Lck Mediates Signal Transmission from CD59 to the TCR/CD3 Pathway in Jurkat T Cells |
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Authors: | Anna M Lipp Kata Juhasz Christian Paar Christoph Ogris Paul Eckerstorfer Roland Thuenauer Jan Hesse Benedikt Nimmervoll Hannes Stockinger Gerhard J Schütz Ulrich Bodenhofer Zsolt Balogi Alois Sonnleitner |
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Institution: | 1. Center for Advanced Bioanalysis GmbH, Linz, Austria.; 2. Institute for Bioinformatics, Johannes Kepler University Linz, Linz, Austria.; 3. Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University Vienna, Vienna, Austria.; 4. Institute of Applied Physics, Vienna University of Technology, Vienna, Austria.; University of New South Wales, Australia, |
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Abstract: | The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 has been implicated in the modulation of T cell responses, but the underlying molecular mechanism of CD59 influencing T cell signaling remained unclear. Here we analyzed Jurkat T cells stimulated via anti-CD3ε- or anti-CD59-coated surfaces, using time-resolved single-cell Ca2+ imaging as a read-out for stimulation. This analysis revealed a heterogeneous Ca2+ response of the cell population in a stimulus-dependent manner. Further analysis of T cell receptor (TCR)/CD3 deficient or overexpressing cells showed that CD59-mediated signaling is strongly dependent on TCR/CD3 surface expression. In protein co-patterning and fluorescence recovery after photobleaching experiments no direct physical interaction was observed between CD59 and CD3 at the plasma membrane upon anti-CD59 stimulation. However, siRNA-mediated protein knock-downs of downstream signaling molecules revealed that the Src family kinase Lck and the adaptor molecule linker of activated T cells (LAT) are essential for both signaling pathways. Furthermore, flow cytometry measurements showed that knock-down of Lck accelerates CD3 re-expression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3ζ completely abolished CD59-triggered Ca2+ signaling, while signaling was still functional upon direct TCR/CD3 stimulation. Altogether, we demonstrate that Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells, and propose that CD59 may act via Lck to modulate T cell responses. |
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