycin A (Biomol). Cells were lysed in ice-cold 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA lysis buffer containing either 1% Triton X-100 plus 0.2% Nonidet P-40 (Nonidet P-40) or 60 mM n-octyl-␤-D-glucopyranoside (Sigma) supplemented with a mixture of protease and phosphatase inhibitors (22). Subcellular Fractionation-Cells were lysed by sonication in ice-cold 10 mM Tris, pH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 2 mM EGTA hypotonic buffer containing the above described mixture of protease and phosphatase inhibitors (buffer A) (34). Lysates were centrifuged to remove nuclei and debris, and particulate membrane-containing and soluble cytoplasm-containing fractions were separated by differential centrifugation for 30 min at 100,000 ϫ g. Membrane fractions were washed with ice-cold buffer A, solubilized by sonication in buffer A supplemented with 150 mM NaCl and either 1% Triton X-100 plus 0.2% Nonidet P-40 or 1% Nonidet P-40, and recentrifuged. Analysis of proteins in membrane fractions was performed after normalization for protein concentration levels.
Immunoprecipitation of Cellular Proteins and in Vitro Binding Assays-If not otherwise indicated, precleared postnuclear cell lysates were normalized for protein concentration levels and immunoprecipitated (3 h, 4°C) with the specific polyclonal Abs or control isotypematched preimmune Ig precoupled to 25 l of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech, Baie d'Urfe, Canada). This was followed by four washes of the precipitates with ice-cold lysis buffer. When membrane proteins were precipitated, the amount of an Ab used was empirically determined to quantitatively precipitate the amount of antigen available. In vitro binding assays were performed after cell disruption in lysis buffer containing 1% Nonidet P-40 plus 1% SDS. Cell lysates were boiled for 2 min, diluted 20-fold with lysis buffer containing 0.5% Nonidet P-40, and incubated with the fusion proteins immobilized on agarose beads.
Gel Electrophoresis and Immunoblotting-Precipitated proteins were solubilized in 2ϫ Laemmli sample buffer, resolved by SDS-PAGE (8 -16% gradient gel, Novex, San Diego, CA) under reducing conditions, transferred to nitrocellulose membrane (Schleicher & Schuell), and immunoblotted with the indicated Abs (22). Signal intensities were quantified using a Molecular Imager System and Molecular Analyst imaging software (Bio-Rad).
Kinase Assays-T cells were incubated for 3-4 h at 37°C in fetal bovine serum-free RPMI 1640 medium before stimulation to reduce background kinase activities to workable levels. Proteins immunoprecipitated from precleared postnuclear lysates were assayed for associated in vitro kinase activity after washing the beads in kinase buffer (25 mM HEPES, pH 7.4, 5 mM MnCl 2 ) by incubation (30 min, 30°C) with [␥-32 P]ATP (15 Ci; NEN Life Science Products) in 25 l of kinase buffer containing 3 g of myelin basic protein (Upstate Biotechnology Inc.) as substrate. Reactions were stopped by boiling with gel sample buffer. Myelin basic protein was resolved by SDS-PAGE, and its phosphorylation was visualized using a phosphor imager (Bio-Rad). Immunoblotting showed that equal amounts of the ERK-1 and PAK proteins were precipitated before and after stimulation of all Jurkat T cell variants.
GEM Fractionation-Purification of the GEM fraction was performed as described (26). Cells were lysed by brief sonication in ice-cold 25 mM MES, pH 6.5, 150 mM NaCl, 5 mM EDTA lysis buffer containing 1% Triton X-100 and supplemented with a mixture of protease and phosphatase inhibitors (22). Lysates were mixed with an equal volume of 80% sucrose made in lysis buffer and were overlaid with 2 ml of 30% sucrose and 1 ml of 5% sucrose. GEM-enriched fractions were collected from the 5%/30% interface following an overnight ultracentrifugation at 200,000 ϫ g. The relative purity of these fractions was monitored by the absence of CD45 in these fractions and its presence in the Triton X-100-soluble membrane fractions.

