In vitro membrane reconstitution of the T-cell receptor proximal signaling network

Nat Struct Mol Biol. 2014 Feb;21(2):133-42. doi: 10.1038/nsmb.2762. Epub 2014 Jan 26.

Abstract

T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45 and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over an ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that co-clustering TCR and Lck or detaching Csk from the membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD3 Complex / metabolism
  • Kinetics
  • Leukocyte Common Antigens / metabolism
  • Lipid Bilayers / metabolism
  • Liposomes / metabolism
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / metabolism
  • Membranes, Artificial
  • Models, Biological
  • Phosphorylation
  • Receptors, Antigen, T-Cell / metabolism*
  • Signal Transduction
  • src-Family Kinases / metabolism

Substances

  • CD3 Complex
  • Lipid Bilayers
  • Liposomes
  • Membranes, Artificial
  • Receptors, Antigen, T-Cell
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
  • src-Family Kinases
  • Leukocyte Common Antigens