TY - JOUR
T1 - Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells
AU - Lipp, Anna M.
AU - Juhasz, Kata
AU - Paar, Christian
AU - Ogris, Christoph
AU - Eckerstorfer, Paul
AU - Thuenauer, Roland
AU - Hesse, Jan
AU - Nimmervoll, Benedikt
AU - Stockinger, Hannes
AU - Schütz, Gerhard J.
AU - Bodenhofer, Ulrich
AU - Balogi, Zsolt
AU - Sonnleitner, Alois
PY - 2014/1/15
Y1 - 2014/1/15
N2 - 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 reexpression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3f 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. Copyright:
AB - 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 reexpression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3f 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. Copyright:
KW - CD3 Complex/metabolism
KW - CD59 Antigens/metabolism
KW - Calcium Signaling
KW - Cell Membrane/metabolism
KW - Humans
KW - Jurkat Cells
KW - Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/physiology
KW - Receptors, Antigen, T-Cell/metabolism
UR - http://www.scopus.com/inward/record.url?scp=84898635046&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0085934
DO - 10.1371/journal.pone.0085934
M3 - Article
C2 - 24454946
AN - SCOPUS:84898635046
SN - 1932-6203
VL - 9
JO - PLoS ONE
JF - PLoS ONE
IS - 1
M1 - e85934
ER -