Requirement of the Src Homology 2 Domain Protein Shb for T Cell Receptor-dependent Activation of the Interleukin-2 Gene Nuclear Factor for Activation of T Cells Element in Jurkat T Cells

Abstract

Stimulation of the T cell antigen receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins. We have recently investigated the role of the adaptor protein Shb in the early events of T cell signaling and observed that Shb associates with Grb2, linker for activation of T cells (LAT) and the TCR -chain in Jurkat cells. We now report that Shb also associates with phospholipase C-␥1 (PLC-␥1) in these cells. Overexpression of Src homology 2 domain defective Shb caused diminished phosphorylation of LAT and consequently the activation of mitogen-activated protein kinases was decreased upon TCR stimulation. In addition, the Shb mutant also blocked phosphorylation of PLC-␥1 and the increase in cytoplasmic Ca 2؉ following TCR stimulation. Nuclear factor for activation of T cells is a major target for Ras and calcium signaling pathways in T cells following TCR stimulation, and the overexpression of the mutant Shb prevented TCR-dependent activation of the nuclear factor for activation of T cells. Consequently, endogenous interleukin-2 production was decreased under these conditions. The results indicate a role for Shb as a link between the TCR and downstream signaling events involving LAT and PLC-␥1 and resulting in the activation of transcription of the interleukin-2 gene.The T cell response is initiated by the presentation of an antigen to the T cell receptor (TCR).1 This is followed by rapid phosphorylation of tyrosines on both the receptor itself and cytoplasmic proteins, the initiation of several signaling cascades, and subsequently the activation of interleukin-2 (IL-2) gene transcription and other T cell immune functions. The early events in T cell signaling involve the activation of several protein tyrosine kinases. After TCR -chain phosphorylation (by the protein tyrosine kinases Lck or Fyn), a Syk family protein tyrosine kinase, ZAP70, binds the chain immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex (1) and consequently becomes phosphorylated and activated. Active ZAP70 is then responsible for phosphorylation and activation of many additional substrates: for example, phospholipase C-␥1 (PLC-␥1), p36/38 linker for activation of T cells (LAT) (2), the guanine nucleotide exchange factor Vav, and the adaptor protein SLP-76 (3). In the course of these events, PLC-␥1, Grb2, and phosphoinositide 3-kinase all associate with tyrosine-phosphorylated p36/38 LAT (4 -7). Phosphorylation of PLC-␥1 activates this enzyme, which then hydrolyzes phosphatidylinositol phosphate to yield diacylglycerol and inositol 1,4,5-tris-phosphate, the latter messenger mobilizing intracellular Ca 2ϩ . The resulting increase in cytoplasmic Ca 2ϩ activates calcineurin, which mediates nuclear translocation and activation of the T cell-specific transcription factor nuclear factor for activation of T cells (NFAT) (8).Activation of the Ras signaling pathway in response to TCR stimulation involves p36/38 LAT, Grb2 and Sos (5). As a consequence, mitogen-activated protein (MAP) kinases are stimulated, caus...

Role of Tyrosine Kinase Signaling for β-Cell Replication and Survival

“…Besides affecting cell survival, Shb overexpression has been shown to enhance NGF-and FGF-2-stimulated neurite outgrowth in the phaeochromocytoma cell line PC12 (9). In the Jurkat T lymphocyte cell line, a functional Shb molecule was required for T cell receptor-dependent stimulation of IL-2 production (15). Shb thus appears to be a versatile signaling protein that interacts with numerous receptors and transmits different signals depending on the stimulus and the responsive cell.…”

“…Shb has the characteristic features of proline-rich sequences in its N-terminus, a central PTB (phosphotyrosine binding) domain, several potential tyrosine phosphorylation sites and a C-terminal SH2 (Src homology 2) domain (22,23). The proline-rich motifs interact with the SH3-domain proteins Src, Eps8, p85 PI3-kinase, Grb2 and PLCy (phospholipase C) (7,15,23), whereas the PTB domain binds p36/38 LAT (15,23). The SH2 domain has been found to associate with the PDGF receptors, the FGFR-1 (7) and the T cell receptor (23).…”

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

“…This interaction is crucial for adhesion and integrin function in T cells (Baker et al 2009;Horn et al 2009;Wang et al 2009). Tyrosine phosphorylated Shb also binds the SH2 domain of SLP-76 and promotes LAT, SLP-76, Vav, and PLC-g1 phosphorylation (Lindholm et al 1999;Lindholm et al 2002). HPK1 is another molecule that binds the SH2 domain of SLP-76 but mediates negative signal transduction.…”

The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

“…Since focal adhesion kinase (FAK) has been shown to regulate Rac activity [12], this signaling intermediate may also be of significance in this context. Shb is a widely expressed Src homology-2 domain containing adaptor protein [13,14] established as a regulator of reproduction [15], glucose homeostasis [16], angiogenesis [17] and T cell function [14,18,19]. Notably, Shb has also been found to regulate the cell cycle in the pancreas and blood system in a cell type dependent manner.…”

“…The Cterminal SH2-domain of Shb [20] facilitates interactions with receptor tyrosine kinases such as vascular endothelial growth factor receptor-2 (VEGFR-2) [21], platelet derived growth factor receptor (PDGFR) [22] and the T cell receptor (TCR) [18,23]. Moreover, Shb associates via its other domains with additional signaling elements such as SH2 domaincontaining leukocyte protein of 76 kDa (Slp76) [23], Vav1 [23], c-Abl [24], FAK [17], phospholipase C-γ (PLCγ), Src, and phosphatidylinositol-3 kinase (PI3K), acting as a scaffold in signaling cascades [20].…”