International Immunology, Vol. 11, No. 6, 943-950,
June 1999
© 1999 Japanese Society for Immunology
Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line
Section on Lymphocyte Signaling, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-5430, USA
1 Department of Immunology, Mayo Clinic and Foundation, Rochester, MN 55905, USA
Correspondence to: L. E. Samelson
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 Ca2+ mobilization and optimal tyrosine phosphorylation of phospholipase C-
1, 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.
Keywords: LAT, TCR, tyrosine phosphorylation, palmitoylation
2 Present address: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
Transmitting editor: J. A. Bluestone
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D. Peters, M. Tsuchida, E. R. Manthei, T. Alam, C. S. Cho, S. J. Knechtle, and M. M. Hamawy Potentiation of CD3-induced expression of the linker for activation of T cells (LAT) by the calcineurin inhibitors cyclosporin A and FK506 Blood, May 1, 2000; 95(9): 2733 - 2741. [Abstract] [Full Text] [PDF]
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S. Lemay, D. Davidson, S. Latour, and A. Veillette Dok-3, a Novel Adapter Molecule Involved in the Negative Regulation of Immunoreceptor Signaling Mol. Cell. Biol., April 15, 2000; 20(8): 2743 - 2754. [Abstract] [Full Text]
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R. Goitsuka, H. Kanazashi, H. Sasanuma, Y.-i. Fujimura, Y. Hidaka, A. Tatsuno, C. Ra, K. Hayashi, and D. Kitamura A BASH/SLP-76-related adaptor protein MIST/Clnk involved in IgE receptor-mediated mast cell degranulation Int. Immunol., April 1, 2000; 12(4): 573 - 580. [Abstract] [Full Text] [PDF]
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X. R. Bustelo Regulatory and Signaling Properties of the Vav Family Mol. Cell. Biol., March 1, 2000; 20(5): 1461 - 1477. [Full Text]
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K. V. Salojin, J. Zhang, C. Meagher, and T. L. Delovitch ZAP-70 Is Essential for the T Cell Antigen Receptor-induced Plasma Membrane Targeting of SOS and Vav in T Cells J. Biol. Chem., February 25, 2000; 275(8): 5966 - 5975. [Abstract] [Full Text] [PDF]
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S. C. Bunnell, M. Diehn, M. B. Yaffe, P. R. Findell, L. C. Cantley, and L. J. Berg Biochemical Interactions Integrating Itk with the T Cell Receptor-initiated Signaling Cascade J. Biol. Chem., January 21, 2000; 275(3): 2219 - 2230. [Abstract] [Full Text] [PDF]
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 L.E. SAMELSON, S.C. BUNNELL, R.P. TRIBLE, T. YAMAZAKI, and W. ZHANG Studies on the Adapter Molecule LAT Cold Spring Harb Symp Quant Biol, January 1, 1999; 64(0): 259 - 264. [Abstract] [PDF]
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W. Zhang, R. P. Trible, M. Zhu, S. K. Liu, C. J. McGlade, and L. E. Samelson Association of Grb2, Gads, and Phospholipase C-gamma 1 with Phosphorylated LAT Tyrosine Residues. EFFECT OF LAT TYROSINE MUTATIONS ON T CELL ANTIGEN RECEPTOR-MEDIATED SIGNALING J. Biol. Chem., July 21, 2000; 275(30): 23355 - 23361. [Abstract] [Full Text] [PDF]
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J. Lin and A. Weiss Identification of the Minimal Tyrosine Residues Required for Linker for Activation of T Cell Function J. Biol. Chem., July 27, 2001; 276(31): 29588 - 29595. [Abstract] [Full Text] [PDF]
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R. Goitsuka, A. Tatsuno, M. Ishiai, T. Kurosaki, and D. Kitamura MIST Functions through Distinct Domains in Immunoreceptor Signaling in the Presence and Absence of LAT J. Biol. Chem., September 14, 2001; 276(38): 36043 - 36050. [Abstract] [Full Text] [PDF]
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M. Zubiaur, O. Fernandez, E. Ferrero, J. Salmeron, B. Malissen, F. Malavasi, and J. Sancho CD38 Is Associated with Lipid Rafts and upon Receptor Stimulation Leads to Akt/Protein Kinase B and Erk Activation in the Absence of the CD3-zeta Immune Receptor Tyrosine-based Activation Motifs J. Biol. Chem., January 4, 2002; 277(1): 13 - 22. [Abstract] [Full Text] [PDF]
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S. Tridandapani, T. W. Lyden, J. L. Smith, J. E. Carter, K. M. Coggeshall, and C. L. Anderson The Adapter Protein LAT Enhances Fcgamma Receptor-mediated Signal Transduction in Myeloid Cells J. Biol. Chem., June 30, 2000; 275(27): 20480 - 20487. [Abstract] [Full Text] [PDF]
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M. Iwashima, M. Takamatsu, H. Yamagishi, Y. Hatanaka, Y.-Y. Huang, C. McGinty, S. Yamasaki, and T. Koike Genetic evidence for Shc requirement in TCR-induced c-Rel nuclear translocation and IL-2 expression PNAS, April 2, 2002; 99(7): 4544 - 4549. [Abstract] [Full Text] [PDF]
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