Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

IA Yudushkin, RD Vale - Proceedings of the National …, 2010 - National Acad Sciences
IA Yudushkin, RD Vale
Proceedings of the National Academy of Sciences, 2010National Acad Sciences
Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-
induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-
specific antibodies, which precludes dynamic measurements. Here, we have developed
genetically encoded, live-cell reporters that enable simultaneous monitoring of the
phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing
subunit of the TcR/CD3. We show that these reporters provide accurate readouts of …
Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-specific antibodies, which precludes dynamic measurements. Here, we have developed genetically encoded, live-cell reporters that enable simultaneous monitoring of the phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing subunit of the TcR/CD3. We show that these reporters provide accurate readouts of TcR/CD3 phosphorylation and are sensitive to the local balance of kinase and phosphatase activities acting upon TcR/CD3. Using these reporters, we demonstrate that, in addition to the expected activation-dependent phosphorylation at the plasma membrane, tyrosine-phosphorylated CD3ζ accumulates on endosomal vesicles distinct from lysosomes. These results suggest that an intracellular pool of phosphorylated CD3ζ may help to sustain TcR/CD3 signaling after the receptor internalization.
National Acad Sciences