TCR Ligation Stimulates the Association of ZAP-70 with
Grb-2, SOS, and Vav-We investigated how SOS and Vav are recruited to the plasma membrane. Initially, we identified those proteins that interact with SOS, Vav, and Grb2 after TCR ligation in Jurkat T cells. As reported (13,24,25,34), TCR cross-linking induced the association of Grb-2 with tyrosinephosphorylated LAT (Fig. 1A). Phospho-LAT was detected in SOS but not Vav immunoprecipitates of TCR-stimulated Jurkat T cell lysates. Interestingly, ZAP-70 was found to interact with SOS, Vav, and Grb-2 in unstimulated T cells, and these associations were significantly enhanced after TCR stimulation. These observations demonstrate for the first time that ZAP-70 interacts with SOS in T cells. Moreover, Grb-2, SOS, and Vav were present in immunoprecipitates of ZAP-70 (data not shown). TCR ligation also stimulated the association of SOS and Vav with Grb-2 as well as the interaction of SOS and Vav.
The relative stoichiometry of interaction induced by TCR ligation between phospho-LAT and SOS was about 10-fold less than that between phospho-LAT and Grb-2, despite the use of equal amounts of anti-SOS and anti-Grb-2 Abs for immunoprecipitation (Fig. 1A). By comparison, a similar relative stoichiometry of interaction between ZAP-70 and either SOS, Vav, or Grb-2 was observed. Most phospho-LAT molecules are palmitoylated and are localized in Triton X-100-insoluble GEM (26). Partitioning of LAT to GEM may therefore diminish the coprecipitation of phospho-LAT with SOS and Vav when these membrane proteins are solubilized in Triton X-100. This notion was supported by the finding that solubilization of these proteins in n-octyl-␤-D-glucopyranoside, a nonionic detergent that dissociates GEM while preserving protein-protein interactions, significantly increased the co-precipitation of phospho-LAT with both SOS and Vav (Fig. 1B). This result suggests that TCR cross-linking induces the partitioning of a significant proportion of SOS and Vav to LAT-containing GEM. In addition, T cell lysis by n-octyl-␤-D-glucopyranoside preserves and does not further enhance Grb-2/ZAP-70, SOS/ZAP-70, and SOS/Vav associations (Fig. 1C), indicating that these interactions are mostly GEM-independent.
We next analyzed whether the SOS/ZAP-70 interaction in T cells depends on the presence of Grb-2. SOS was immunoprecipitated from T cell lysates after several rounds of immunodepletion of Grb-2 with an anti-Grb-2 Ab. The high efficiency of Grb-2 immunodepletion was confirmed by immunoblotting of depleted cell lysates with anti-Grb-2 (data not shown). If such interaction depends on the presence of Grb-2, immunodepletion of Grb-2, which bridges the binding of SOS with ZAP-70 or LAT, would be expected to remove ZAP-70 and LAT from SOS immunoprecipitates. Fig. 1D shows that TCR stimulation significantly increased the amounts of ZAP-70 and LAT that coimmunoprecipitated with SOS in nondepleted T cell lysates. Conversely, sequential Grb-2 immunodepletion rendered the SOS immunoprecipitates virtually devoid of detectable ZAP-70, LAT, and Grb-2. These findings are consistent with the formation of TCR-induced trimeric SOS⅐Grb-2⅐LAT complexes and, more importantly, provide the first demonstration of SOS/Grb-2/ZAP-70 interactions in TCR-stimulated T cells.
Inducible Association of ZAP-70 with SOS and Grb-2 Is Phosphotyrosine-and Lck-dependent-The requirement of TCR-induced tyrosine phosphorylation for the association of ZAP-70 with Grb-2⅐SOS complexes was analyzed. We deter- mined the relative amounts of ZAP-70 in Grb-2 and SOS immunoprecipitates from TCR-activated Jurkat T cells treated for 3 h prior to stimulation with 5 M herbimycin A, a potent inhibitor of tyrosine phosphorylation (40). Herbimycin A treatment markedly reduced the TCR-stimulated binding of ZAP-70 to Grb-2 and SOS but did not significantly affect the constitutive association of these molecules ( Fig. 2A). Moreover, analysis of the Lck-deficient JCaM1.6 T cells demonstrated that Lck, which is required for tyrosine phosphorylation of ZAP-70 and LAT (25,31), is essential for the inducible binding of ZAP-70 and LAT to Grb-2 in the plasma membrane of Jurkat T cells (Fig. 2B). This result, coupled with the induced formation of Grb-2⅐ZAP-70 and SOS⅐ZAP-70 complexes, strongly suggests that a phosphotyrosine-dependent mechanism controls the binding of ZAP-70 to Grb-2 and SOS in T cells.
