Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment.
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.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA.
Ivan A
IA
Yudushkin
Ronald D
RD
Vale
D017252:CD3 Complex / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011992:Endosomes / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D008247:Lysosomes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010766:Phosphorylation / Q000235:genetics / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine