ZAP-70 Is Essential for the T Cell Antigen Receptor-induced Plasma Membrane Targeting of SOS and Vav in T Cells

ABSTRACT The Csk tyrosine kinase negatively regulates the Src family kinases Lck and Fyn in T cells. Engagement of the T-cell antigen receptor results in a removal of Csk from the lipid raft-associated transmembrane protein PAG/Cbp. Instead, Csk becomes associated with an ∼72-kDa tyrosine-phosphorylated protein, which we identify here as G3BP, a phosphoprotein reported to bind the SH3 domain of Ras GTPase-activating protein. G3BP reduced the ability of Csk to phosphorylate Lck at Y505 by decreasing the amount of Csk in lipid rafts. As a consequence, G3BP augmented T-cell activation as measured by interleukin-2 gene activation. Conversely, elimination of endogenous G3BP by RNA interference increased Lck Y505 phosphorylation and reduced TCR signaling. In antigen-specific T cells, endogenous G3BP moved into a intracellular location adjacent to the immune synapse, but deeper inside the cell, upon antigen recognition. Csk colocalization with G3BP occurred in this “parasynaptic” location. We conclude that G3BP is a new player in T-cell-antigen receptor signaling and acts to reduce the amount of Csk in the immune synapse.

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Susceptibility to cell death is a dominant phenotype: triggering of activation-driven T-cell death independent of the T-cell antigen receptor complex

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.379-385.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 379-385

Author(s):

G Nickas ◽

J Meyers ◽

L D Hebshi ◽

J D Ashwell ◽

D P Gold ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Interleukin 2 ◽

Antigen Receptor ◽

Alternative Activation ◽

Receptor Complex ◽

T Cell Antigen Receptor ◽

Cell Antigen Receptor ◽

Cell Antigen

The failure of Thy-1 and Ly-6 to trigger interleukin-2 production in the absence of surface T-cell antigen receptor complex (TCR) expression has been interpreted to suggest that functional signalling via these phosphatidylinositol-linked alternative activation molecules is dependent on the TCR. We find, in contrast, that stimulation of T cells via Thy-1 or Ly-6 in the absence of TCR expression does trigger a biological response, the cell suicide process of activation-driven cell death. Activation-driven cell death is a process of physiological cell death that likely represents the mechanism of negative selection of T cells. The absence of the TCR further reveals that signalling leading to activation-driven cell death and to lymphokine production are distinct and dissociable. In turn, the ability of alternative activation molecules to function in the absence of the TCR raises another issue: why immature T cells, thymomas, and hybrids fail to undergo activation-driven cell death in response to stimulation via Thy-1 and Ly-6. One possibility is that these activation molecules on immature T cells are defective. Alternatively, susceptibility to activation-driven cell death may be developmentally regulated by TCR-independent factors. We have explored these possibilities with somatic cell hybrids between mature and immature T cells, in which Thy-1 and Ly-6 are contributed exclusively by the immature partner. The hybrid cells exhibit sensitivity to activation-driven cell death triggered via Thy-1 and Ly-6. Thus, the Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 triggering, the mature phenotype of sensitivity to cell death is genetically dominant.

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Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817142115 ◽

2018 ◽

Vol 115 (51) ◽

pp. E11914-E11923 ◽

Cited By ~ 7

Author(s):

Asit Manna ◽

Huaying Zhao ◽

Junya Wada ◽

Lakshmi Balagopalan ◽

Harichandra D. Tagad ◽

...

Keyword(s):

Plasma Membrane ◽

T Cell ◽

Protein Complex ◽

Antigen Receptor ◽

Receptor Activation ◽

T Cell Antigen Receptor ◽

Cell Antigen Receptor ◽

Cell Antigen ◽

Phospholipase Cγ1 ◽

Quaternary Complex

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy–entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.

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Rosmarinic acid inhibits Ca2+-dependent pathways of T-cell antigen receptor-mediated signaling by inhibiting the PLC-γ1 and Itk activity

Blood ◽

10.1182/blood-2002-07-1992 ◽

2003 ◽

Vol 101 (9) ◽

pp. 3534-3542 ◽

Cited By ~ 30

Author(s):

Mi-Ae Kang ◽

Su-Young Yun ◽

Jonghwa Won

Keyword(s):

T Cells ◽

T Cell ◽

Signaling Pathways ◽

Tyrosine Phosphorylation ◽

Rosmarinic Acid ◽

Antigen Receptor ◽

Tcr Signaling ◽

T Cell Antigen Receptor ◽

Cell Antigen Receptor ◽

Cell Antigen

Rosmarinic acid (RosA) is a hydroxylated compound frequently found in herbal plants and is mostly responsible for anti-inflammatory and antioxidative activity. Previously, we observed that RosA inhibited T-cell antigen receptor (TCR)– induced interleukin 2 (IL-2) expression and subsequent T-cell proliferation in vitro. In this study, we investigated in detail inhibitory mechanism of RosA on TCR signaling, which ultimately activates IL-2 promoter by activating transcription factors, such as nuclear factor of activated T cells (NF-AT) and activating protein-1 (AP-1). Interestingly, RosA inhibited NF-AT activation but not AP-1, suggesting that RosA inhibits Ca2+- dependent signaling pathways only. Signaling events upstream of NF-AT activation, such as the generation of inositol 1,4,5-triphosphate and Ca2+ mobilization, and tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) were strongly inhibited by RosA. Tyrosine phosphorylation of PLC-γ1 is largely dependent on 3 kinds of protein tyrosine kinases (PTKs), ie, Lck, ZAP-70, and Itk. We found that RosA efficiently inhibited TCR-induced tyrosine phosphorylation and subsequent activation of Itk but did not inhibit Lck or ZAP-70. ZAP-70–dependent signaling pathways such as the tyrosine phosphorylation of LAT and SLP-76 and serine/threonine phosphorylation of mitogen-activated protein kinases (MAPKs) were intact in the presence of RosA, confirming that RosA suppresses TCR signaling in a ZAP-70–independent manner. Therefore, we conclude that RosA inhibits TCR signaling leading to Ca2+ mobilization and NF-AT activation by blocking membrane-proximal events, specifically, the tyrosine phosphorylation of inducible T cells kinase (Itk) and PLC-γ1.

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The role of Raf-1 in the regulation of extracellular signal-regulated kinase 2 by the T cell antigen receptor.

Journal of Experimental Medicine ◽

10.1084/jem.180.1.401 ◽

1994 ◽

Vol 180 (1) ◽

pp. 401-406 ◽

Cited By ~ 43

Author(s):

M Izquierdo ◽

S Bowden ◽

D Cantrell

Keyword(s):

T Cells ◽

T Cell ◽

Map Kinases ◽

Interleukin 2 ◽

Antigen Receptor ◽

T Cell Antigen Receptor ◽

Cell Antigen Receptor ◽

Cell Antigen ◽

Constitutively Active

Triggering of the T cell antigen receptor (TCR) complex activates the serine/threonine kinase Raf-1 whose function is necessary for TCR induction of the interleukin 2 gene. Raf-1 has been identified as a candidate mitogen-activated protein (MAP) kinase kinase kinase (MKKK) and thus has the potential to couple the TCR to the activation of the MAP kinases such as ERK2. In the present study, the role of Raf-1 in ERK2 regulation of ERK2 in T cells has been explored. A constitutively active Raf-1 kinase, v-raf, or a dominant inhibitory Raf-1 mutant were expressed transiently from the pEF BOS vector in Jurkat cells and the effects of these Raf-1 mutants on a coexpressed ERK2 reporter was assessed. The action of the constitutively active Raf-1 was to stimulate the ERK2 kinase, whereas the dominant negative version of Raf-1 inhibited the ERK2 activation induced by triggering of the TCR. These data indicate a role for Raf-1 in the regulation of ERK2 in T cells.

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