Docking Protein Gab2 Is Phosphorylated by ZAP-70 and Negatively Regulates T Cell Receptor Signaling by Recruitment of Inhibitory Molecules

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 CD3, 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. T cells recognize antigens (Ag)1 presented by the major histocompatibility complex on antigen-presenting cells (APC). An Ag recognition signal is then transmitted to the cytoplasm via the T cell antigen receptor (TCR) complex and induces T cell activation, leading to various cellular events such as proliferation, apoptosis, anergy, and differentiation as well as a variety of effector functions.To evoke appropriate T cell responses and maintain immune equilibrium, these stimulatory signals must be regulated by negative signals delivered via inhibitory molecules (1). In contrast to positive regulation, however, the precise mechanism of negative regulation of T cells has not as yet been widely characterized. In B cells and NK cells, several inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motif such as Fc␥RIIB, CD22, CD72, killer inhibitory receptor, and Ly-49 have been reported (2). Upon stimulation by ligand binding, these inhibitory receptors become tyrosine-phosphorylated and recruit Src homology 2 (SH2) domain-containing tyrosine phosphatases (SHP-1 and SHP-2) and/or Src homology 2 domain-containing inositol-5-phosphatase, resulting in the inhibition of antigen receptor-mediated effector function and cytokine expression. In T cells, it has been well established that CTLA-4 and PD-1 are induced upon T cell activation and function as an inhibitory co-receptor at a late activation phase (3, 4). However, there has been little investigation of the inhibitory machineries through typical inhibitory receptors in T cells, suggesting the possibility that another inhibitory...

Abnormal hematopoiesis in Gab2 mutant mice

“…34 In contrast, Gab2 recruits negative regulators of PI-3K signaling downstream of the T-cell receptor. 35,36 While we did not find evidence for a negative regulatory function in lymphocyte development from transplanted HSCs, our experiments did not explore T-or B-cell receptor signaling in detail. It is possible that Gab2 mediates a balance between positive and negative signals in hematopoiesis; this may also account for the milder phenotypes overall.…”

Adaptive Tolerance and Clonal Anergy Are Distinct Biochemical States

“…In a third potential mechanism, the docking protein Gab2 has been shown to recruit the phosphatase SHP-2 to the TCR complex in activated cells. This requires Zap70 phosphorylation of LAT and Gab2 to initiate and, thus, could represent a negative feedback loop on the activity of Zap70 (46). By a different mechanism of negative feedback, the Cbl molecules c-Cbl and Cbl-b have been implicated in down-regulating TCR signaling through E3 ligase ubiquitination (47,48).…”

CEACAM1 Dynamics during Neisseria gonorrhoeae Suppression of CD4+ T Lymphocyte Activation

“…50), and B and T lymphocyte attenuator (BTLA; Ref 51). Because SHP-2 has been implicated in both inhibitory (52,53) and stimulatory (54,55) signaling cascades, its contribution to CEACAM1-dependent effects must still be defined.…”