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

Proc Natl Acad Sci U S A. 2010 Dec 21;107(51):22128-33. doi: 10.1073/pnas.1016388108. Epub 2010 Dec 6.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD3 Complex / genetics
  • CD3 Complex / immunology
  • CD3 Complex / metabolism*
  • Endosomes / genetics
  • Endosomes / immunology
  • Endosomes / metabolism*
  • 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
  • Receptors, Antigen, T-Cell / metabolism*
  • Signal Transduction / physiology*
  • Tyrosine / genetics
  • Tyrosine / immunology
  • Tyrosine / metabolism

Substances

  • CD3 Complex
  • CD3 antigen, zeta chain
  • Receptors, Antigen, T-Cell
  • Tyrosine