Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line

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1 International Immunology, Vol. 11, No. 6, pp Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line Weiguo Zhang, Brenda J. Irvin 1,2, Ronald P. Trible, Robert T. Abraham 1,2 and Lawrence E. Samelson Section on Lymphocyte Signaling, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD , USA 1 Department of Immunology, Mayo Clinic and Foundation, Rochester, MN 55905, USA 2 Present address: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA Keywords: LAT, TCR, tyrosine phosphorylation, palmitoylation Abstract The adaptor molecule LAT (linker for activation of T cells) is a palmitoylated integral membrane protein that localizes to the glycolipid-enriched microdomains in the plasma membrane. Upon TCR engagement, LAT becomes phosphorylated on multiple tyrosine residues and then binds several critical signaling molecules. Here, we describe the generation and characterization of a LAT-deficient cell line. Using this cell line, we demonstrate that LAT is required for TCR-mediated Ca 2 mobilization and optimal tyrosine phosphorylation of phospholipase C-γ1, Vav and SLP-76. LAT is also required for Erk activation, CD69 up-regulation, and AP- and NFAT-mediated gene transcription. We also demonstrate, by reconstituting this cell line with LAT mutants, that the LAT transmembrane domain and palmitoylation at Cys26, but not Cys29, are required for LAT function and TCR signaling. These studies provide further evidence for the crucial role of the LAT molecule, and demonstrate the use of a LAT-deficient cell line for the analysis of LAT structure and function. Introduction Many of the early biochemical events that result from engagement of the TCR are now well characterized (1,2). Following antigen stimulation or anti-tcr antibody cross-linking, protein tyrosine kinases (PTK) of the Src family of PTK, Lck and/or Fyn, phosphorylate tyrosine residues within immunoreceptor tyrosine-based activation motifs in the cytosolic tails of the TCRζ or CD3 subunits. These immunoreceptor tyrosine-based activation motifs are characterized by two repeated sequences, Tyr X X Leu/Ile, separated by amino acids. Phosphorylation of these tyrosines creates a binding site for the tandem SH2 domains of the ZAP-70 PTK. Subsequent phosphorylation of ZAP-70 by the Src kinases results in activation of ZAP-70. Following these rapid events, the activated PTK at the engaged TCR phosphorylate a number of proteins that can be divided into two categories: linker or adaptor proteins that bind other proteins either constitutively or following modification, such as tyrosine phosphorylation and enzymes that are regulated by tyrosine phosphorylation (3,4). The LAT (linker for activation of T cells) molecule is a critical linker molecule phosphorylated on multiple tyrosine residues after TCR engagement (5). LAT is an integral membrane protein, and its phosphorylation results in recruitment and binding by the SH2 domain of the cytosolic Grb2 molecule. Bound to the SH3 domains of Grb2 are a number of other linkers and enzymes including SOS (an activator of Ras), Cbl, a complex of proteins including SLP-76, SLAP, Vav (an activator of Rac) and others. Some of these proteins are themselves brought together by specific phosphorylation induced by the activated PTK. LAT also binds phospholipase C (PLC)-γ1 and phosphatidylinositol-3-kinase. The binding of these proteins to LAT appears to be critical to TCR-mediated signaling. Mutation of two tyrosine residues in the cytosolic tail of LAT inhibits binding of all the proteins identified above. Overexpression of this LAT mutant blocks TCR-induced activation as measured by an inhibition of activation of two transcription factors, NFAT and AP-1, critical to induction of IL-2 production (5). Correspondence to: L. E. Samelson Transmitting editor: J. A. Bluestone Received 9 February 1999, accepted 26 February 1999

