Activation of T lymphocytes and the role of the adapter ...

Activation of T lymphocytes and the role of the adapter LAT

Enrique Aguado*, Mario Martínez-Florensa and Pedro Aparicio

Departamento de Bioquímica, Biología Molecular B e Inmunología. Facultad de Medicina. Universidad de Murcia. 30100 Murcia, Spain.

* To whom correspondence should be addressed: Enrique Aguado, Departamento de Bioquímica, Biología Molecular B e Inmunología, Facultad de Medicina, Campus de Espinardo, Apartado de Correos 4021, Universidad de Murcia, 30100-Murcia, Spain. Tel.: +34 968 363960. Fax: 34 968 364150. E-mail: [email protected]

Abstract The adapter molecule LAT (Linker for the Activation of T cells) is a membrane protein that becomes phosphorylated on conserved tyrosine residues upon TCR/CD3 complex engagement in T lymphocytes. Tyrosine phosphorylation of this adapter recruits to the membrane many signaling proteins through the interaction with the phosphotyrosine binding domains of these proteins, allowing the activation of several intracellular signaling pathways. Initial studies performed in T cell lines suggested that the adapter LAT acts primarily as a platform for the distribution of activation signals coming from the TCR/CD3 complex, and the phenotype of LAT deficient mice, in which T cell development is arrested at an early stage, supported this “activatory” function. However, the analysis of several knock in mice strains in which some tyrosine residues have been mutated, has revealed the development of lymphoproliferative disorders caused by polyclonal T lymphocytes producing high titers of T helper-type 2 (TH2)cytokines. Very recently, it has been demonstrated that raft localization of LAT is altered in anergic T lymphocytes. Therefore, LAT show unexpected regulatory functions in T cell development and homeostasis.

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LAT is a transmembrane adapter transducing activation signals. T lymphocytes recognize foreign antigens presented by Antigen Presenting Cells (APC), and for this purpose they express at their surface an antigen receptor composed of the TCR and - chains, and several invariant polypeptides (the CD3-, -, - and - polypeptides) responsible for intracellular signal transduction. Antigen engagement by the TCR leads to Lck activation, which in turn, phosphorylates the tyrosine residues found in the immunoreceptor tyrosine-based activation motifs (ITAM) in the CD3 chains of the TCR (1). Phosphorylation of these motifs recruits the ZAP-70 tyrosine kinase to the TCR/CD3 complex in the membrane, leading to its activation by Lck and, consequently, to the phosphorylation of downstream targets. The adapter protein LAT is one of the most prominently tyrosine-phosphorylated substrates of ZAP-70, and was identified as an integral transmembrane protein of 36-38 kDa (2). LAT presents a single region of about 20 hydrophobic amino acids that constitutes the transmembrane segment and is expressed in the membrane of T lymphocytes, NK and mast cells, platelets, and pre-B cells (2). As the majority of adapters LAT lacks any enzymatic or transcriptional activity. LAT presents in its amino acid sequence nine conserved tyrosine residues (Figure 1) that, upon phosphorylation, are potential docking sites for different proteins. Indeed, from the initial studies, it was demonstrated that in T lymphocytes LAT is tyrosine phosphorylated by Lck-activated ZAP-70, leading to the binding with Grb2, Grap, Gads-SLP-76, PLC-1, Vav, Cbl and the regulatory subunit of PI-3K (2). Consequently, upon their binding to LAT, these proteins can themselves be activated by tyrosine phosphorylation and find higher concentrations of their substrates in the plasma membrane. In vitro analysis performed in LAT deficient cell lines demonstrated the essential role of this adapter protein in the transduction of intracellular signals coupled to the TCR/CD3 complex (3;4). LAT deficient cells show no deficiency in proximal signaling events, but downstream pathways triggered by TCR engagement, as Ca2+ influx generation and NFAT activation or MAPK activation, were prevented in these cell lines. Importantly, the re-expression of LAT in these cell lines restored the normal activation of the downstream signaling pathways triggered upon TCR engagement. Moreover, the characterization of these LAT deficient cell lines allowed the individual analysis of the different tyrosine residues of LAT, by means of generating stable transfectants of

