Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells.
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 Ca(2+) imaging as a read-out for stimulation. This analysis revealed a heterogeneous Ca(2+) 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 Ca(2+) 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.
Center for Advanced Bioanalysis GmbH, Linz, Austria.
Anna M
AM
Lipp
Center for Advanced Bioanalysis GmbH, Linz, Austria.
Kata
K
Juhasz
Center for Advanced Bioanalysis GmbH, Linz, Austria.
Christian
C
Paar
Institute for Bioinformatics, Johannes Kepler University Linz, Linz, Austria.
Christoph
C
Ogris
Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University Vienna, Vienna, Austria.
Paul
P
Eckerstorfer
Center for Advanced Bioanalysis GmbH, Linz, Austria.
Roland
R
Thuenauer
Center for Advanced Bioanalysis GmbH, Linz, Austria.
Jan
J
Hesse
Center for Advanced Bioanalysis GmbH, Linz, Austria.
Benedikt
B
Nimmervoll
Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University Vienna, Vienna, Austria.
Hannes
H
Stockinger
Institute of Applied Physics, Vienna University of Technology, Vienna, Austria.
Gerhard J
GJ
Schütz
Institute for Bioinformatics, Johannes Kepler University Linz, Linz, Austria.
Ulrich
U
Bodenhofer
Center for Advanced Bioanalysis GmbH, Linz, Austria.
Zsolt
Z
Balogi
Center for Advanced Bioanalysis GmbH, Linz, Austria.
Alois
A
Sonnleitner
D017252:CD3 Complex / Q000378:metabolism*; D018957:CD59 Antigens / Q000378:metabolism*; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000502:physiology*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*
D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D018957:CD59 Antigens; D011948:Receptors, Antigen, T-Cell; C051590:CD59 protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)