2 944 Reconstitution of a LAT-deficient T cell line A recent set of observations confirms and extends these conclusions (6). The J.CaM2 variant of Jurkat was generated by mutagenesis over a decade ago (7). Engagement of its TCR results in tyrosine phosphorylation of the TCR and some substrates such as Cbl, while other proteins such as PLC-γ1 and SLP-76 show deficient phosphorylation. The cells fail to show an elevation of intracellular calcium, activation of Ras or Erk, and transcriptional activation of NFAT and AP-1 does not occur. These cells were shown to lack LAT protein and expression of LAT resulted in reconstitution of all of the deficient signaling events. Further analysis of the LAT molecule reveals that it is palmitoylated at two cysteine residues near the transmembrane region (Cys26 and Cys29) (8). The combination of its transmembrane domain and palmitoylation, especially at Cys26, targets the LAT molecule to specialized subdomains of the plasma membrane known as membrane rafts or glycolipidenriched microdomains. LAT mutants that are not palmitoylated at Cys26 do not target to these microdomains. The functional significance of raft targeting is demonstrated by the failure of this LAT mutant to be phosphorylated following TCR engagement. While the J.CaM2 cell was under investigation, we performed an independent mutagenesis and screening experiment in the hope of finding LAT-deficient variants of Jurkat. In this study we describe such a LAT-deficient T cell line, ANJ3, and show that it has a similar phenotype as J.CaM2. It is severely compromised in signaling events mediated by the TCR and we demonstrate that reconstitution with wildtype LAT restores normal responses. We also use this cell line to test the hypothesis that LAT targeting to plasma membrane microdomains is central to normal signaling processes. For this we stably express LAT mutants that are not appropriately palmitoylated and show that they fail to reconstitute TCR-mediated signaling. Methods Cell culture, antibodies and immunoprecipitation Jurkat cells (E6.1) and LAT-deficient cells (ANJ3) were maintained in RPMI 1640 supplemented with 10% FCS. Procedures for random chemical mutagenesis of the Jurkat E6.1 T cell line, as well as selection of mutants deficient in calcium signaling, were defined previously (9). The antibodies used in the experiments were: rabbit polyclonal anti-lat (5), anti- ZAP-70 (10), anti-slp-76 (a gift from Dr A. Chan), anti-cd3ε (OKT3), monoclonal anti-myc (9E10), monoclonal anti-plcγ1 (a gift from Dr. S. Rhee), anti-erk2 from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-vav and anti-phosphotyrosine (4G10) from Upstate Biotechnology (Lake Placid, NY). For immunoprecipitation, Jurkat cells (10 8 cells/ml) were either stimulated with OKT3 ascites (1:100) for 2 min or left untreated. Cells were lysed in ice-cold lysis buffer (1% Brij, 25 mm Tris, 150 mm NaCl and 5 mm EDTA) before immunoprecipitation. Protein samples were resolved on SDS PAGE, transferred to nitrocellulose and immunoblotted with mab or antisera. Immunoreactive proteins were detected with horseradish peroxidase-coupled secondary antibody followed by ECL (Amersham, Arlington Heights, IL). Transfection of Jurkat cells For stable transfection of Jurkat cells (ANJ3), 10 7 cells in 0.4 ml RPMI 1640, 25 mm HEPES and 2 mm glutamine were incubated with 15 µg of plasmid DNA, electroporated with a BioRad GenePulser (310V, 960 µf) and selected in the presence of 1.5 mg/ml G418. Analysis of CD3 and CD69 expression