Structural Requirements for the Assembly of the ZAP-70⅐Grb-2⅐SOS Complex in T Cells-We next determined how the ZAP-70⅐Grb-2⅐SOS complex is formed. Since ZAP-70 does not possess an SH3 domain and SOS is not tyrosine-phosphorylated upon TCR stimulation, we considered the possibility that phospho-ZAP-70 interacts indirectly with SOS through the SH2 domain of Grb-2. The ability of an immobilized GST-Grb-2 fusion protein to pull down ZAP-70 from primary T cell lysates was determined after the lysates were boiled in SDS-containing lysis buffer to disrupt any preexisting associations. Wildtype full-length GST-Grb-2 and GST-Grb-2 SH2 domain fusion proteins precipitated significant amounts of ZAP-70 from TCR/ CD4-activated C57BL/6J spleen T cells (Fig. 2C). Negligible binding of ZAP-70 to the GST-Grb-2 mutant containing the SH2 domain loss-of-binding mutation (R86K) was observed. Conversely, the NH 2 -terminal SH3 domain (P49L) and COOHterminal SH3 domain (G203R) mutations did not alter the binding of ZAP-70 to immobilized GST-Grb-2. The binding of ZAP-70, LAT, and Vav to the Grb-2-SH2 domain was also evident in a pull-down assay of membrane-enriched fractions from unstimulated or TCR/CD4-stimulated C57BL/6J thymocytes solubilized by sonication in 1% Nonidet P-40 buffer under nondenaturing conditions (in the absence of SDS) (Fig. 2D).
ZAP-70 Is Required for the Membrane Translocation of SOS and Vav-To analyze whether ZAP-70 controls the recruitment of SOS and Vav to the plasma membrane, we compared the subcellular redistribution of SOS and Vav in wild-type Jurkat T cells with that in ZAP-70-deficient P116 Jurkat T cells. In unstimulated Jurkat T cells, SOS and Vav are constitutively associated with the plasma membrane (Fig. 3, A and B). kDa) and Vav (95 kDa) are among the most prominent tyrosine phosphoproteins associated with SOS in the membrane of wildtype Jurkat T cells (Fig. 3A, bottom). The identity of the SOSassociated 62-kDa phosphoprotein is presently unknown and does not appear to be the membrane-localized RasGAP-associated phosphoprotein p62 dok (42). TCR cross-linking induced the tyrosine phosphorylation of membrane-bound Vav and its association with the ZAP-70 and CD3⑀ phosphoproteins as well as Grb-2 and SOS in wild-type Jurkat T cells (Fig. 3B). In contrast, the extent of TCR-induced tyrosine phosphorylation of Vav and the amounts of Vav-associated proteins were reduced significantly in ZAP-70-deficient P116 T cells. The association of SOS with ZAP-70 and Vav is relatively specific, given the absence of other protein-tyrosine kinases (e.g. Fyn and Lck) and GDP/GTP exchange factors (e.g. C3G) in the SOS and Vav immunoprecipitates. 2 In addition, LAT was found in SOS and Vav immunoprecipitates from plasma membrane fractions solubilized in n-octyl-␤-D-glucopyranoside, which dissociates GEM (Fig. 3, A and B). Note also that while TCR stimulation significantly increased the association of SOS and Vav with Grb-2 in the membrane of wild-type Jurkat T cells (Fig. 3, A and B), such complexes were absent from membrane fractions of ZAP-70-deficient P116 T cells. Taken together, these data indicate that ZAP-70 is essential for the membrane targeting of SOS and Vav.

ZAP-70 Is Essential for the Assembly of Grb-2⅐SOS(Vav), Grb-2⅐LAT, and Grb-2⅐TCR Membrane Complexes-Analyses
of membrane-associated Grb-2 illustrated that Grb-2 is constitutively present in similar amounts in membrane fractions obtained from both resting and TCR-activated wild-type and ZAP-70-deficient Jurkat T cells (Fig. 3C). Thus, the impaired membrane recruitment of SOS and Vav in ZAP-70-deficient Jurkat T cells does not result from either the reduced ability or failure of Grb-2 to be recruited to the plasma membrane.
