A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse.
Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters include a notion of relative spatial and temporal control of particular biochemical steps involved in the process. An essential step in T-cell receptor (TCR) mediated signalling is the activation of the protein tyrosine kinase ZAP-70. ZAP-70 is recruited to the TCR upon receptor engagement and, once activated, is responsible for the phosphorylation of the protein adaptor, Linker for Activation of T-cells, or LAT. LAT phosphorylation results in the recruitment of a signalosome including PLCgamma1, Grb2/SOS, GADS and SLP-76. In order to examine the real time spatial and temporal evolution of ZAP-70 activity following TCR engagement in the immune synapse, we have developed ROZA, a novel FRET-based biosensor whose function is dependent upon ZAP-70 activity. This new probe not only provides a measurement of the kinetics of ZAP-70 activity, but also reveals the subcellular localization of the activity as well. Unexpectedly, ZAP-70 dependent FRET was observed not only at the T-cell -APC interface, but also at the opposite pole of the cell or "antisynapse".
Randriamampita
Clotilde
C
Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.
Mouchacca
Pierre
P
Malissen
Bernard
B
Marguet
Didier
D
Trautmann
Alain
A
Lellouch
Annemarie Coffman
AC
D015374:Biosensing Techniques; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013569:Synapses / Q000276:immunology / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000378:metabolism / Q000502:physiology*
D011494:Protein Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase