Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.
T lymphocytes expressing the γδ T cell receptor (γδ TCR) can recognize antigens expressed by tumor cells and subsequently kill these cells. γδ T cells are indeed used in cancer immunotherapy clinical trials. The anti-CD3ε antibody UCHT1 enhanced the in vitro tumor killing activity of human γδ T cells by an unknown molecular mechanism. Here, we demonstrate that Fab fragments of UCHT1, which only bind monovalently to the γδ TCR, also enhanced tumor killing by expanded human Vγ9Vδ2 γδ T cells or pan-γδ T cells of the peripheral blood. The Fab fragments induced Nck recruitment to the γδ TCR, suggesting that they stabilized the γδ TCR in an active CD3ε conformation. However, blocking the Nck-CD3ε interaction in γδ T cells using the small molecule inhibitor AX-024 neither reduced the γδ T cells’ natural nor the Fab-enhanced tumor killing activity. Likewise, Nck recruitment to CD3ε was not required for intracellular signaling, CD69 and CD25 up-regulation, or cytokine secretion by γδ T cells. Thus, the Nck-CD3ε interaction seems to be dispensable in γδ T cells.
Signal transduction regulates the proper function of T cells in an immune response. Upon binding to its specific ligand associated with major histocompatibility complex (MHC) molecules on an antigen presenting cell, the T cell receptor (TCR) initiates intracellular signaling that leads to extensive actin polymerization. Wiskott-Aldrich syndrome protein (WASp) is one of the actin nucleation factors that is recruited to TCR microclusters, where it is activated and regulates actin network formation. Here we highlight the research that has focused on WASp-deficient T cells from both human and mice in TCR-mediated signal transduction. We discuss the role of WASp in proximal TCR signaling as well as in the Ras/Rac-MAPK (mitogen-activated protein kinase), PKC (protein kinase C) and Ca2+-mediated signaling pathways.
Following T-cell antigen receptor (TCR) engagement, rearrangement of the actin cytoskeleton supports intracellular signal transduction and T-cell activation. The non-catalytic region of the tyrosine kinase (Nck) molecule is an adapter protein implicated in TCR-induced actin polymerization. Further, Nck is recruited to the CD3e subunit of the TCR upon TCR triggering. Here we examine the role of actin polymerization in the recruitment of Nck to the TCR. To this end, Nck binding to CD3e was quantified in Jurkat cells using the proximity ligation assay. We show that inhibition of actin polymerization using cytochalasin D delayed the recruitment of Nck1 to the TCR upon TCR triggering. Interestingly, CD3e phosphorylation was also delayed. These findings suggest that actin polymerization promotes the recruitment of Nck to the TCR, enhancing downstream signaling, such as phosphorylation of CD3e.Abbreviations: CD3e, cluster of differentiation 3e; CREB, cyclic AMP-response element binding protein; CytD, cytochalasin D; Erk, extracellular signal-regulated kinase; F-actin, filamentous actin; LAT, linker of activated T cells; Lck, lymphocyte-specific protein tyrosine kinase; MHC, major histocompatibility complex; Nck, non-catalytic region of tyrosine kinase ; PLA, proximity ligation assay; PRS, proline-rich sequence; SH domain, Src homology domain; SLP-76, SH domain containing leukocyte protein of 76 000 MW; TCR, T-cell receptor; WAsP, Wiskott--Aldrich syndrome protein; ZAP70, f chain-associated protein kinase of 70 000 MW ª
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