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

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.

Figure 1

Recruitment of SLP-76 and Gads to GEMs after TCR ligation. Jurkat T cells were either left unstimulated (US) or stimulated with C305 for 5 min, followed by lysis in MES lysis buffer plus protease and phosphatase inhibitors. Lysates were subjected to sucrose gradient ultracentrifugation for GEM purification. Sequential fractions were removed starting from the top of the gradient and are indicated as fraction number. The gradient fractions were separated by SDS-PAGE, followed by detection of GM1 using horseradish peroxidase–conjugated cholera toxin B subunit (A) or immunoblot analysis using anti-LAT (B), anti–SLP-76 (C), or anti-Gads (D).

Figure 2

SLP-76 amino acids 224–244 are required for recruitment of SLP-76 to GEMs. (A) Schematic of the constructs encoding wild-type SLP-76 (WT), the SLP-76 mutant with tyrosines 113, 128, and 145 altered to phenylalanine (Y3F), the SLP-76 mutant incapable of binding Gads (Δ224–244), and the SLP-76 mutant with a nonfunctional SH2 domain (R448K). (B) Jurkat T cells were transfected with the various constructs and then left unstimulated or stimulated with C305, followed by lysis and preparation of GEM fractions as described for Fig. 1. A Triton-soluble fraction (fraction 11) and a GEM fraction (fraction 3) were separated by SDS-PAGE, followed by immunoblot analysis using anti-Flag (top) or anti–SLP-76 (bottom). Note that in the anti–SLP-76 blot, the band in the unstimulated Δ224–244 lane is broader than all other SLP-76 bands. This is due to immunoreactivity of both the endogenous wild-type SLP-76 and the Δ224–244 mutant. Also, note that in the stimulated lanes, only the slower migrating species (wild-type SLP-76) appears.

Figure 2

SLP-76 amino acids 224–244 are required for recruitment of SLP-76 to GEMs. (A) Schematic of the constructs encoding wild-type SLP-76 (WT), the SLP-76 mutant with tyrosines 113, 128, and 145 altered to phenylalanine (Y3F), the SLP-76 mutant incapable of binding Gads (Δ224–244), and the SLP-76 mutant with a nonfunctional SH2 domain (R448K). (B) Jurkat T cells were transfected with the various constructs and then left unstimulated or stimulated with C305, followed by lysis and preparation of GEM fractions as described for Fig. 1. A Triton-soluble fraction (fraction 11) and a GEM fraction (fraction 3) were separated by SDS-PAGE, followed by immunoblot analysis using anti-Flag (top) or anti–SLP-76 (bottom). Note that in the anti–SLP-76 blot, the band in the unstimulated Δ224–244 lane is broader than all other SLP-76 bands. This is due to immunoreactivity of both the endogenous wild-type SLP-76 and the Δ224–244 mutant. Also, note that in the stimulated lanes, only the slower migrating species (wild-type SLP-76) appears.

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).

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).

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).

Figure 4

Targeting of SLP-76 to GEMs supports reconstitution of NFAT promoter activity in LAT-deficient T cells. J.CaM2 cells were transfected with NFAT-luc and pCMV/β-gal plus the indicated constructs. 24 h after transfection, cells were left unstimulated (US), stimulated with C305, or stimulated with PMA plus ionomycin for 16 h. Samples were assayed for luciferase activity (normalized to β-gal; left panel). PMA plus ionomycin responses were similar (∼100,000 relative light units) for each sample. This experiment is representative of 10 independent transfections. Expression of the transfected proteins for this experiment was determined by examining whole cell lysates by immunoblot analysis with anti–SLP-76 (to detect transfected SLP-76 or the chimera) or anti-Myc (to detect transfected LAT) antibodies (right panel).