TCR stimulation induced the co-precipitation of membranebound Grb-2 with many phosphoproteins, including ZAP-70, LAT, the TCR⅐CD3 complex and SLP-76 (Fig. 3C). ZAP-70 was essential for optimal tyrosine phosphorylation of Grb-2-associated LAT, SLP-76, and Vav. Importantly, TCR stimulation effectively induced the recruitment of SOS and Vav to membrane-bound Grb-2 in wild-type but not ZAP-70-deficient Jurkat T cells (Fig. 3C). In addition, although TCR stimulation enhanced the tyrosine phosphorylation and amount of the Grb-2-associated TCR⅐CD3 complex in wild-type Jurkat T cells, this was not evident in ZAP-70-deficient Jurkat T cells. The latter finding confirms the association of Grb-2 with TCR in the plasma membrane (34) and suggests that ZAP-70 controls the basal and TCR-induced association of Grb-2 with phosphorylated CD3 components of the TCR complex. These observations provide direct evidence for an essential role for ZAP-70 in the assembly of Grb-2⅐SOS(Vav), Grb-2⅐TCR, and LAT⅐Grb-2 membrane complexes in TCR-stimulated T cells.
LAT Influences the Recruitment of SOS and Vav to GEM in the Plasma Membrane-LAT-deficient J.CaM2 T cells are defective in several TCR-mediated signaling events downstream of TCR and ZAP-70, such as ERK-1 activation, Vav tyrosine phosphorylation, calcium-dependent signaling, and interleukin-2 gene expression (27). Nonetheless, the role of LAT in the plasma membrane targeting of SOS and Vav is not presently known. It is possible that, in addition to the assembly with Grb-2, SOS, and Vav, ZAP-70 promotes the plasma membrane targeting of SOS and Vav by phosphorylating LAT, a downstream effector of ZAP-70. Consistent with this hypothesis, we found that J.CaM2 T cells are defective in the TCR-induced membrane targeting of Vav (Fig. 4A). In contrast, the presence of LAT is not absolutely required for optimal plasma membrane targeting of SOS. Furthermore, similar amounts of Grb-2 were present in the plasma membranes of Jurkat and J.CaM2 T cells (data not shown).
To  Jurkat T cells markedly reduces the binding of Grb-2 to LAT and blocks the transcriptional activity of NFAT and AP-1 (25). Mutation of LAT Cys-26 and Cys-29 blocks the palmitoylation, translocation to GEM, and TCR-induced tyrosine phosphorylation of LAT (26). Fig. 4B shows that the expression of LAT: WT, LAT:C26A/C29A, and LAT:Y171F/Y191F in J.CaM2 transfectants was about 6-, 2-, and 4-fold higher, respectively, than that of endogenous LAT in wild-type Jurkat T cells. All stable transfectants displayed equivalent protein levels of ZAP-70, SOS, Vav, and Grb-2 ( Fig. 4B; only ZAP-70 immunoblotting is shown). In agreement with a previous report (26), wild-type LAT and LAT:C26A/C29A and LAT:Y171F/Y191F mutants were predominantly partitioned to the plasma membrane. 2 A basal level of tyrosine phosphorylation of LAT:WT, LAT:C26A/ C29A, and LAT:Y171F/Y191F was noted in J.CaM2 transfectants, and TCR stimulation induced a significant increase in tyrosine phosphorylation of LAT:WT and, to a lesser extent, LAT:Y171F/Y191F (Fig. 4C). The level of tyrosine phosphorylation of LAT:C26A/C29A was not enhanced upon TCR stimulation. Immunoblotting with relevant mAbs revealed that LAT:WT but not LAT:C26A/C29A can be induced to associate with SOS, Vav, Grb-2, and ZAP-70 and that the binding of LAT:Y171F/Y191F to Grb-2 is markedly reduced. Consistent with the assembly of a SOS⅐Grb-2⅐LAT trimolecular complex, the LAT:Y171F/Y191F mutation also completely disrupted the binding of SOS to LAT. Conversely, this mutation did not alter interaction of Vav and ZAP-70 with LAT. Thus, TCR stimulation significantly enhances the binding of LAT to SOS, Vav, Grb-2, and ZAP-70. Furthermore, LAT palmitoylation at Cys-26 and Cys-29 is essential for the interaction of phospho-LAT with SOS, Vav, ZAP-70, and Grb, and Tyr-171 and Tyr-191 are required for phospho-LAT to associate with Grb-2 and SOS.
