In vitro membrane reconstitution of the T-cell receptor proximal signaling network.
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.
1] The Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA. [2] Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.
Enfu
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Hui
1] The Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA. [2] Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.
Ronald D
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Vale
D017252:CD3 Complex / Q000378:metabolism; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D008567:Membranes, Artificial; D008954:Models, Biological; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism
D017252:CD3 Complex; D008051:Lipid Bilayers; D008081:Liposomes; D008567:Membranes, Artificial; D011948:Receptors, Antigen, T-Cell; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases; D017493:Leukocyte Common Antigens