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J.CaM2 (which are LAT deficient) expressing different forms of this adapter in which individual tyrosines or combinations of them were mutated to phenylalanines (5). This kind of analysis established that tyrosines 7, 8 and 9 share the responsibility of binding to the SH2 domain of Grb2, tyrosine 6 of LAT binds upon its phosphorylation to the Cterminal SH2 domain of PLC-1, and the Gads-SLP-76 complex binds to phosphorylated tyrosines 7 and 8 (5). Therefore, the four distal tyrosine residues of LAT are essential for the association with critical signaling molecules, and thus for TCR signals transduction. Using the same kind of approach Lin and Weiss expanded the analysis about the function of LAT tyrosines, and demonstrated that tyrosine residues 6, 7 and 9 were sufficient for Ca2+ mobilization (6). However, full reconstitution of TCR-dependent Erk activation also required the presence of tyrosines 4 and 9. Very interestingly, this article also demonstrated that reconstitution of tyrosine residues in “Trans” is not enough to reconstitute Erk activation upon TCR engagement. This means that, at least, tyrosines 6, 7 and 8 must be on the same molecule of LAT for a proper transduction of intracellular signals. Phenotype of LAT mutant murine strains reveals a regulatory function for this adapter protein. Given that LAT is strongly expressed by thymocytes, it was of interest to analyze the role of this adapter during T cell development. Therefore, LAT deficient mice were generated and showed normal B and NK cell compartments, but a total absence of T lymphocytes in the periphery (7). Adult thymi from LAT deficient mice are smaller and thymic development is blocked at the double negative (DN) stage, demonstrating a crucial role for LAT in T cell development by transducing intracellular signals from the pre-TCR that allow DN to double positive (DP) transition. Interestingly, although LAT is expressed by NK cells and platelets, no obvious abnormality of NK or platelet function and development is observed in LAT-/- mice. In order to elucidate whether the tyrosines of LAT have different functions in vivo, several Knock In animals harboring individual mutations of these residues have been developed. In agreement with data obtained in LAT deficient cell lines, tyrosine residues 6, 7, 8 and 9 show in vivo a role of transduction of activation signals, since

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mice having a mutation of the three (LATY7/8/9F) or four (LATY6/7/8/9F) distal tyrosines of LAT have small thymi and thymic development is also blocked at the DN stage (8;9). Also, knock-in mice in which tyrosine number 6 has been replaced by phenylalanine (LATY6F) had reduced numbers of thymocytes, demonstrating in vivo a positive function for this aminoacid during T cell development (10;11). However, LATY6F mice older than 7 weeks showed a population of polyclonal CD4 T lymphocytes that expanded in the periphery and infiltrated the thymus. This population of cells produced high amounts of TH2 type cytokines and had a phenotype (CD44high, CD62Llow, CD69+, and CD24-) resembling activated/memory T cells. The cytokines produced by this population are responsible for thymic eosinophilia observed in the thymi and periphery of these mice and the observed augmented population of activated B cells producing high amounts of IgG1 and IgE. In a similar way, despite a total block of αβ T cell development, LATY7/8/9F mice older than 20 weeks showed in their peripheral lymphoid organs a proliferating population of γδ T cells producing also high amounts of TH2 type cytokines (8). Therefore, the populations of CD4  and  T proliferating in LATY6F and LATY7/8/9F mice, respectively, initiate a strikingly similar TH2-type disorder characterized by hypergammaglobulinemia E and G1. These unexpected phenotypes suggest that LAT not only acts transducing activatory signals, but also may regulate the signaling cassettes operated by the TCR/pre-TCR complexes. LAT as a negative regulator of intracellular signaling. Upon tyrosine phosphorylation LAT molecules induce the activation of downstream signaling pathways, but also are likely to trigger negative regulatory loop(s) acting at later time points. In this way, the delayed induction of the negative loop(s) would only allow a transient transduction of activatory signals, preventing the harmful effects of excessive activation of T cells. It is possible that, upon tyrosine phosphorylation, mutant LATY6F and LATY7/8/9F molecules transduce weak intracellular signals that are enough to trigger the development of a few αβ or γδ T cells, but would fall below the threshold required to trigger the negative regulatory loop expected to turn off the TCR operated signaling cassette. In this way, the tonic signals generated by the encounter of the TCR with self peptide/MHC complexes, and that are required for T cell survival, would be greatly enhanced and would lead naïve CD4 T cells to a TH2 differentiation program.