The fact that LAT palmitoylation at Cys-26 and Cys-29 is required both for the translocation of LAT into GEM and the binding of LAT to SOS and Vav raised the possibility that LAT is necessary for TCR-induced targeting of SOS and Vav into GEM. Consistent with a previous report (26), LAT:WT but not LAT:C26A/C29A was localized to a GEM-enriched fraction of J.CaM2 transfectants (Fig. 4D). TCR-induced targeting of SOS and Vav to GEM was also significantly impaired in the J.CaM2 T cells and LAT:C26A/C29A J.CaM2 transfectants (Fig. 4E). tions downstream of Ras in the SOS-Ras-Raf-ERK pathway (19), and PAK controls the activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase as well as cytoskeleton organization downstream of the Vav-Rac pathway (2,4,43). Fig. 5A shows that TCR-induced ERK-1 and PAK kinase activities were virtually undetectable in both ZAP-70deficient P116 and LAT-deficient J.CaM2 T cells relative to that of wild type Jurkat T cells. Stable transfection of LAT:WT, but not LAT:C26A/C29A, into J.CaM2 T cells restored ERK-1 and PAK activation. In addition, analysis of the [ 32 P]orthophosphate-labeled guanine nucleotides bound to Ras revealed that TCR stimulation enhances GDP/GTP exchange on Ras in wildtype but not ZAP-70-deficient Jurkat T cells (Fig. 5B). Thus, ZAP-70 and LAT are required to regulate the TCR-induced SOS-Ras-Raf-ERK and Vav-Rac-PAK-c-Jun N-terminal kinase (p38 mitogen-activated protein kinase) signaling pathways. Moreover, failure of LAT:C26A/C29A-transfected J.CaM2 T cells to up-regulate ERK-1 and PAK activities upon TCR stimulation demonstrates that Cys-26-and Cys-29-dependent partitioning of LAT into GEM is required for activation of ERK-1 and PAK.
Interactions of ZAP-70 with Grb-2, SOS, and Vav Occur Independently of LAT-Consistent with the function of LAT downstream of ZAP-70, we found that the binding of ZAP-70 to GST-Grb-2 as well as SOS and Vav was unaltered in unstimulated and TCR-activated J.CaM2 T cells (Fig. 5, C and D). Basal and TCR-induced association of Vav with SOS were unimpaired in the absence of LAT. In contrast, in parallel to the down-regulation of TCR-induced tyrosine phosphorylation of Vav in P116 and J.CaM2 cells (27), the binding of Vav to GST-Grb-2 was significantly diminished in lysates from both LAT-and ZAP-70-deficient T cells. These results demonstrate that ZAP-70 interacts with Grb-2, SOS, and Vav upstream of LAT and suggest that there is another level of regulation of the formation of SOS-and Vav-dependent multimeric complexes. This regulation is mediated by the assembly of ZAP-70⅐Grb2⅐SOS⅐Vav complexes upstream of LAT.
