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Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation.

Tanimura N, Nagafuku M, Minaki Y, Umeda Y, Hayashi F, Sakakura J, Kato A, Liddicoat DR, Ogata M, Hamaoka T, Kosugi A - J. Cell Biol. (2003)

Bottom Line: Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions.The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT.Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

View Article: PubMed Central - PubMed

Affiliation: School of Allied Health Sciences, Faculty of Medicine, Osaka University, Suita, Japan.

ABSTRACT
Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

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The PLCγ1 binding site of LAT is important for the reduced mobility of LAT in aggregated rafts. (A) Schematic representations of the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP fusion proteins. A LAT-GFP chimera was constructed by attaching EGFP to the full length of mouse LAT. Y136 is a binding site for PLCγ1, and Y175, Y195, and Y235 are binding sites for Grb2/Gads in mouse LAT. Mutated amino acids are shown for each construct. (B) Association of LAT-GFP with PLCγ1 or Grb2. The LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were stimulated with OKT3 for 5 min. Cell lysates were immunoprecipitated with either anti-PLCγ1 or anti-Grb2 antibody, and immunoprecipitates were analyzed by immunoblotting with anti-GFP and antibodies specific for the immunoprecipitated protein. (C) After the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were mixed with anti-CD3 beads for 20 min, a selected area (2-μm square) on the patches was photobleached, and fluorescence recovery was monitored. Images at representative time points are shown. (D) Bleaching recovery kinetics is represented as the percentage of FRAP in patches. Data are representative of three individual experiments.
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fig8: The PLCγ1 binding site of LAT is important for the reduced mobility of LAT in aggregated rafts. (A) Schematic representations of the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP fusion proteins. A LAT-GFP chimera was constructed by attaching EGFP to the full length of mouse LAT. Y136 is a binding site for PLCγ1, and Y175, Y195, and Y235 are binding sites for Grb2/Gads in mouse LAT. Mutated amino acids are shown for each construct. (B) Association of LAT-GFP with PLCγ1 or Grb2. The LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were stimulated with OKT3 for 5 min. Cell lysates were immunoprecipitated with either anti-PLCγ1 or anti-Grb2 antibody, and immunoprecipitates were analyzed by immunoblotting with anti-GFP and antibodies specific for the immunoprecipitated protein. (C) After the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were mixed with anti-CD3 beads for 20 min, a selected area (2-μm square) on the patches was photobleached, and fluorescence recovery was monitored. Images at representative time points are shown. (D) Bleaching recovery kinetics is represented as the percentage of FRAP in patches. Data are representative of three individual experiments.

Mentions: After TCR engagement, LAT is tyrosine-phosphorylated by ZAP-70, creating docking sites for multiple downstream effector proteins. It has been shown that the distal four tyrosine residues of LAT bind PLCγ1, Grb2, and Gads, and these interactions are essential for TCR signal transduction (Zhang et al., 2000; Sommers et al., 2001). Next, we wanted to determine which molecular interactions influence LAT mobility in aggregated rafts. We established the following Jurkat-derived transfectants expressing different LAT-GFP mutants containing substitutions of critical tyrosines with phenylalanines in the LAT cytoplasmic domain (Fig. 8 A): LAT(Y136F)-GFP with a mutation at Tyr136, a site for PLCγ1 binding; LAT(3YF)-GFP with mutations at Tyr175, Tyr195, and Tyr235, sites for Grb2/Gads binding; and LAT(4YF)-GFP with mutations at the above four tyrosine residues that abolish both PLCγ1- and Grb2/Gads-binding. To confirm the association of LAT-GFP with PLCγ1 or Grb2, we activated the transfectants with OKT3, immunoprecipitated PLCγ1, or Grb2 with specific antibodies, and detected LAT-GFP association by anti-GFP blotting (Fig. 8 B). Although PLCγ1 and Grb2 were clearly associated with LAT-GFP in the wild-type LAT-GFP transfectant, mutations of the distal four tyrosines in the LAT(4YF)-GFP transfectant completely abolished these interactions. As expected, a mutation at Tyr136 abrogated PLCγ1 binding with LAT, but not Grb2 binding. In the LAT(3YF)-GFP mutant, the association of Grb2 with LAT was almost absent, and that of PLCγ1 with LAT was also undetectable (unpublished data). This is consistent with previous findings (Zhang et al., 2000), and we did not further analyze the LAT(3YF)-GFP transfectant because it was impossible to distinguish LAT(3YF)-GFP from LAT(4YF)-GFP in terms of protein interactions.


Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation.