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In agreement with this negative regulatory function, it has been demonstrated that, upon its tyrosine phosphorylation LAT is able to bind to Gab2, resulting in the recruitment of the tyrosine phosphatase SHP-2 (12). Interestingly, the possible recruitment of inhibitory molecules by LAT fits well with the recessive nature of the LATY6F and LATY7/8/9F mutations: in heterozygous mice, the chronic signals expected to be delivered by the mutant forms of LAT are likely blunted by dominant, negative signals originating from wild-type LAT molecules. In this context, it has been recently shown that upon TCR engagement LAT becomes phosphorylated on serine and threonine residues by Erk kinase (13). Phosphorylation of threonine 155 of human LAT negatively regulates PLC-γ1 binding and Erk activation, showing that the activation of the Ras-Erk pathway (in which LAT has an essential role) also triggers a negative regulatory loop attenuating TCR signaling. However, since threonine 155 residue is not conserved in all the other species analyzed, it remains to be determined whether LAT Ser/Thr-phosphorylation by Erk constitutes a general negative regulatory mechanism controlling TCR signals. Localization of LAT in GEMs and its role in anergy. Lipid rafts, also known as GEMs (glycolipid-enriched membrane domains) are plasma membrane domains enriched in sphingolipids, cholesterol and glycosyl-phosphatidylinositol (GPI)-anchored proteins that play critical functions in immune receptor signaling. LAT shows in its amino acid sequence two conserved cysteine residues in the juxtamembrane region that are palmitoylated, allowing the localization of LAT in the lipid rafts (14). Interestingly, mutation of these amino acids prevents the partitioning of LAT into the rafts, preventing the transduction of signals coming from the TCR. However, it has been recently shown that a chimeric LAT protein containing the transmembrane domain of a non-raft protein effectively restores TCR associated signaling in JCaM2 cells (15). Therefore, it is possible that LAT does not directly require palmitoylation for its interaction with TCR in the lipid rafts, but for an interaction with another raft-associated regulatory protein, and the presence of an appropriate transmembrane segment would substitute this requirement. The relevance of raft localization of LAT has been recently highlighted by the finding that its localization in the GEMs is defective in anergic CD4 T cells (16). By generating anergic T cells with two different approaches, Altman and co-workers have 6

demonstrated that, although the most proximal signaling events triggered upon TCR engagement (as ζ-chain phosphorylation or ZAP-70 activation) are intact, tyrosine phosphorylation of LAT and its ability to recruit and activate PLC-γ1 are greatly diminished in anergic T cells. Strikingly, these defects of LAT functions are the result of the selective impairment of its palmitoylation, and this decrease was selective since palmitoylation of Fyn, which is also a palmitoylated protein, was not affected by the same treatment. Therefore, these data reveal that LAT is the most upstream signaling unit altered in anergic T cells. It remains to be determined the regulation of the process by which LAT becomes palmitoylated and localized to the membrane rafts, but the selective nature of the palmitoylation defect suggests a deficiency in the expression and/or function of a palmitoyltransferase with a selective activity toward LAT. A more detailed study, including “in vivo” analysis would be of help for a better understanding of this phenomenon. Concluding remarks. The transmembrane adapter LAT has a major role in the transduction of activation signals coming from the TCR/CD3 complex. In vitro experiments have revealed the importance of several of its tyrosine residues in the distribution of signals to different downstream signaling pathways. Nevertheless, the study of two animal models harboring point mutations in several of its tyrosine residues has revealed unexpected regulatory functions for this adaptor whose nature remains poorly defined. From the analysis of either LATY6F or LATY7/8/9F mice it seems that LAT contributes to a noncharacterized negative loop of intracellular signaling. In this context, the finding that threonine phosphorylation of the human form of LAT can negatively affect the TCR generated signals constitutes a possible model for explaining the regulatory functions played by this adapter, as revealed by the phenotype of LAT Knock In models. Also, the recent finding that localization of LAT in the lipid rafts is defective in anergic T cells confers it a new unpredicted function. Therefore, new approaches intending to discover interaction of LAT with other molecules, and also new “in vivo” analysis could bring some light in this puzzling issue.

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Figure 1. Aminoacid sequences of the human, rat and mouse LAT molecules. Aminoacid sequences of human (h), rat (r) and mouse (m) are shown, and the conserved residues are shadowed. The transmembrane segments are indicated together with the tyrosine (Y) residues found within the cytoplasmic region. The conserved tyrosines of LAT have been numbered 1 to 9 by beginning at the membrane proximal tyrosine. Conserved juxtamembrane palmitoylated cysteines (C) are also shown.