For CD3 expression, Jurkat cells were stained with anti-cd3ε antibody (OKT3) for 30 min, washed and then stained with FITC-conjugated anti-mouse antibody for 30 min. These cells were washed again and analyzed by FACScan (Becton Dickinson, San Jose, CA). For CD69 expression, cells were either left unstimulated or stimulated with OKT3 for 16 h. Cells were stained with FITC-conjugated anti-cd69 antibody, washed and analyzed by FACScan. Ca 2 flux, luciferase assay and Erk kinase assay The measurement for intracellular free Ca 2 and luciferase assay were performed as described (9). The AP-1 reporter plasmid was kindly provided by Dr D. McKean (Mayo Clinic). For the Erk kinase assay, Jurkat cells were stimulated with OKT3 for 15 min or left unstimulated. Anti-Erk2 immunoprecipitates were resuspended in the kinase reaction buffer (20 mm Tris HCl, ph 7.6, 13 mm MgCl 2 and 1.5 mm EGTA). The kinase reaction was performed using 10 µg myelin basic protein (MBP) and 5 µci [γ- 32 P]ATP per reaction. Results Isolation and reconstitution of a LAT-deficient Jurkat clone Mutagenesis of Jurkat T cells followed by selection for cells that fail to flux calcium following TCR or pervanadate treatment has led to the identification of a number of mutants with defects in proteins critical to TCR-mediated signaling (7,9,11) This approach was also used to identify Jurkat variants deficient in LAT. Wild-type Jurkat cells were mutagenized with the frame shift mutagen, ICR-191, and cells that failed to mobilize Ca 2 in response to pervanadate were selected. The resulting bulk population was subcloned and individual clones were screened by blotting with an anti-lat antibody. Three clones were non-responsive to pervanadate and deficient in LAT expression (not shown). One of these, ANJ3, was chosen for further analysis. Stimulation with the anti-cd3ε antibody OKT3 (Fig. 1A and B) failed to induce calcium mobilization and it was deficient in LAT expression (Fig. 2, lanes 3 and 4, lower panel). To confirm that the failure of Ca 2 mobilization was due to loss of LAT, ANJ3 cells were transfected with an expression vector containing a myctagged LAT cdna. Stably transfected clones with a similar level of LAT expression were selected for further analysis (Fig. 2, lane 5 and 6, lower panel). TCR expression was confirmed by flow cytometry (Fig. 3A). myc-tagged LAT migrated on SDS PAGE as a doublet of kda. Reconstitution of ANJ3 with wild-type LAT, designated as ANJ3 LAT(wt), restored the Ca 2 mobilization response to anti-cd3 antibody stimulation (Fig. 1C).

3 Reconstitution of a LAT-deficient T cell line 945 Fig. 1. Ca 2 mobilization in wild-type Jurkat cells (E6.1), LAT-deficient Jurkat cells (ANJ3) and ANJ3 cells reconstituted with wild-type and different LAT mutants. Jurkat cells were loaded with Indo-1 and stimulated with anti-cd3ε antibody (OKT3). The ratio of the fluorescence emission of Ca 2 -bound and -free form is plotted as a function of time after stimulation. The cells tested in each panel are the following: wild-type Jurkat (E6.1), LAT-deficient (ANJ3), ANJ3 reconstituted with wild-type LAT, ANJ3 reconstituted with LAT C26A, C29A and transmembrane domain truncation mutation ( tm). LAT reconstitution restores the optimal tyrosine phosphorylation of PLC-γ1, Vav and SLP-76 To confirm the role of LAT in regulating the tyrosine phosphorylation of intracellular proteins during TCR engagement, Jurkat (E6.1), ANJ3 and ANJ3 transfectants reconstituted with wild-type LAT were stimulated with OKT3, and PTK substrates were detected by probing with anti-phosphotyrosine antibody (Fig. 2, top panel). The tyrosine phosphorylated LAT molecule was not detected in cellular lysates prepared from ANJ3 cells (Fig. 2, lane 4), but this