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Recruitment of SLP-76 to the membrane and glycolipid-enriched membrane microdomains replaces the requirement for linker for activation of T cells in T cell receptor signaling.

Boerth NJ, Sadler JJ, Bauer DE, Clements JL, Gheith SM, Koretzky GA - J. Exp. Med. (2000)

Bottom Line: Expression of this construct reconstitutes TCR-inducted phospholipase Cgamma1 phosphorylation, extracellular signal-regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2).Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling.Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Program, The Leonard and Madlyn Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, 19104-6160, USA.

ABSTRACT
Two hematopoietic-specific adapters, src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224-244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cgamma1 phosphorylation, extracellular signal-regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224-244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.

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Targeting of SLP-76 to GEMs reconstitutes proximal TCR signaling in LAT-deficient T cells. (A) Schematic of the LAT/SLP-76 chimeric construct used to target SLP-76 to GEMs. The NH2 terminus contains LAT amino acids 1–35, including the LAT extracellular domain (EC), transmembrane domain (TM), and amino acids surrounding cysteines 26 and 29 (CC). (B) J.CaM2 cells were transfected with HLA-A2 (control), Myc-LAT, Flag–SLP-76, or the LAT/SLP-76 chimera and then left unstimulated (US), stimulated with C305 (TCR), or stimulated with PV for 5 min. Lysates were subjected to immunoprecipitation with anti-PLCγ1. Immune complexes were analyzed by SDS-PAGE and immunoblot analysis with 4G10 (top) or anti-PLCγ1 (bottom). (C) J.CaM2 cells were transfected with HLA-A2 (control), Myc-LAT, Flag–SLP-76, or the LAT/SLP-76 chimera and then left unstimulated (US), stimulated with C305 (TCR), or stimulated with PMA for 5 min. Lysates were subjected to SDS-PAGE, followed by immunoblot analysis with antiphospho-ERK (top) and with anti-ERK to ensure equal loading of lanes (bottom).
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Figure 3: Targeting of SLP-76 to GEMs reconstitutes proximal TCR signaling in LAT-deficient T cells. (A) Schematic of the LAT/SLP-76 chimeric construct used to target SLP-76 to GEMs. The NH2 terminus contains LAT amino acids 1–35, including the LAT extracellular domain (EC), transmembrane domain (TM), and amino acids surrounding cysteines 26 and 29 (CC). (B) J.CaM2 cells were transfected with HLA-A2 (control), Myc-LAT, Flag–SLP-76, or the LAT/SLP-76 chimera and then left unstimulated (US), stimulated with C305 (TCR), or stimulated with PV for 5 min. Lysates were subjected to immunoprecipitation with anti-PLCγ1. Immune complexes were analyzed by SDS-PAGE and immunoblot analysis with 4G10 (top) or anti-PLCγ1 (bottom). (C) J.CaM2 cells were transfected with HLA-A2 (control), Myc-LAT, Flag–SLP-76, or the LAT/SLP-76 chimera and then left unstimulated (US), stimulated with C305 (TCR), or stimulated with PMA for 5 min. Lysates were subjected to SDS-PAGE, followed by immunoblot analysis with antiphospho-ERK (top) and with anti-ERK to ensure equal loading of lanes (bottom).

Mentions: Others have shown that the tyrosines within the cytoplasmic domain of LAT are critical for TCR signaling function, presumably because they recruit effector proteins to a multimolecular signaling complex 34. We addressed the importance of SLP-76 as one of these effectors by asking if LAT could function without its tyrosine residues if SLP-76 was constitutively targeted to GEMs. This was accomplished by expressing a chimeric protein including regions of both LAT and SLP-76. As illustrated in Fig. 3 A, the NH2 terminus of the chimera consists of the extracellular and transmembrane domains and a limited portion of the cytoplasmic tail of LAT, including the two cysteine residues (C26 and C29) previously shown to be necessary and sufficient for GEM localization of LAT 3536. COOH-terminal to the cysteines, the chimera consists of full length, wild-type SLP-76. Note that the chimera contains none of the LAT tyrosine residues shown to be phosphorylated after TCR engagement.


