ABSTRACT
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.
Subject(s)
CD3 Complex/metabolism , Endosomes/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/physiology , CD3 Complex/genetics , CD3 Complex/immunology , Endosomes/genetics , Endosomes/immunology , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Jurkat Cells , Lysosomes/genetics , Lysosomes/immunology , Lysosomes/metabolism , Microscopy, Fluorescence , Phosphorylation/genetics , Phosphorylation/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Tyrosine/genetics , Tyrosine/immunology , Tyrosine/metabolismABSTRACT
Endoplasmic reticulum-localized protein-tyrosine phosphatase PTP1B terminates growth factor signal transduction by dephosphorylation of receptor tyrosine kinases (RTKs). But how PTP1B allows for RTK signaling in the cytoplasm is unclear. In order to test whether PTP1B activity is spatially regulated, we developed a method based on Förster resonant energy transfer for imaging enzyme-substrate (ES) intermediates in live cells. We observed the establishment of a steady-state ES gradient across the cell. This gradient exhibited robustness to cell-to-cell variability, growth factor activation, and RTK localization, which demonstrated spatial regulation of PTP1B activity. Such regulation may be important for generating distinct cellular environments that permit RTK signal transduction and that mediate its eventual termination.