species was observed in the reconstituted cells (Fig. 2, lane 6). Tyrosine phosphorylation of proteins likely to be PLC-γ1 (135 kda),vav (100 kda) and SLP-76 (76 kda) was also reduced in ANJ3 cells. Tyrosine phosphorylation of other cellular proteins such as Cbl and ZAP-70 seemed normal. Importantly, reconstitution of LAT in ANJ3 restored the tyrosine phosphorylation of these proteins observed in activated cells (Fig. 2, lane 5 and 6). Direct immunoprecipitation of these proteins was then performed to provide definitive identification. As shown in Fig. 4(A), OKT3 stimulation induced the tyrosine phosphorylation of LAT from normal Jurkat (E6.1) and ANJ3 reconstituted with LAT. Surprisingly, there was a small amount of tyrosine phosphorylated LAT immunoprecipitated from ANJ3, indicating that ANJ3 was not totally deficient in LAT expression. Immunoprecipitation of ZAP-70 showed that the tyrosine phosphorylation of ZAP-70 upon stimulation was similar in these cells, but basal tyrosine phosphorylation of ZAP-70 in ANJ3 cells was higher (Fig. 4B). Immunoprecipitation of Vav, SLP-76 and PLC-γ1 from stimulated ANJ3 cells showed reduced tyrosine phosphorylation (Fig. 4C E). The basal

4 946 Reconstitution of a LAT-deficient T cell line expression of CD69 in ANJ3 cells could be induced following reconstitution of this cell with the LAT molecule (Fig. 3D). We next examined the effect of LAT deficiency on Erk activation by performing in vitro kinase assays on anti-erk2 immunoprecipitates from unstimulated or OKT3-stimulated E6.1, ANJ3 and ANJ3 LAT(wt). As shown in Fig. 5, Erk was activated in normal Jurkat T cells (Fig. 5, lane 1 and 2), but there was very little Erk activation in ANJ3 cells (Fig. 5, lane 3 and 4). Reconstitution of ANJ3 with LAT restored receptormediated Erk activation (Fig. 5, lane 5 and 6). Together the CD69 and Erk studies show that these pathways, both downstream of Ras activation in T cells, are dependent on LAT. Fig. 2. Tyrosine phosphorylation of cellular proteins in wild-type Jurkat cells (E6.1), ANJ3 and ANJ3 reconstituted with wild-type LAT. Jurkat cells were either unstimulated ( ) or stimulated with OKT3 for 2 min. Cells were lysed in 1% Brij lysis buffer. Post-nuclear lysates were loaded on SDS PAGE and analyzed by immunoblotting with antiphosphotyrosine antibody (4G10, top) and anti-lat antibody (bottom). phosphorylation of SLP-76 was also reduced in ANJ3. Reconstitution with LAT restored the tyrosine phosphorylation of these proteins following TCR ligation. Immunoprecipitation of Vav also revealed association of SLP-76 as previously described (Fig. 4C). The impact of LAT expression on tyrosine phosphorylation of PLC-γ1 in ANJ3 was demonstrated in Fig. 4(E). PLC-γ1 could still be phosphorylated in ANJ3 upon OKT3 stimulation, but at a reduced level compared to wildtype Jurkat. The defect in PLC-γ1 phosphorylation was fully reversed in ANJ3 reconstituted with LAT. Immunoprecipitation of PLC-γ1 also showed the presence of associated tyrosine phosphorylated LAT upon stimulation as previously described. There was no tyrosine phosphorylated LAT found in anti- PLC-γ1 immunoprecipitates from ANJ3 cells. The tagged transfected LAT co-precipitated with PLC-γ1 in the reconstituted cells. LAT involvement in CD69 up-regulation and Erk activation after TCR stimulation CD69 is a marker for T cell activation and its expression is induced in a Ras-dependent manner following anti-tcr or phorbol myristate acetate (PMA) stimulation (12). Since LAT forms a complex with Grb2 and SOS, we reasoned that the activation of Ras pathway might be affected by the absence of LAT. We first tested whether LAT is involved in up-regulation of CD69. As shown in Fig. 3, anti-cd3 stimulation induced CD69 