Tanimura N, Nagafuku M, Minaki Y, Umeda Y, Hayashi F, Sakakura J, Kato A, Liddicoat DR, Ogata M, Hamaoka T, Kosugi A - J. Cell Biol. (2003)

The PLCγ1 binding site of LAT is important for the reduced mobility of LAT in aggregated rafts. (A) Schematic representations of the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP fusion proteins. A LAT-GFP chimera was constructed by attaching EGFP to the full length of mouse LAT. Y136 is a binding site for PLCγ1, and Y175, Y195, and Y235 are binding sites for Grb2/Gads in mouse LAT. Mutated amino acids are shown for each construct. (B) Association of LAT-GFP with PLCγ1 or Grb2. The LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were stimulated with OKT3 for 5 min. Cell lysates were immunoprecipitated with either anti-PLCγ1 or anti-Grb2 antibody, and immunoprecipitates were analyzed by immunoblotting with anti-GFP and antibodies specific for the immunoprecipitated protein. (C) After the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were mixed with anti-CD3 beads for 20 min, a selected area (2-μm square) on the patches was photobleached, and fluorescence recovery was monitored. Images at representative time points are shown. (D) Bleaching recovery kinetics is represented as the percentage of FRAP in patches. Data are representative of three individual experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172749&req=5

fig8: The PLCγ1 binding site of LAT is important for the reduced mobility of LAT in aggregated rafts. (A) Schematic representations of the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP fusion proteins. A LAT-GFP chimera was constructed by attaching EGFP to the full length of mouse LAT. Y136 is a binding site for PLCγ1, and Y175, Y195, and Y235 are binding sites for Grb2/Gads in mouse LAT. Mutated amino acids are shown for each construct. (B) Association of LAT-GFP with PLCγ1 or Grb2. The LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were stimulated with OKT3 for 5 min. Cell lysates were immunoprecipitated with either anti-PLCγ1 or anti-Grb2 antibody, and immunoprecipitates were analyzed by immunoblotting with anti-GFP and antibodies specific for the immunoprecipitated protein. (C) After the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were mixed with anti-CD3 beads for 20 min, a selected area (2-μm square) on the patches was photobleached, and fluorescence recovery was monitored. Images at representative time points are shown. (D) Bleaching recovery kinetics is represented as the percentage of FRAP in patches. Data are representative of three individual experiments.
Mentions: After TCR engagement, LAT is tyrosine-phosphorylated by ZAP-70, creating docking sites for multiple downstream effector proteins. It has been shown that the distal four tyrosine residues of LAT bind PLCγ1, Grb2, and Gads, and these interactions are essential for TCR signal transduction (Zhang et al., 2000; Sommers et al., 2001). Next, we wanted to determine which molecular interactions influence LAT mobility in aggregated rafts. We established the following Jurkat-derived transfectants expressing different LAT-GFP mutants containing substitutions of critical tyrosines with phenylalanines in the LAT cytoplasmic domain (Fig. 8 A): LAT(Y136F)-GFP with a mutation at Tyr136, a site for PLCγ1 binding; LAT(3YF)-GFP with mutations at Tyr175, Tyr195, and Tyr235, sites for Grb2/Gads binding; and LAT(4YF)-GFP with mutations at the above four tyrosine residues that abolish both PLCγ1- and Grb2/Gads-binding. To confirm the association of LAT-GFP with PLCγ1 or Grb2, we activated the transfectants with OKT3, immunoprecipitated PLCγ1, or Grb2 with specific antibodies, and detected LAT-GFP association by anti-GFP blotting (Fig. 8 B). Although PLCγ1 and Grb2 were clearly associated with LAT-GFP in the wild-type LAT-GFP transfectant, mutations of the distal four tyrosines in the LAT(4YF)-GFP transfectant completely abolished these interactions. As expected, a mutation at Tyr136 abrogated PLCγ1 binding with LAT, but not Grb2 binding. In the LAT(3YF)-GFP mutant, the association of Grb2 with LAT was almost absent, and that of PLCγ1 with LAT was also undetectable (unpublished data). This is consistent with previous findings (Zhang et al., 2000), and we did not further analyze the LAT(3YF)-GFP transfectant because it was impossible to distinguish LAT(3YF)-GFP from LAT(4YF)-GFP in terms of protein interactions.

Bottom Line: Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions.The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT.Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

View Article: PubMed Central - PubMed

Affiliation: School of Allied Health Sciences, Faculty of Medicine, Osaka University, Suita, Japan.

ABSTRACT
Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

Show MeSH
Related in: MedlinePlus