Docking protein Gab2 is phosphorylated by ZAP-70 and negatively regulates T cell receptor signaling by recruitment of inhibitory molecules

To maintain various T cell responses and immune equilibrium, activation sig nals triggered by T cell antigen receptor (TCR) must be regulated by inhibi tory signals. Gab2, an adaptor protein of the insulin receptor substrate-1 family, has been shown to be involved in the downstream signaling from cyto kine 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 CD3 zeta, upon TCR stimulat ion. Overexpression of Gab2 in Jurkat cells or antigen-specific T cell hybr idomas resulted in the inhibition of NF-AT activation, interleukin-2 produc tion, and tyrosine phosphorylation. The structure-function relationship of Gab2 was analyzed by mutants of Gab2. The Gab2 mutants lacking SHP-2-bindin g sites mostly abrogated the inhibitory activity of Gab2, but its inhibitor y function was restored by fusing to active SHP-2 as a chimeric protein. A mutant with defective phosphatidylinositol 3-kinase binding capacity also i mpaired the inhibitory activity, and the pleckstrin homology domain-deletio n mutant revealed a crucial function of the pleckstrin homology domain for localization to the plasma membrane. These results suggest that Gab2 is a s ubstrate of ZAP-70 and functions as a switch molecule toward inhibition of TCR signal transduction by mediating the recruitment of inhibitory molecule s to the TCR signaling complex.