up-regulation in Jurkat E6.1 cells (Fig. 3B), but failed to induce CD69 expression in ANJ3 cells (Fig. 3C). However, LAT in TCR-dependent transcriptional activation Engagement of the TCR leads to activation of Ras and Ca 2 pathways and eventually leads to activation of AP-1 and NFATmediated transcriptional activation. Because LAT resulted in a marked impairment of calcium mobilization and pathways coupled to Ras activation, we predicted that transcriptional activation would also be inhibited. To confirm that LAT is involved in TCR-mediated activation of AP-1 and NFAT, we transiently transfected E6.1, ANJ3 and ANJ3 LAT(wt) with pap-1-luc or pnfat-luc reporter plasmids which contain multiple copies of AP-1 or NFAT binding sites. Transfected cells were either left unstimulated, or stimulated with OKT3, or with PMA ionomycin. Luciferase activity was normalized to the level of PMA ionomycin stimulation. As shown in Fig. 6, there was no AP-1 or NFAT transcriptional activation following OKT3 stimulation of ANJ3 cells. These defects were corrected by reconstitution of these cells with LAT. Tyrosine phosphorylation of LAT with mutations at palmitoylation sites in ANJ3 cells Our previous studies showed that LAT palmitoylation at C26 is required for targeting LAT to glycolipid-enriched microdomains and LAT tyrosine phosphorylation (8). To further study the functional role of LAT palmitoylation in TCR signaling, we used ANJ3 cells to make stable transfectants with the LAT C26A, C29A and transmembrane domain-truncation ( tm) mutants. As shown in Fig. 7A, we selected clones expressing amounts of LAT similar to wild-type Jurkat E6.1 by blotting the whole cell lysates from those transfectants with anti-lat antibody. These clones also express similar amounts of CD3 (not shown). Interestingly, unlike wild-type or C29A LAT which can be observed to migrate on SDS PAGE as two species, the C26A mutant was predominantly found in one 38 kda form (Fig. 7A), suggesting that palmitoylation at C26 is required for generation of the p36 form of LAT. After the stable transfectants were either stimulated with OKT3 or left unstimulated, LAT was immunoprecipitated using the antimyc antibody. As shown in Fig. 7B, wild-type and C29A LAT were tyrosine phosphorylated after stimulation. The tyrosine phosphorylation of cellular proteins in C29A was indistinguishable from that in wild-type Jurkat (not shown). In contrast, cells expressing C26A and LAT tm showed either no increase or very little increase in tyrosine phosphorylation of the mutant LAT. This result is consistent with our previous result using transient transfection to express these mutants in Jurkat cells.

5 Reconstitution of a LAT-deficient T cell line 947 Fig. 3. CD3ε expression and CD69 up-regulation in Jurkat E6.1, ANJ3 and ANJ3 reconstituted with wild-type LAT. (A) CD3ε expression of the cell lines tested. The x-axis in the histograms is the fluorescence intensity. The y-axis is the relative cell number. Cells labeled with arrows were stained with OKT3. E6.1 cells stained with a FITC-conjugated control antibody were used a control (dotted line). (B D) CD69 expression in E6.1, ANJ3 and ANJ3 LAT(wt). Cells were either treated or treated with OKT3 as indicated. Fig. 4. Tyrosine phosphorylation of LAT, ZAP-70, Vav, SLP-76 and PLC-γ1 in Jurkat E6.1, ANJ3 and ANJ3 reconstituted with wild-type LAT. LAT, ZAP-70, Vav, SLP-76 and PLC-γ1 were immunoprecipitated from either unstimulated ( ) or OKT3-stimulated ( ) E6.1, ANJ3 and ANJ3 reconstituted with wild-type LAT with antibodies against each individual protein. Immunoprecipitates were resolved on SDS PAGE and analyzed by immunoblotting with anti-phosphotyrosine antibody and antibodies against each individual protein.

