A genetically selective inhibitor demonstrates a function for the kinase Zap70 in regulatory T cells independent of its catalytic activity.
ABSTRACT To investigate the role of the kinase Zap70 in T cells, we generated mice expressing a Zap70 mutant whose catalytic activity can be selectively blocked by a small-molecule inhibitor. We found that conventional naive, effector and memory T cells were dependent on the kinase activity of Zap70 for their activation, which demonstrated a nonredundant role for Zap70 in signals induced by the T cell antigen receptor (TCR). In contrast, the catalytic activity of Zap70 was not required for activation of the GTPase Rap1 and inside-out signals that promote integrin adhesion. This Zap70 kinase-independent pathway was sufficient for the suppressive activity of regulatory T cells (T(reg) cells), which was unperturbed by inhibition of the catalytic activity of Zap70. Our results indicate Zap70 is a likely therapeutic target.
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ABSTRACT: The interaction of CLEC-2 on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signalling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signalling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the centre of the platelet to form a single structure. Fluorescence life-time imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilised Podoplanin using direct stochastic optical reconstruction microscopy (dSTORM). These findings provide mechanistic insight by which CLEC-2 signalling promotes adhesion to Podoplanin and regulation of Podoplanin signalling thereby contributing to lymphatic vasculature development.Journal of Biological Chemistry 11/2014; 289(52). DOI:10.1074/jbc.M114.584284 · 4.60 Impact Factor
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ABSTRACT: T-cell antigen receptor (TCR) signaling is essential for activation, proliferation, and effector function of T cells. Modulation of both intensity and duration of TCR signaling can regulate these events. However, it remains unclear how individual T cells integrate such signals over time to make critical cell-fate decisions. We have previously developed an engineered mutant allele of the critical T-cell kinase zeta-chain-associated protein kinase 70 kDa (Zap70) that is catalytically inhibited by a small molecule inhibitor, thereby blocking TCR signaling specifically and efficiently. We have also characterized a fluorescent reporter Nur77-eGFP transgenic mouse line in which T cells up-regulate GFP uniquely in response to TCR stimulation. The combination of these technologies unmasked a sharp TCR signaling threshold for commitment to cell division both in vitro and in vivo. Further, we demonstrate that this threshold is independent of both the magnitude of the TCR stimulus and Interleukin 2. Similarly, we identify a temporal threshold of TCR signaling that is required for commitment to proliferation, after which T cells are able to proliferate in a Zap70 kinase-independent manner. Taken together, our studies reveal a sharp threshold for the magnitude and duration of TCR signaling required for commitment of T cells to proliferation. These results have important implications for understanding T-cell responses to infection and optimizing strategies for immunomodulatory drug delivery.Proceedings of the National Academy of Sciences 08/2014; 111(35). DOI:10.1073/pnas.1413726111 · 9.81 Impact Factor
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ABSTRACT: Foxp3(+) regulatory T cells (Treg cells) maintain immunological tolerance, and their deficiency results in fatal multiorgan autoimmunity. Although heightened signaling via the T cell antigen receptor (TCR) is critical for the differentiation of Treg cells, the role of TCR signaling in Treg cell function remains largely unknown. Here we demonstrated that inducible ablation of the TCR resulted in Treg cell dysfunction that could not be attributed to impaired expression of the transcription factor Foxp3, decreased expression of Treg cell signature genes or altered ability to sense and consume interleukin 2 (IL-2). Instead, TCR signaling was required for maintaining the expression of a limited subset of genes comprising 25% of the activated Treg cell transcriptional signature. Our results reveal a critical role for the TCR in the suppressor capacity of Treg cells.Nature Immunology 09/2014; 15(11). DOI:10.1038/ni.3004 · 24.97 Impact Factor