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The four distal tyrosines are required for LAT-dependent signaling in FcepsilonRI-mediated mast cell activation.

Saitoh S, Odom S, Gomez G, Sommers CL, Young HA, Rivera J, Samelson LE - J. Exp. Med. (2003)

Bottom Line: Examination of bone marrow-derived mast cells expressing various tyrosine to phenylalanine mutants in LAT demonstrates a differential requirement for these different binding sites.In these studies, assays of biochemical pathways, degranulation, and cytokine and chemokine release were performed.Deletion of all four distal tyrosines, and in particular, loss of the primary phospholipase C-gamma-binding tyrosine had a significant effect on antigen-induced histamine release.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.

ABSTRACT
The linker for activation of T cells (LAT) is an adaptor protein critical for Fc epsilon RI-mediated mast cell activation. LAT is a substrate of the tyrosine kinases activated after TCR and Fc epsilon RI engagement. After phosphorylation of the cytosolic domain of LAT, multiple signaling molecules such as phospholipase C-gamma1, Grb2, and Gads associate with phosphorylated LAT via their SH2 domains. The essential role of the four distal tyrosines in TCR-mediated signaling and T cell development has been demonstrated by experiments using LAT-deficient cell lines and genetically modified mice. To investigate the role of these four tyrosines of LAT in Fc epsilon RI-mediated mast cell activation, bone marrow-derived mast cells from LAT-deficient mice were infected with retroviral vectors designed to express wild-type or mutant LAT. Examination of bone marrow-derived mast cells expressing various tyrosine to phenylalanine mutants in LAT demonstrates a differential requirement for these different binding sites. In these studies, assays of biochemical pathways, degranulation, and cytokine and chemokine release were performed. Finally, the role of these tyrosines was also evaluated in vivo using genetically modified animals. Deletion of all four distal tyrosines, and in particular, loss of the primary phospholipase C-gamma-binding tyrosine had a significant effect on antigen-induced histamine release.

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MAP kinase activity in BMMCs after antigen stimulation. (A and B) ERK and JNK activities in BMMCs after antigen stimulation. BMMCs were sensitized with 1 μg/ml anti-DNP IgE and stimulated with 100 ng/ml DNP-HSA for 10 min. (A) JNK1 kinase activity was measured by in vitro kinase assay. (top) An autoradiograph of 32P-incorporated into c-Jun–GST after its phosphorylation by JNK1 immunoprecipitates from BMMCs. The same filter was probed with an anti-JNK1 antibody. (bottom) The JNK1 protein amount is shown. (B) ERK2 kinase activity was measured by in vitro kinase assay. (top) An autoradiograph of 32P-incorporated into myelin basic protein (MBP) after its phosphorylation by ERK2 immunoprecipitates from BMMCs. (bottom) The amount of ERK2 protein in the same cell lysates by anti-ERK2 immunoblot. (C) p38 phosphorylation in BMMCs after antigen stimulation. BMMCs were sensitized with 1 μg/ml anti-DNP IgE and stimulated with 100 ng/ml DNP-HSA for 5 and 10 min. Proteins in cell lysates from 2 × 105 BMMCs were separated by SDS-PAGE, and were transferred to nitrocellulose membrane filters. The membrane was immunoblotted with anti–phospho-p38 antibody. After stripping, the same membrane was immunoblotted with anti-p38. Representative data from three independent experiments are shown.
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fig6: MAP kinase activity in BMMCs after antigen stimulation. (A and B) ERK and JNK activities in BMMCs after antigen stimulation. BMMCs were sensitized with 1 μg/ml anti-DNP IgE and stimulated with 100 ng/ml DNP-HSA for 10 min. (A) JNK1 kinase activity was measured by in vitro kinase assay. (top) An autoradiograph of 32P-incorporated into c-Jun–GST after its phosphorylation by JNK1 immunoprecipitates from BMMCs. The same filter was probed with an anti-JNK1 antibody. (bottom) The JNK1 protein amount is shown. (B) ERK2 kinase activity was measured by in vitro kinase assay. (top) An autoradiograph of 32P-incorporated into myelin basic protein (MBP) after its phosphorylation by ERK2 immunoprecipitates from BMMCs. (bottom) The amount of ERK2 protein in the same cell lysates by anti-ERK2 immunoblot. (C) p38 phosphorylation in BMMCs after antigen stimulation. BMMCs were sensitized with 1 μg/ml anti-DNP IgE and stimulated with 100 ng/ml DNP-HSA for 5 and 10 min. Proteins in cell lysates from 2 × 105 BMMCs were separated by SDS-PAGE, and were transferred to nitrocellulose membrane filters. The membrane was immunoblotted with anti–phospho-p38 antibody. After stripping, the same membrane was immunoblotted with anti-p38. Representative data from three independent experiments are shown.