6 948 Reconstitution of a LAT-deficient T cell line Fig. 5. Erk activation in Jurkat E6.1 and ANJ3-derived clones. Cells were either left unstimulated or stimulated with OKT3 before lysis. Erk was immunoprecipitated from these lysates using anti-erk2 antibody. The kinase reaction was performed using MBP as an in vitro substrate. The relative intensity of each band was quantitated by using a densitometer. LAT palmitoylation and TCR-mediated signaling These stable transfectants were then tested to determine whether TCR engagement induced normal signaling events. We predicted that the LAT C29A mutant would have a similar response as wild-type, but that the C26A and LAT tm would be defective. We first examined the tyrosine phosphorylation of PLC-γ1 (Fig. 7C). As shown previously, the LAT-deficient ANJ3 showed some increase in phosphorylation of this protein following TCR engagement, but far less than in Jurkat E6.1 or in LAT-reconstituted cells. Transfection of ANJ3 with C29A also restored tyrosine phosphorylation to the level seen in wild-type E6.1 and ANJ3 transfected with wild-type LAT. Transfection of C26A and LAT tm failed to result in enhanced PLC-γ1 tyrosine phosphorylation after TCR engagement. Ca 2 mobilization in these transfectants was then tested and results were as predicted from these PLC-γ1 phosphorylation data. As shown in Fig. 1E, expression of the C29A mutant LAT could restore Ca 2 mobilization in ANJ3 cells, while C26A and LAT tm could not (Fig. 1D and F). Events coupled to the Ras pathway were also examined in these stable transfectants. Only those clones expressing wildtype or the C29A LAT mutant showed CD69 expression upon OKT3 stimulation (data not shown). As shown in Fig. 5, only reconstitution with the wild-type or C29A LAT allowed an increase in Erk kinase activity (Fig. 5, lanes 9 and 10). There was very little if any increase of Erk activation in ANJ3 cells or ANJ3 cells transfected with C26A and LAT tm (Fig. 5, lanes 7, 8, 11, and 12). Fig. 6. Reconstitution of AP-1 and NFAT promoter-dependent transcription in E6.1, ANJ3 and ANJ3 reconstituted with wild-type LAT. Cells were transfected with 10 µg pap-1-luc or pnfat-luc. Cells were either unstimulated, stimulated with OKT3 or stimulated with PMA ionomycin. Cell extracts were assayed for luciferase activity. Percent of maximum response from PMA ionomycin stimulation is shown on the y-axis. Discussion For many years the pp36 38 molecule, now known as LAT, had been thought to have an important function as a linker molecule that, after phosphorylation, was capable of binding several signaling proteins, and coupling Ras and Ca 2 pathways to the activated TCR (13 15). Cloning and further characterization of LAT allowed us to test this hypothesis (5). Upon tyrosine phosphorylation, LAT interacts with PLC-γ1, phophotidylinositol-3-kinase, Grb2 and other signaling molecules. The mutation of two critical tyrosine residues in LAT resulted in inhibition of the interaction with these molecules. The critical role of LAT and its ability to bind signaling molecules were shown by the inhibition of T cell activation produced by expressing this mutant form of LAT. Additional evidence in support of LAT function was sought by generating and characterizing Jurkat cell line lacking the LAT protein. As discussed above, a mutant variant of Jurkat with a profound signaling defect, J.CaM2, was recently demonstrated to lack LAT (6). While that cell line was under investigation, in parallel as a screen for such LAT-deficient cell lines, mutagenized Jurkat cells were activated with the tyrosine phosphatase inhibitor pervanadate, which increases the level of phosphorylated tyrosines and leads to an increase in intracellular calcium. Since receptor-mediated calcium

7 Reconstitution of a LAT-deficient T cell line 949 Fig. 7. Tyrosine phosphorylation of LAT and PLC-γ1 in ANJ3-derived clones. (A) Anti-LAT blot of lysates from wild-type Jurkat and ANJ3 transfectants showing relative expression of LAT. (B) LAT was immunoprecipitated from unstimulated ( ) and OKT3-stimulated ( ) cells with anti-myc antibody (9E10), and analyzed by immunoblotting with anti-phosphotyrosine and anti-lat antibodies. (C) PLC-γ1 was immunoprecipitated from unstimulated ( ) and OKT3-stimulated cells, and analyzed by immunoblotting with anti-phosphotyrosine and anti- PLC-γ1 antibodies. elevation is dependent on both the tyrosine phosphorylation of PLC-γ1 and recruitment of this enzyme to LAT, we assumed that we would find mutant cell lines