Recruitment of SLP-76 to the membrane and glycolipid-enriched membrane microdomains replaces the requirement for linker for activation of T cells in T cell receptor signaling.

Boerth NJ, Sadler JJ, Bauer DE, Clements JL, Gheith SM, Koretzky GA - J. Exp. Med. (2000)

Targeting of SLP-76 to GEMs reconstitutes proximal TCR signaling in LAT-deficient T cells. (A) Schematic of the LAT/SLP-76 chimeric construct used to target SLP-76 to GEMs. The NH2 terminus contains LAT amino acids 1–35, including the LAT extracellular domain (EC), transmembrane domain (TM), and amino acids surrounding cysteines 26 and 29 (CC). (B) J.CaM2 cells were transfected with HLA-A2 (control), Myc-LAT, Flag–SLP-76, or the LAT/SLP-76 chimera and then left unstimulated (US), stimulated with C305 (TCR), or stimulated with PV for 5 min. Lysates were subjected to immunoprecipitation with anti-PLCγ1. Immune complexes were analyzed by SDS-PAGE and immunoblot analysis with 4G10 (top) or anti-PLCγ1 (bottom). (C) J.CaM2 cells were transfected with HLA-A2 (control), Myc-LAT, Flag–SLP-76, or the LAT/SLP-76 chimera and then left unstimulated (US), stimulated with C305 (TCR), or stimulated with PMA for 5 min. Lysates were subjected to SDS-PAGE, followed by immunoblot analysis with antiphospho-ERK (top) and with anti-ERK to ensure equal loading of lanes (bottom).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193307&req=5

Figure 3: Targeting of SLP-76 to GEMs reconstitutes proximal TCR signaling in LAT-deficient T cells. (A) Schematic of the LAT/SLP-76 chimeric construct used to target SLP-76 to GEMs. The NH2 terminus contains LAT amino acids 1–35, including the LAT extracellular domain (EC), transmembrane domain (TM), and amino acids surrounding cysteines 26 and 29 (CC). (B) J.CaM2 cells were transfected with HLA-A2 (control), Myc-LAT, Flag–SLP-76, or the LAT/SLP-76 chimera and then left unstimulated (US), stimulated with C305 (TCR), or stimulated with PV for 5 min. Lysates were subjected to immunoprecipitation with anti-PLCγ1. Immune complexes were analyzed by SDS-PAGE and immunoblot analysis with 4G10 (top) or anti-PLCγ1 (bottom). (C) J.CaM2 cells were transfected with HLA-A2 (control), Myc-LAT, Flag–SLP-76, or the LAT/SLP-76 chimera and then left unstimulated (US), stimulated with C305 (TCR), or stimulated with PMA for 5 min. Lysates were subjected to SDS-PAGE, followed by immunoblot analysis with antiphospho-ERK (top) and with anti-ERK to ensure equal loading of lanes (bottom).
Mentions: Others have shown that the tyrosines within the cytoplasmic domain of LAT are critical for TCR signaling function, presumably because they recruit effector proteins to a multimolecular signaling complex 34. We addressed the importance of SLP-76 as one of these effectors by asking if LAT could function without its tyrosine residues if SLP-76 was constitutively targeted to GEMs. This was accomplished by expressing a chimeric protein including regions of both LAT and SLP-76. As illustrated in Fig. 3 A, the NH2 terminus of the chimera consists of the extracellular and transmembrane domains and a limited portion of the cytoplasmic tail of LAT, including the two cysteine residues (C26 and C29) previously shown to be necessary and sufficient for GEM localization of LAT 3536. COOH-terminal to the cysteines, the chimera consists of full length, wild-type SLP-76. Note that the chimera contains none of the LAT tyrosine residues shown to be phosphorylated after TCR engagement.

Bottom Line: Expression of this construct reconstitutes TCR-inducted phospholipase Cgamma1 phosphorylation, extracellular signal-regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2).Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling.Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Program, The Leonard and Madlyn Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, 19104-6160, USA.

ABSTRACT
Two hematopoietic-specific adapters, src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224-244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cgamma1 phosphorylation, extracellular signal-regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224-244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.

Show MeSH
Related in: MedlinePlus