Mentions: MAP kinase activation is central to transcriptional activation of cytokines and chemokines in mast cells. We reported previously that mast cells deficient in LAT demonstrated impaired ERK and JNK activation (13). JNK1 activation in the reconstituted BMMCs was measured by in vitro kinase assay using c-Jun–GST as a substrate. As shown in Fig. 6 A, JNK1 activation in BMMCs expressing LAT-WT was robust and comparable or greater than in LAT+/+ BMMCs. JNK1 activation was comparable in cells expressing LAT-4YF and LAT−/− BMMCs. JNK1 activation was not fully reconstituted by expression of any of the four mutant LAT constructs. JNK1 activation in cells expressing LAT-2YF was decreased by 55–60% compared with those cells with LAT-WT. JNK1 activation in cells expressing LAT-1YF and LAT-3YF was decreased by ∼78%. JNK1 activation in BMMCs with LAT-4YF was decreased by 90%. These results demonstrate that the four distal tyrosines are critical for JNK1 activation and that both the Y136-binding site and the three Grb2-binding sites are essential for JNK1 activation after FcɛRI engagement.


The four distal tyrosines are required for LAT-dependent signaling in FcepsilonRI-mediated mast cell activation.

Saitoh S, Odom S, Gomez G, Sommers CL, Young HA, Rivera J, Samelson LE - J. Exp. Med. (2003)

MAP kinase activity in BMMCs after antigen stimulation. (A and B) ERK and JNK activities in BMMCs after antigen stimulation. BMMCs were sensitized with 1 μg/ml anti-DNP IgE and stimulated with 100 ng/ml DNP-HSA for 10 min. (A) JNK1 kinase activity was measured by in vitro kinase assay. (top) An autoradiograph of 32P-incorporated into c-Jun–GST after its phosphorylation by JNK1 immunoprecipitates from BMMCs. The same filter was probed with an anti-JNK1 antibody. (bottom) The JNK1 protein amount is shown. (B) ERK2 kinase activity was measured by in vitro kinase assay. (top) An autoradiograph of 32P-incorporated into myelin basic protein (MBP) after its phosphorylation by ERK2 immunoprecipitates from BMMCs. (bottom) The amount of ERK2 protein in the same cell lysates by anti-ERK2 immunoblot. (C) p38 phosphorylation in BMMCs after antigen stimulation. BMMCs were sensitized with 1 μg/ml anti-DNP IgE and stimulated with 100 ng/ml DNP-HSA for 5 and 10 min. Proteins in cell lysates from 2 × 105 BMMCs were separated by SDS-PAGE, and were transferred to nitrocellulose membrane filters. The membrane was immunoblotted with anti–phospho-p38 antibody. After stripping, the same membrane was immunoblotted with anti-p38. Representative data from three independent experiments are shown.
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Related In: Results  -  Collection