lacking LAT that would not flux calcium upon TCR engagement. Several such lines were established and one, ANJ3, was selected for further testing. LAT levels, determined by Western blotting, were at least 50 times less than the parental Jurkat (E6.1) and TCR engagement of this clone failed to produce calcium elevation. Further analysis of these cells revealed that, though ZAP-70 tyrosine phosphorylation was induced by receptor activation, phosphorylation of several substrates including PLC-γ1, SLP-76 and Vav was dramatically reduced. The restoration of tyrosine phosphorylation of these proteins in ANJ3 cells by reintroduction of LAT demonstrates that the signaling apparatus, including the TCR and PTK, function normally. In addition to the inhibition of calcium elevation, the functional consequences of LAT deficiency include a decrease in ERK activation and CD69 induction. These events are dependent on the activation of Ras. Previous work demonstrates that LAT is responsible for recruitment of the Ras activator Sos via its interaction with Grb2. Without LAT the Ras pathway is not appropriately activated. That and the failure of calcium elevation results in the lack of AP-1 and NFAT activation. These transcription factors, whose activation is required for production of IL-2, depend on intact Ras and calcium pathways. The results obtained in the ANJ3 cell line are identical to those seen in J.CaM2 (6). Thus analysis of two independently derived LAT-deficient mutants of Jurkat provide strong evidence for the central function of this molecule. A new area of research in the study of signaling via immunoreceptors is investigation of the function of plasma membrane microdomains (16,17). This membrane heterogeneity has been defined as detergent insolubility, although recently imaging techniques have been used to demonstrate these microdomains in living cells (18 20). The heterogeneity is due to self-association of glycosphingolipids and cholesterol, resulting in localized enrichment of particular lipids and proteins, which often are themselves acylated. These proteins include the Src family kinases, Lck and Fyn, which are involved in TCR activation (21,22). Two immunoreceptors have been shown to localize to such microdomains upon activation. These are the FcεRI and, in the hands of some investigators, the TCR (16,17,23). Additionally, activation via the TCR results in enhanced recruitment and tyrosine phosphorylation of signaling molecules within the domains (8). Studies addressing LAT intracellular localization revealed that the molecule is not only membrane localized, but also targeted to microdomains (8). Targeting is mediated by both a region likely to be a transmembrane domain, and dual cysteines at positions 26 and 29. A cysteine to alanine mutation at position 26 prevented LAT microdomain targeting, while mutation at 29 decreased it. The C26A mutation also resulted in a failure of LAT tyrosine phosphorylation following TCR cross-linking. The existence of a LAT-deficient cell line allowed us to test the hypothesis that LAT targeting to microdomains and subsequent tyrosine phosphorylation is critical to TCR-mediated signaling. These cells were transfected with LAT containing the C26 and C29 single mutations, and a mutant lacking the transmembrane domain. Reconstitution with the C29A mutant resulted in LAT tyrosine phosphorylation and restoration of TCR-mediated signaling. In contrast, neither the C26A nor the transmembrane deletion mutant demonstrated enhanced tyrosine phosphorylation upon TCR activation. Substrate phosphorylation was also impaired. PLC-γ1 showed a slight increase in tyrosine phosphorylation following TCR activation in ANJ3 cells. The enhanced PLC-γ1 tyrosine phosphorylation seen with activation in cells reconstituted with wild-type LAT was not observed in cells reconstituted with C26A or tm mutants. Signaling via PLC-γ1 in cells expressing these two mutants was markedly inhibited as demonstrated by the lack of calcium flux following TCR engagement. Similarly these two mutants failed to reconstitute Ras pathway function as shown by the minimal increase in ERK activation and the failure of CD69 induction following anti-cd3 stimulation. These results provide further evidence that LAT targeting to membrane microdomains is critical to its function as a linker molecule. Its failure to be tyrosine phosphorylated when it is not appropriately targeted results in a failure of recruitment of signaling molecules to appropriate intracellular locations.