Figure 5

The LAT/SLP-76 chimera is more efficient at restoring TCR signaling in J.CaM2 than wild-type LAT. (A) J.CaM2 cells were transfected with NFAT-luc and pCMV/β-gal plus the indicated constructs. 40 μg of plasmid was used for both the A2 and SLP-76 controls. Varying amounts of plasmid (from 5 to 40 μg, as shown) encoding LAT or the LAT/SLP-76 chimera were used. 24 h after transfection, cells were left unstimulated (US), stimulated with C305, or stimulated with PMA plus ionomycin for 16 h. Samples were assayed for luciferase (normalized to β-gal activity; left panel). PMA plus ionomycin responses were similar (∼120,000 relative light units) for each sample. This experiment is representative of four independent transfections. Expression of the transfected proteins for this experiment was determined by examining whole cell lysates by immunoblot analysis with anti–SLP-76 (to detect transfected SLP-76 or the chimera) or anti-Myc (to detect transfected LAT) antibodies (right panel). (B) J.CaM2 cells were transfected with NFAT-luc and pCMV/β-gal plus the indicated constructs. 24 h after transfection, cells were left unstimulated (US), stimulated with C305, or stimulated with PMA plus ionomycin for 16 h. Samples were assayed for luciferase (normalized to β-gal activity; left panel). PMA plus ionomycin responses were similar (∼120,000 relative light units) for each sample. This experiment is representative of three independent transfections. Note the doublet in the SLP-76 blot examining expression of the LAT/SLP-76 chimera (panel A, right). The slower migrating species represents the chimera, while the faster migrating band is endogenous wild-type SLP-76.

Figure 5

The LAT/SLP-76 chimera is more efficient at restoring TCR signaling in J.CaM2 than wild-type LAT. (A) J.CaM2 cells were transfected with NFAT-luc and pCMV/β-gal plus the indicated constructs. 40 μg of plasmid was used for both the A2 and SLP-76 controls. Varying amounts of plasmid (from 5 to 40 μg, as shown) encoding LAT or the LAT/SLP-76 chimera were used. 24 h after transfection, cells were left unstimulated (US), stimulated with C305, or stimulated with PMA plus ionomycin for 16 h. Samples were assayed for luciferase (normalized to β-gal activity; left panel). PMA plus ionomycin responses were similar (∼120,000 relative light units) for each sample. This experiment is representative of four independent transfections. Expression of the transfected proteins for this experiment was determined by examining whole cell lysates by immunoblot analysis with anti–SLP-76 (to detect transfected SLP-76 or the chimera) or anti-Myc (to detect transfected LAT) antibodies (right panel). (B) J.CaM2 cells were transfected with NFAT-luc and pCMV/β-gal plus the indicated constructs. 24 h after transfection, cells were left unstimulated (US), stimulated with C305, or stimulated with PMA plus ionomycin for 16 h. Samples were assayed for luciferase (normalized to β-gal activity; left panel). PMA plus ionomycin responses were similar (∼120,000 relative light units) for each sample. This experiment is representative of three independent transfections. Note the doublet in the SLP-76 blot examining expression of the LAT/SLP-76 chimera (panel A, right). The slower migrating species represents the chimera, while the faster migrating band is endogenous wild-type SLP-76.

Figure 6

Tethering of SLP-76 to the membrane is sufficient to reconstitute signaling in LAT-deficient T cells. (A) Schematic of the LAT/SLP-76 chimera with point mutations altering cysteines 26 and 29 to serine. (B) J.14-v-29 cells were transfected with LAT/SLP-76 wild type, LAT/SLP-76 CS, or FLAG–SLP-76 and then lysed for GEM purification. The Triton-soluble fractions (fraction 11) and GEM fractions (fraction 3) were separated by SDS-PAGE, followed by immunoblot analysis using anti-SLP-76. (C) J.CaM2 cells were transfected with NFAT-luc, pCMV/β-gal, and the indicated constructs. 24 h after transfection, cells were left unstimulated (US), stimulated with C305, or stimulated with PMA plus ionomycin for 16 h. The samples were assayed for luciferase activity, which is normalized to the β-gal activity (left panel). PMA plus ionomycin responses were similar (∼250,000 relative light units) for each condition. This experiment is representative of four independent transfections. Expression of the transfected proteins in the experiment shown was determined by immunoblot analysis of whole cell lysates with anti–SLP-76 (to detect transfected SLP-76 or the chimera) or anti-Myc (to detect transfected LAT) antibodies (right panel).

Figure 6

Tethering of SLP-76 to the membrane is sufficient to reconstitute signaling in LAT-deficient T cells. (A) Schematic of the LAT/SLP-76 chimera with point mutations altering cysteines 26 and 29 to serine. (B) J.14-v-29 cells were transfected with LAT/SLP-76 wild type, LAT/SLP-76 CS, or FLAG–SLP-76 and then lysed for GEM purification. The Triton-soluble fractions (fraction 11) and GEM fractions (fraction 3) were separated by SDS-PAGE, followed by immunoblot analysis using anti-SLP-76. (C) J.CaM2 cells were transfected with NFAT-luc, pCMV/β-gal, and the indicated constructs. 24 h after transfection, cells were left unstimulated (US), stimulated with C305, or stimulated with PMA plus ionomycin for 16 h. The samples were assayed for luciferase activity, which is normalized to the β-gal activity (left panel). PMA plus ionomycin responses were similar (∼250,000 relative light units) for each condition. This experiment is representative of four independent transfections. Expression of the transfected proteins in the experiment shown was determined by immunoblot analysis of whole cell lysates with anti–SLP-76 (to detect transfected SLP-76 or the chimera) or anti-Myc (to detect transfected LAT) antibodies (right panel).