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fig6: MAP kinase activity in BMMCs after antigen stimulation. (A and B) ERK and JNK activities in BMMCs after antigen stimulation. BMMCs were sensitized with 1 μg/ml anti-DNP IgE and stimulated with 100 ng/ml DNP-HSA for 10 min. (A) JNK1 kinase activity was measured by in vitro kinase assay. (top) An autoradiograph of 32P-incorporated into c-Jun–GST after its phosphorylation by JNK1 immunoprecipitates from BMMCs. The same filter was probed with an anti-JNK1 antibody. (bottom) The JNK1 protein amount is shown. (B) ERK2 kinase activity was measured by in vitro kinase assay. (top) An autoradiograph of 32P-incorporated into myelin basic protein (MBP) after its phosphorylation by ERK2 immunoprecipitates from BMMCs. (bottom) The amount of ERK2 protein in the same cell lysates by anti-ERK2 immunoblot. (C) p38 phosphorylation in BMMCs after antigen stimulation. BMMCs were sensitized with 1 μg/ml anti-DNP IgE and stimulated with 100 ng/ml DNP-HSA for 5 and 10 min. Proteins in cell lysates from 2 × 105 BMMCs were separated by SDS-PAGE, and were transferred to nitrocellulose membrane filters. The membrane was immunoblotted with anti–phospho-p38 antibody. After stripping, the same membrane was immunoblotted with anti-p38. Representative data from three independent experiments are shown.
Mentions: MAP kinase activation is central to transcriptional activation of cytokines and chemokines in mast cells. We reported previously that mast cells deficient in LAT demonstrated impaired ERK and JNK activation (13). JNK1 activation in the reconstituted BMMCs was measured by in vitro kinase assay using c-Jun–GST as a substrate. As shown in Fig. 6 A, JNK1 activation in BMMCs expressing LAT-WT was robust and comparable or greater than in LAT+/+ BMMCs. JNK1 activation was comparable in cells expressing LAT-4YF and LAT−/− BMMCs. JNK1 activation was not fully reconstituted by expression of any of the four mutant LAT constructs. JNK1 activation in cells expressing LAT-2YF was decreased by 55–60% compared with those cells with LAT-WT. JNK1 activation in cells expressing LAT-1YF and LAT-3YF was decreased by ∼78%. JNK1 activation in BMMCs with LAT-4YF was decreased by 90%. These results demonstrate that the four distal tyrosines are critical for JNK1 activation and that both the Y136-binding site and the three Grb2-binding sites are essential for JNK1 activation after FcɛRI engagement.

Bottom Line: Examination of bone marrow-derived mast cells expressing various tyrosine to phenylalanine mutants in LAT demonstrates a differential requirement for these different binding sites.In these studies, assays of biochemical pathways, degranulation, and cytokine and chemokine release were performed.Deletion of all four distal tyrosines, and in particular, loss of the primary phospholipase C-gamma-binding tyrosine had a significant effect on antigen-induced histamine release.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.

ABSTRACT
The linker for activation of T cells (LAT) is an adaptor protein critical for Fc epsilon RI-mediated mast cell activation. LAT is a substrate of the tyrosine kinases activated after TCR and Fc epsilon RI engagement. After phosphorylation of the cytosolic domain of LAT, multiple signaling molecules such as phospholipase C-gamma1, Grb2, and Gads associate with phosphorylated LAT via their SH2 domains. The essential role of the four distal tyrosines in TCR-mediated signaling and T cell development has been demonstrated by experiments using LAT-deficient cell lines and genetically modified mice. To investigate the role of these four tyrosines of LAT in Fc epsilon RI-mediated mast cell activation, bone marrow-derived mast cells from LAT-deficient mice were infected with retroviral vectors designed to express wild-type or mutant LAT. Examination of bone marrow-derived mast cells expressing various tyrosine to phenylalanine mutants in LAT demonstrates a differential requirement for these different binding sites. In these studies, assays of biochemical pathways, degranulation, and cytokine and chemokine release were performed. Finally, the role of these tyrosines was also evaluated in vivo using genetically modified animals. Deletion of all four distal tyrosines, and in particular, loss of the primary phospholipase C-gamma-binding tyrosine had a significant effect on antigen-induced histamine release.

Show MeSH