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A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse.

Randriamampita C, Mouchacca P, Malissen B, Marguet D, Trautmann A, Lellouch AC - PLoS ONE (2008)

Bottom Line: 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.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".

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.

ABSTRACT
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".

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ROZA is specific for ZAP-70.(A) WT J77cl20 or cells transiently expressing either ROZA or ROZA YF (5×106 cells/point) were preincubated for 30 minutes with inhibitors (A. 10 µM PP2 or B 200 µM piceatannol) or medium alone, followed by a 2 minute activation with 5 mM freshly prepared PV. ROZA was immunoprecpitated with anti-GFP antibody. Upper panel: anti-phosphoTyr (4G10) western blot. Lower panel: anti-GFP western blot. (B) PV induces an increase in ZAP-70 activity in Jurkat T-cells expressing ROZA. No change in FRET ratio was observed in ZAP-70-deficient Jurkat T-cells (P116) cells expressing ROZA or in normal Jurkat T-cells transfected with the mutated probe (YF).
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pone-0001521-g003: ROZA is specific for ZAP-70.(A) WT J77cl20 or cells transiently expressing either ROZA or ROZA YF (5×106 cells/point) were preincubated for 30 minutes with inhibitors (A. 10 µM PP2 or B 200 µM piceatannol) or medium alone, followed by a 2 minute activation with 5 mM freshly prepared PV. ROZA was immunoprecpitated with anti-GFP antibody. Upper panel: anti-phosphoTyr (4G10) western blot. Lower panel: anti-GFP western blot. (B) PV induces an increase in ZAP-70 activity in Jurkat T-cells expressing ROZA. No change in FRET ratio was observed in ZAP-70-deficient Jurkat T-cells (P116) cells expressing ROZA or in normal Jurkat T-cells transfected with the mutated probe (YF).

Mentions: In order to establish that the observed FRET changes were due to a ZAP-70-dependent phosphorylation of the probe, Jurkat T-cells transiently expressing ROZA were stimulated, lysed, and subsequently subjected to anti-phosphotyrosine immunoprecipitation and anti-GFP immunoblotting (Fig. 3A). The resulting immunoblots of the transfected Jurkat T-cell samples displayed an activation-dependent phosphorylated species with the expected molecular weight of the ROZA probe (69 kD). Pre-treatment of transfected cells with the Syk family inhibitor piceatannol, or with the Src kinase inhibitor PP2, resulted in inhibition of ROZA phosphorylation. Similarly, mutation of the probe's target tyrosine residue LAT 175 to phenylalanine resulted in significant inhibition of the probe phosphorylation (Fig. 3A upper gel). Stripping and reblotting with an anti-GFP antibody confirmed that the most prevalent GFP containing species had a molecular weight consistent with the full length ROZA (Fig. 3A lower gel).


A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse.

Randriamampita C, Mouchacca P, Malissen B, Marguet D, Trautmann A, Lellouch AC - PLoS ONE (2008)

ROZA is specific for ZAP-70.(A) WT J77cl20 or cells transiently expressing either ROZA or ROZA YF (5×106 cells/point) were preincubated for 30 minutes with inhibitors (A. 10 µM PP2 or B 200 µM piceatannol) or medium alone, followed by a 2 minute activation with 5 mM freshly prepared PV. ROZA was immunoprecpitated with anti-GFP antibody. Upper panel: anti-phosphoTyr (4G10) western blot. Lower panel: anti-GFP western blot. (B) PV induces an increase in ZAP-70 activity in Jurkat T-cells expressing ROZA. No change in FRET ratio was observed in ZAP-70-deficient Jurkat T-cells (P116) cells expressing ROZA or in normal Jurkat T-cells transfected with the mutated probe (YF).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211399&req=5

pone-0001521-g003: ROZA is specific for ZAP-70.(A) WT J77cl20 or cells transiently expressing either ROZA or ROZA YF (5×106 cells/point) were preincubated for 30 minutes with inhibitors (A. 10 µM PP2 or B 200 µM piceatannol) or medium alone, followed by a 2 minute activation with 5 mM freshly prepared PV. ROZA was immunoprecpitated with anti-GFP antibody. Upper panel: anti-phosphoTyr (4G10) western blot. Lower panel: anti-GFP western blot. (B) PV induces an increase in ZAP-70 activity in Jurkat T-cells expressing ROZA. No change in FRET ratio was observed in ZAP-70-deficient Jurkat T-cells (P116) cells expressing ROZA or in normal Jurkat T-cells transfected with the mutated probe (YF).
Mentions: In order to establish that the observed FRET changes were due to a ZAP-70-dependent phosphorylation of the probe, Jurkat T-cells transiently expressing ROZA were stimulated, lysed, and subsequently subjected to anti-phosphotyrosine immunoprecipitation and anti-GFP immunoblotting (Fig. 3A). The resulting immunoblots of the transfected Jurkat T-cell samples displayed an activation-dependent phosphorylated species with the expected molecular weight of the ROZA probe (69 kD). Pre-treatment of transfected cells with the Syk family inhibitor piceatannol, or with the Src kinase inhibitor PP2, resulted in inhibition of ROZA phosphorylation. Similarly, mutation of the probe's target tyrosine residue LAT 175 to phenylalanine resulted in significant inhibition of the probe phosphorylation (Fig. 3A upper gel). Stripping and reblotting with an anti-GFP antibody confirmed that the most prevalent GFP containing species had a molecular weight consistent with the full length ROZA (Fig. 3A lower gel).

Bottom Line: 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.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".

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.

ABSTRACT
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".

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