8 950 Reconstitution of a LAT-deficient T cell line Acknowledgements W. Z. is a fellow of the Leukemia Society of America. B. J. I and R. T. A. are supported by an NIH grant, GM Abbreviations MBP PMA PLC PTK References myelin basic protein phorbol myristate acetate phospholipase C protein tyrosine kinase 1 Weiss, A. and Littman, D. R Signal transduction by lymphocyte antigen receptors. Cell 76: Wange, R. L. and Samelson, L. E Complex complexes: signaling at the TCR. Immunity 5: Peterson, E. J., Clements, J. L., Fang, N. and Koretzky, G. A Adaptor proteins in lymphocyte antigen-receptor signaling. Curr. Opin. Immunol. 10: Samelson, L. E Adaptor proteins and T cell antigen receptor signaling. Prog. Biophys. Mol. Biol Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P. and Samelson, L. E LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92:83. 6 Finco, T. S., Kadlecek, T., Zhang, W., Samelson, L. E. and Weiss, A LAT is required for TCR-mediated activation of PLCγ1 and the ras pathway. Immunity 9: Goldsmith, M. A., Dazin, P. F. and Weiss, A At least two nonantigen-binding molecules are required for signal transduction by the T-cell antigen receptor. Proc. Natl Acad. Sci. USA 85: Zhang, W., Trible, R. P. and Samelson, L. E LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation. Immunity 9: Williams, B. L., Schreiber, K. L., Zhang, W., Wange, R. L., Samelson, L. E., Leibson, P. J. and Abraham, R. T Genetic evidence for differential coupling of Syk family kinases to the T- cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line. Mol. Cell. Biol. 18: Wange, R. L., Guitian, R., Isakov, N., Watts, J. D., Aebersold, R. and Samelson, L. E Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70. J. Biol. Chem. 270: Goldsmith, M. A. and Weiss, A Isolation and characterization of a T-lymphocyte somatic mutant with altered signal transduction by the antigen receptor. Proc. Natl Acad. Sci. USA 84: D Ambrosio, D., Cantrell, D. A., Frati, L., Santoni, A. and Testi, R Involvement of p21 ras activation in T cell CD69 expression. Eur. J. Immunol. 24: Gilliland, L. K., Schieven, G. L., Norris, N. A., Kanner, S. A., Aruffo, A. and Ledbetter, J. A Lymphocyte lineagerestricted tyrosine-phosphorylated proteins that bind PLCγ SH2 domains. J. Biol. Chem. 267: Weber, J. R., Bell, G. M., Han, M. Y., Pawson, T. and Imboden, J. B Association of the tyrosine kinase LCK with phospholipase C-γ1 after stimulation of the T cell antigen receptor. J. Exp. Med. 176: Sieh, M., Batzer, A., Schlessinger, J. and Weiss, A GRB2 and phospholipase C-gamma 1 associate with a 36- to 38- kilodalton phosphotyrosine protein after T-cell receptor stimulation. Mol. Cell. Biol. 14: Xavier, R., Brennan, T., Li, Q., McCormack, C. and Seed, B Membrane compartmentation is required for efficient T cell activation. Immunity 8: Montixi, C., Langlet, C., Bernard, A. M., Thimonier, J., Dubois, C., Wurbel, M. A., Chauvin, J. P., Pierres, M. and He, H. T Engagement of T cell receptor triggers its recruitment to lowdensity detergent-insoluble membrane domains. EMBO J. 17: Brown, D. A. and London, E Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes? Biochem. Biophys. Res. Commun. 240:1. 19 Brown, R. E Sphingolipid organization in biomembranes: what physical studies of model membranes reveal. J. Cell Sci. 111:1. 20 Varma, R. and Mayor, S GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394: Kabouridis, P. S., Magee, A. I. and Ley, S. C S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes. EMBO J. 16: van t Hof, W. and Resh, M. D Rapid plasma membrane anchoring of newly synthesized p59 fyn : selective requirement for NH 2 -terminal myristoylation and palmitoylation at cysteine-3. J. Cell Biol. 136: Field, K. A., Holowka, D. and Baird, B Compartmentalized activation of the high affinity immunoglobulin E receptor within membrane domains. J. Biol. Chem. 272:4276.