Membrane Translocation of SOS and Vav Requires TCR ITAMs-To test the possibility that TCR ITAMs function as docking sites for a ZAP-70⅐Grb-2⅐SOS(Vav) signaling complex, we assayed the TCR-induced membrane translocation of SOS and Vav in T cells from TCR Ϫ/Ϫ mice reconstituted with the TCR transgene encoding TCR molecules in which all three ITAMs were deleted (TCR-D67-150-Tg) (35). Although this deletion results in a signaling-deficient TCR that is unable to recruit ZAP-70 to the TCR⅐CD3 complex, TCR-D67-150-Tg promotes TCR surface expression and T cell maturation (35). We found that TCR cross-linking enhances the translocation of SOS and Vav to the plasma membrane in control C57BL/6 but not TCR-D67-150-Tg thymocytes (Fig. 6A). Similar to that found in human Jurkat T cells, immunoblotting with anti-ZAP-70 and anti-Vav mAbs demonstrated that membrane-associated SOS binds ZAP-70 and Vav following TCR stimulation in mouse thymocytes. By comparison, significantly lower amounts of ZAP-70 were associated with membrane-bound SOS and Vav in TCR-D67-150-Tg mice due to the impaired recruitment of SOS and Vav to the plasma membrane. The association of SOS and Vav was also noticeably decreased in TCR-stimulated TCR-D67-150-Tg thymocytes. Note that the impaired translocation of SOS was accompanied by increased amounts of SOS and SOS-associated Vav and ZAP-70 in the cytoplasm of the TCR-D67-150-Tg mice (Fig. 6B). In contrast, the amounts of membrane-associated Grb-2 in unstimulated and stimulated C57BL/6 and TCR-D67-150-Tg thymocytes were comparable (Fig. 6C). These data suggest that deficient membrane recruitment of SOS and Vav to the TCR complex does not result from the decreased translocation of Grb-2 to the plasma membrane but rather reflects the impaired recruitment of ZAP-70 to the TCR ITAMs. Consistent with this notion, translocation of ZAP-70 to the plasma membrane is deficient in TCR-D67-150-Tg thymocytes (Fig. 6C). DISCUSSION A protein-tyrosine kinase-dependent mode of translocation of the SOS and Vav GDP/GTP exchange factors proximal to Ras and Rac GTPase-containing signaling complexes in the plasma membrane is known to be a pivotal step required for TCRinduced T cell activation and function. However, the mechanism by which this translocation occurs was not previously elucidated. The observation that ZAP-70 is recruited to the plasma membrane upon TCR stimulation and that activated Lck enhances the plasma membrane accumulation of ZAP-70 (30) raises the question of whether ZAP-70 influences the plasma membrane targeting of ZAP-70 downstream effector proteins that function as a scaffold and recruit SH2 domaincontaining signaling proteins (32). In this study, we investigated the role of ZAP-70, LAT and their associated signaling proteins in the translocation of SOS and Vav from the cytoplasm to the plasma membrane after TCR ligation. Using ZAP-70-deficient Jurkat T cells, we found that ZAP-70 is essential for the constitutive and TCR-induced membrane targeting of SOS and Vav as well as the formation of SOS⅐Grb-2, Vav⅐Grb-2, SOS⅐Vav, and SOS(Vav)⅐ZAP-70 complexes in the plasma membrane. Our data indicate that ZAP-70 controls the membrane translocation of SOS and Vav by 1) binding directly to the Grb-2 SH2 domain in Grb-2⅐SOS and Grb-2⅐Vav complexes; 2) increasing the tyrosine phosphorylation of Vav, which promotes the association of Vav with ZAP-70, Grb-2, and SOS in the plasma membrane; and 3) enhancing the tyrosine phosphorylation of membrane-localized LAT and its association with Grb-2⅐SOS and Grb-2⅐Vav complexes.
The use of LAT-deficient J.CaM2 T cells revealed that LAT is essential for the TCR-induced recruitment of SOS and Vav into GEM. This observation provides a biochemical basis for the rapidly emerging concept of sphingolipid-cholesterol-rich microdomains or GEM that cluster critical signaling molecules, including Ras and Rac GTPases, in the plasma membrane of activated T cells (26,28,29).