Figure 6

Tethering of SLP-76 to the membrane is sufficient to reconstitute signaling in LAT-deficient T cells. (A) Schematic of the LAT/SLP-76 chimera with point mutations altering cysteines 26 and 29 to serine. (B) J.14-v-29 cells were transfected with LAT/SLP-76 wild type, LAT/SLP-76 CS, or FLAG–SLP-76 and then lysed for GEM purification. The Triton-soluble fractions (fraction 11) and GEM fractions (fraction 3) were separated by SDS-PAGE, followed by immunoblot analysis using anti-SLP-76. (C) J.CaM2 cells were transfected with NFAT-luc, pCMV/β-gal, and the indicated constructs. 24 h after transfection, cells were left unstimulated (US), stimulated with C305, or stimulated with PMA plus ionomycin for 16 h. The samples were assayed for luciferase activity, which is normalized to the β-gal activity (left panel). PMA plus ionomycin responses were similar (∼250,000 relative light units) for each condition. This experiment is representative of four independent transfections. Expression of the transfected proteins in the experiment shown was determined by immunoblot analysis of whole cell lysates with anti–SLP-76 (to detect transfected SLP-76 or the chimera) or anti-Myc (to detect transfected LAT) antibodies (right panel).

Figure 7

Structure/function analysis of SLP-76 domains required to support TCR-induced NFAT activity in LAT-deficient cells. (A) Schematic of the LAT/SLP-76 chimeric constructs used in this experiment. Y3F contains three point mutations altering tyrosines 113, 128, and 145 to phenylalanine, abrogating tyrosine phosphorylation of the chimera; Δ224–244 includes a 20–amino acid deletion eliminating the Gads binding site; and R448K contains a point mutation in arginine 448, eliminating function of the SLP-76 SH2 domain. (B) J.CaM2 cells were transfected with NFAT-luc, pCMV/β-gal, and the indicated constructs. 24 h after transfection, cells were left unstimulated (US), stimulated with C305, or stimulated with PMA plus ionomycin for 16 h. The samples were assayed for luciferase activity, which is normalized to the β-gal activity (left panel). The PMA plus ionomycin response was similar (∼200,000 relative light units) for each condition. This experiment is representative of five independent transfections. Expression of the transfected proteins in the experiment shown was determined by immunoblot analysis of whole cell lysates with anti–SLP-76 (to detect transfected SLP-76 or the chimera) or anti-Myc (to detect transfected LAT) antibodies (right panel).

Figure 8

Structure/function analysis of SLP-76 domains required to support TCR-induced PLCγ1 and ERK activity in LAT-deficient cells. (A) J.CaM2 cells were transfected with the indicated constructs. 24 h later, cells were left unstimulated (US) or were stimulated with C305 (TCR) or PV for 5 min and then lysed and subjected to immunoprecipitation using anti-PLCγ1. The immune complexes were analyzed for phosphotyrosine contain (4G10, top) and amount of PLCγ1 (bottom). (B) Transfected J.CaM2 cells were stimulated and then analyzed for phospho-ERK (top) and with anti-ERK to ensure equal loading of lanes (bottom) as described for Fig. 3.

Figure 8

Structure/function analysis of SLP-76 domains required to support TCR-induced PLCγ1 and ERK activity in LAT-deficient cells. (A) J.CaM2 cells were transfected with the indicated constructs. 24 h later, cells were left unstimulated (US) or were stimulated with C305 (TCR) or PV for 5 min and then lysed and subjected to immunoprecipitation using anti-PLCγ1. The immune complexes were analyzed for phosphotyrosine contain (4G10, top) and amount of PLCγ1 (bottom). (B) Transfected J.CaM2 cells were stimulated and then analyzed for phospho-ERK (top) and with anti-ERK to ensure equal loading of lanes (bottom) as described for Fig. 3.