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Reference List 1. Lin, J. and Weiss, A. (2001) J.Cell Sci. 114, 243-244 2. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P., and Samelson, L. E. (1998) Cell 92, 8392 3. Finco, T. S., Kadlecek, T., Zhang, W., Samelson, L. E., and Weiss, A. (1998) Immunity. 9, 617626 4. Zhang, W., Irvin, B. J., Trible, R. P., Abraham, R. T., and Samelson, L. E. (1999) Int.Immunol. 11, 943-950 5. Zhang, W., Trible, R. P., Zhu, M., Liu, S. K., McGlade, C. J., and Samelson, L. E. (2000) J.Biol.Chem. 275, 23355-23361 6. Lin, J. and Weiss, A. (2001) J.Biol.Chem. 276, 29588-29595 7. Zhang, W., Sommers, C. L., Burshtyn, D. N., Stebbins, C. C., DeJarnette, J. B., Trible, R. P., Grinberg, A., Tsay, H. C., Jacobs, H. M., Kessler, C. M., Long, E. O., Love, P. E., and Samelson, L. E. (1999) Immunity. 10, 323-332 8. Nunez-Cruz, S., Aguado, E., Richelme, S., Chetaille, B., Mura, A. M., Richelme, M., Pouyet, L., Jouvin-Marche, E., Xerri, L., Malissen, B., and Malissen, M. (2003) Nat.Immunol. 4, 999-1008 9. Sommers, C. L., Menon, R. K., Grinberg, A., Zhang, W., Samelson, L. E., and Love, P. E. (2001) J.Exp.Med. 194, 135-142 10. Aguado, E., Richelme, S., Nunez-Cruz, S., Miazek, A., Mura, A. M., Richelme, M., Guo, X. J., Sainty, D., He, H. T., Malissen, B., and Malissen, M. (2002) Science 296, 2036-2040 11. Sommers, C. L., Park, C. S., Lee, J., Feng, C., Fuller, C. L., Grinberg, A., Hildebrand, J. A., Lacana, E., Menon, R. K., Shores, E. W., Samelson, L. E., and Love, P. E. (2002) Science 296, 2040-2043 12. Yamasaki, S., Nishida, K., Sakuma, M., Berry, D., McGlade, C. J., Hirano, T., and Saito, T. (2003) Mol.Cell Biol. 23, 2515-2529 13. Matsuda, S., Miwa, Y., Hirata, Y., Minowa, A., Tanaka, J., Nishida, E., and Koyasu, S. (2004) EMBO J. 23, 2577-2585 14. Zhang, W., Trible, R. P., and Samelson, L. E. (1998) Immunity. 9, 239-246 15. Zhu, M., Shen, S., Liu, Y., Granillo, O., and Zhang, W. (2005) J.Immunol. 174, 31-35 16. Hundt, M., Tabata, H., Jeon, M. S., Hayashi, K., Tanaka, Y., Krishna, R., De Giorgio, L., Liu, Y. C., Fukata, M., and Altman, A. (2006) Immunity. 24, 513-522

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Transmembrane segment h r m

Palmitoylation residues

MEEA I LVPCVLGLLLLPI LAMLMA . L CVH C HRLPGS YDS TS S DS LYP RGIQ FKRPH TVAP : 59 MEAD ALS PVELGLLLLPF VVMLLAAL CVR C RELPAS YDS AS TES LYP RS IL I KP PQ I TVP : 60 MEAD ALS PVGLGLLLLPF LVT LLAAL CVR C RELPVS YDS TS TES LYP RS IL I KP PQ I TVP : 60

1 h r m

WPPA. . YP PVTS YP PLSQ PDLLPI PRS PQPLGGSHRT PSS R R DS DGANSVAS YEN EEPAC :117 RT PAT S YP LVTS FP PLRQ PDLLPI PRS PQPLGGSHRMPSS R QNS DDANSVAS YEN QEPAR :120 RT PAVS YP LVTS FP PLRQ PDLLPI PRS PQPLGGSHRMPSS Q QNS DDANSVAS YEN QEPAC :120

3 h r m

4

E. . DADEDEDDYHNPG YLVV LPDS TPATS TAAP SAPALS T P GI RDS AFSMES I DD YVNV P :175 KNVDEDEDEDDYP E . G YLVV LPDS S PAAVPVVS SAPVPS NP DLGDS AFSMES CED YVNV P :179 KNVDADEDEDDYP N . G YLVV LPDS S PAAVPVVS SAPVPS NP DLGDS AFSV ES CED YVNV P :179

5 h r m

6

7

ES GES AEAS LDGS RE YVNVS QELHPGAAKTEPAALS S Q EAE. EVEEEG. . . . . APD YEN L :229 ES EES AEAS LDGS RE YVNVS QDAQP. VI RAELASVTS Q EVEDE . EEEDVDGEEAPD YEN L :237 ES EES AEAS LDGS RE YVNVS P EQQP. VTRAELASVNS Q EVEDEGEEEGVDGEEAPD YEN L :238

8 h r m

2

9

Q E LN :233 Q E LN :241 Q E LN :242

Figure 1