Our results favor a model in which the plasma membrane targeting of SOS or Vav begins with the assembly of ZAP-70 with Grb-2, SOS, and Vav, which occurs independently of the subsequent tyrosine phosphorylation of LAT (Fig. 7). According to this model, upon TCR stimulation ZAP-70 is tyrosine-phosphorylated by Lck, translocated to the plasma membrane to associate with TCR (step 1), and by functioning as a docking phosphoprotein potentiates the formation of a complex between SOS and Vav with Grb-2 (steps 2 and 3). Support for such a role for ZAP-70 is also provided by the finding that a membranetargeted CD2/ZAP-70 chimera induces late signaling events in the absence of TCR stimulation but in the presence of a functional kinase-active Lck (44). Subsequent tyrosine phosphoryl- ation of membrane-localized LAT by ZAP-70 (step 4) may result in a shift of the ZAP-70⅐Grb-2⅐SOS^SOS⅐Grb-2⅐LAT equilibrium and exchange of SOS between ZAP-70-containing complexes in the Triton X-100-soluble fraction and GEM-associated LAT complexes (step 5). This exchange is compatible with our observations that 1) following TCR cross-linking, a significant proportion of SOS and Vav may be partitioned to GEM; 2) TCR-induced GEM targeting of SOS and Vav is significantly impaired in LAT-deficient J.Cam2 T cells; 3) LAT C26 and C29, which are critical for targeting of LAT to GEM (26), are essential for interaction of LAT with SOS, Vav, and Grb-2; and 4) palmitoylation at Cys-26 is required for TCR downstream signaling including tyrosine phosphorylation of phospholipase C␥1, ERK activation, and Ca 2ϩ mobilization (45). Therefore, a pathway leading from TCR through Ras and Rac to ERK-1 and PAK requires ZAP-70 and its downstream effector LAT, which are indispensable for the recruitment of SOS and Vav to the plasma membrane into GEM containing Ras and Rac.
Whereas Grb-2 is predominantly a cytoplasmic protein, we found that Grb-2 is constitutively present in similar amounts in membrane fractions obtained from both resting and TCRactivated T cells. Accordingly, in ZAP-70-deficient Jurkat T cells, the attenuation of Grb-2/SOS and Grb-2/Vav interactions and the observed block in GTPase-mediated amplification of TCR signaling may result not from the decreased translocation of Grb-2 to the plasma membrane but rather from the sequestration of SOS and Vav from the plasma membrane.
To examine the role of TCR in the membrane recruitment of SOS and Vav, TCR Ϫ/Ϫ mice reconstituted with TCR lacking all three ITAMs (TCR-D67-150-Tg) were used to demonstrate that membrane translocation of SOS and Vav requires the presence of TCR ITAMs. Uncoupling of the membrane recruitment of SOS and Vav from TCR-proximal signaling observed in TCR-D67-150-Tg mice does not result from the reduced recruitment of Grb-2 to the plasma membrane. Rather, signal amplification by TCR may be impaired as a result of the uncoupling of ZAP-70 from TCR ITAMs and SOS and of Vav from ZAP-70-containing complexes. These findings may explain the direct relationship between the number of TCR ITAMs within the TCR⅐CD3 complex and the efficiency of negative and positive thymic selection (35); i.e. quantitative differences in the strength of TCR signaling, which depend on the number of TCR ITAMs, may be a reflection of reduced TCR-ZAP-70-dependent membrane recruitment of SOS and Vav. Moreover, this model provides a basis for understanding the inability of Ras to amplify TCR signals and interleukin-2 production in anergic T-cells. Indeed, the diminished abilities of Grb-2 and ZAP-70 to interact with TCR, described in anergic T cells (22), may preclude the initial assembly of ZAP-70⅐Grb-2⅐SOS(Vav) complexes and subsequently interfere with the targeting of Grb-2⅐SOS and Grb-2⅐Vav complexes to plasma membrane/GEM-localized Ras and Rac.
We have demonstrated the presence of Vav in a membraneassociated SOS-containing complex. Since Vav-deficient mice display a block in the activation of the SOS-mediated pathway, including activation of mitogen-activated protein kinase and the NF-B transcription factor (18), the specific contribution of Vav to SOS function is of interest and is currently under investigation.
In conclusion, our results demonstrate a pivotal role of ZAP-70 in TCR-induced translocation of SOS and Vav to the plasma membrane and GEM in T cells. ZAP-70 controls this process through the assembly of ZAP-70⅐Grb-2⅐SOS(Vav) complexes upstream of LAT and by the subsequent phosphorylation of LAT. LAT is critical for the recruitment of SOS and Vav into plasma membrane GEM. The TCR⅐ZAP-70⅐LAT signaling module emerges as an essential part of the complex mechanism that regulates the recruitment of SOS and Vav to the plasma membrane and promotes the activation of the mitogen-activated protein kinase and PAK signaling cascades involved in T cell activation, proliferation, and cytokine production.