Abstract
To maintain various T cell responses and immune equilibrium, activation signals triggered by T cell antigen receptor (TCR) must be regulated by inhibitory signals. Gab2, an adaptor protein of the insulin receptor substrate-1 family, has been shown to be involved in the downstream signaling from cytokine receptors. We investigated the functional role of Gab2 in TCR-mediated signal transduction. Gab2 was phosphorylated by ZAP-70 and co-precipitated with phosphoproteins, such as ZAP-70, LAT, and CD3zeta, upon TCR stimulation. Overexpression of Gab2 in Jurkat cells or antigen-specific T cell hybridomas resulted in the inhibition of NF-AT activation, interleukin-2 production, and tyrosine phosphorylation. The structure-function relationship of Gab2 was analyzed by mutants of Gab2. The Gab2 mutants lacking SHP-2-binding sites mostly abrogated the inhibitory activity of Gab2, but its inhibitory function was restored by fusing to active SHP-2 as a chimeric protein. A mutant with defective phosphatidylinositol 3-kinase binding capacity also impaired the inhibitory activity, and the pleckstrin homology domain-deletion mutant revealed a crucial function of the pleckstrin homology domain for localization to the plasma membrane. These results suggest that Gab2 is a substrate of ZAP-70 and functions as a switch molecule toward inhibition of TCR signal transduction by mediating the recruitment of inhibitory molecules to the TCR signaling complex.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Animals
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Antigens, CD / biosynthesis
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Antigens, Differentiation, T-Lymphocyte / biosynthesis
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Binding Sites
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Blotting, Western
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CD3 Complex / metabolism
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Carrier Proteins / metabolism
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Cell Line
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Cytokines / metabolism
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DNA / metabolism
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Dose-Response Relationship, Drug
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Flow Cytometry
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Humans
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Hybridomas / metabolism
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Interleukin-2 / biosynthesis
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Intracellular Signaling Peptides and Proteins
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Jurkat Cells
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Lectins, C-Type
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Luciferases / metabolism
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Lymphocyte Activation
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Membrane Proteins*
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Mice
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Mutation
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphoproteins / metabolism*
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Phosphorylation*
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Precipitin Tests
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Protein Binding
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Protein Structure, Tertiary
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Protein Tyrosine Phosphatase, Non-Receptor Type 11
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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Protein Tyrosine Phosphatases / metabolism
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Protein-Tyrosine Kinases / metabolism*
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Receptors, Antigen, T-Cell / metabolism*
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SH2 Domain-Containing Protein Tyrosine Phosphatases
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Signal Transduction*
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Structure-Activity Relationship
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Transfection
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Tyrosine / metabolism
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ZAP-70 Protein-Tyrosine Kinase
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src Homology Domains
Substances
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Adaptor Proteins, Signal Transducing
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Antigens, CD
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Antigens, Differentiation, T-Lymphocyte
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CD3 Complex
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CD3 antigen, zeta chain
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CD69 antigen
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Carrier Proteins
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Cytokines
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GAB2 protein, human
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Gab2 protein, mouse
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Interleukin-2
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Intracellular Signaling Peptides and Proteins
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LAT protein, human
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Lat protein, mouse
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Lectins, C-Type
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Membrane Proteins
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Phosphoproteins
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Receptors, Antigen, T-Cell
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Tyrosine
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DNA
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Luciferases
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Phosphatidylinositol 3-Kinases
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Protein-Tyrosine Kinases
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ZAP-70 Protein-Tyrosine Kinase
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ZAP70 protein, human
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Zap70 protein, mouse
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PTPN11 protein, human
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PTPN6 protein, human
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Protein Tyrosine Phosphatase, Non-Receptor Type 11
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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Protein Tyrosine Phosphatases
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Ptpn11 protein, mouse
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Ptpn6 protein, mouse
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SH2 Domain-Containing Protein Tyrosine Phosphatases