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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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(A) In ROZA-expressing Jurkat T-cells, PV induces changes in the 436→535 and in the 436→470 signals (top) and in their ratio (bottom).Traces correspond to the mean of 3 different T-cells. (B) In Jurkat T-cells (top), anti-CD3 induces a partial activation of ZAP-70 that can be completed by PV. In peripheral blood T-cells (bottom), anti-CD3 triggers a large and rapid FRET decrease. Inset: ZAP-70 activity is shown as the inverse of the FRET signals. Traces correspond to the average of 8–17 individual cells. (C) In Jurkat T-cells stably expressing ROZA and stimulated with PV or anti-CD3, ZAP-70 (site 319) as well as endogenous LAT (site 175) are phosphorylated after a 2 minute stimulation.
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pone-0001521-g002: (A) In ROZA-expressing Jurkat T-cells, PV induces changes in the 436→535 and in the 436→470 signals (top) and in their ratio (bottom).Traces correspond to the mean of 3 different T-cells. (B) In Jurkat T-cells (top), anti-CD3 induces a partial activation of ZAP-70 that can be completed by PV. In peripheral blood T-cells (bottom), anti-CD3 triggers a large and rapid FRET decrease. Inset: ZAP-70 activity is shown as the inverse of the FRET signals. Traces correspond to the average of 8–17 individual cells. (C) In Jurkat T-cells stably expressing ROZA and stimulated with PV or anti-CD3, ZAP-70 (site 319) as well as endogenous LAT (site 175) are phosphorylated after a 2 minute stimulation.

Mentions: Stimulation of ROZA-expressing Jurkat T-cells with pervanadate (PV), a phosphatase inhibitor that unveils constitutive tyrosine kinase activity, triggered a simultaneous increase of the 436→470 nm signal and a decrease of the 436→535 nm signal (Fig. 2A upper panel). Stimulation thus triggers a decrease of the 535 nm/470 nm ratio, i.e., of the FRET signal (Fig. 2A lower panel). Changes in FRET signals could also be detected in Jurkat T-cells (Fig. 2B upper panel) or human PBT (Fig. 2B lower panel) after stimulation with anti-CD3. The ROZA expressing Jurkat T-cells were similarly stimulated by either anti-CD3 or PV and subsequently lysed and subjected to western blot analysis. Both phosphorylated forms of ZAP-70 and endogenous LAT where readily detectable 2 minutes (120 seconds) after stimulation, confirming that ZAP-70 is indeed activated under these conditions in the ROZA expressing cells (Fig. 2C). The stimulation dependent decrease in FRET is compatible with a model in which the autofluorescent proteins of the non-phosphorylated form of the probe have a relative distance or orientation which allows FRET, whereas the phosphorylated form of the probe assumes a conformation less compatible with FRET. Other FRET based kinase activity probes described in the literature function with a phosphorylation dependent decrease in FRET capacity [17], [18]. The most relevant example is the second generation Src kinase reporter [18], which like ROZA incorporates an SH2 phospho-tyrosine binding domain, and also undergoes a phosphorylation dependent decrease in FRET. The authors postulate that the close positioning of the N and C termini of the SH2 domain would allow a relative proximity of the linked autofluorescent protein domains and thus FRET in the probe's basal state. This relative proximity would be disrupted during the phosphorylation dependent intramolecular binding event and the FRET ratio would decrease. The N and C termini of the SH2 domain of Grb2 are also found on the same face of the domain in its crystal structure which is consistent with the FRET decrease observed in ROZA [19]. Fig 1B illustrates such a model, taking into account the possibility that in the basal state, the probe may adopt several conformations. In the following figures, ZAP-70 activity is arbitrarily expressed as the inverse of FRET ratio (Fig. 2B inset). Using this representation a FRET decrease corresponds to an increase in net ZAP-70 dependent activity.


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)

(A) In ROZA-expressing Jurkat T-cells, PV induces changes in the 436→535 and in the 436→470 signals (top) and in their ratio (bottom).Traces correspond to the mean of 3 different T-cells. (B) In Jurkat T-cells (top), anti-CD3 induces a partial activation of ZAP-70 that can be completed by PV. In peripheral blood T-cells (bottom), anti-CD3 triggers a large and rapid FRET decrease. Inset: ZAP-70 activity is shown as the inverse of the FRET signals. Traces correspond to the average of 8–17 individual cells. (C) In Jurkat T-cells stably expressing ROZA and stimulated with PV or anti-CD3, ZAP-70 (site 319) as well as endogenous LAT (site 175) are phosphorylated after a 2 minute stimulation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211399&req=5

pone-0001521-g002: (A) In ROZA-expressing Jurkat T-cells, PV induces changes in the 436→535 and in the 436→470 signals (top) and in their ratio (bottom).Traces correspond to the mean of 3 different T-cells. (B) In Jurkat T-cells (top), anti-CD3 induces a partial activation of ZAP-70 that can be completed by PV. In peripheral blood T-cells (bottom), anti-CD3 triggers a large and rapid FRET decrease. Inset: ZAP-70 activity is shown as the inverse of the FRET signals. Traces correspond to the average of 8–17 individual cells. (C) In Jurkat T-cells stably expressing ROZA and stimulated with PV or anti-CD3, ZAP-70 (site 319) as well as endogenous LAT (site 175) are phosphorylated after a 2 minute stimulation.
Mentions: Stimulation of ROZA-expressing Jurkat T-cells with pervanadate (PV), a phosphatase inhibitor that unveils constitutive tyrosine kinase activity, triggered a simultaneous increase of the 436→470 nm signal and a decrease of the 436→535 nm signal (Fig. 2A upper panel). Stimulation thus triggers a decrease of the 535 nm/470 nm ratio, i.e., of the FRET signal (Fig. 2A lower panel). Changes in FRET signals could also be detected in Jurkat T-cells (Fig. 2B upper panel) or human PBT (Fig. 2B lower panel) after stimulation with anti-CD3. The ROZA expressing Jurkat T-cells were similarly stimulated by either anti-CD3 or PV and subsequently lysed and subjected to western blot analysis. Both phosphorylated forms of ZAP-70 and endogenous LAT where readily detectable 2 minutes (120 seconds) after stimulation, confirming that ZAP-70 is indeed activated under these conditions in the ROZA expressing cells (Fig. 2C). The stimulation dependent decrease in FRET is compatible with a model in which the autofluorescent proteins of the non-phosphorylated form of the probe have a relative distance or orientation which allows FRET, whereas the phosphorylated form of the probe assumes a conformation less compatible with FRET. Other FRET based kinase activity probes described in the literature function with a phosphorylation dependent decrease in FRET capacity [17], [18]. The most relevant example is the second generation Src kinase reporter [18], which like ROZA incorporates an SH2 phospho-tyrosine binding domain, and also undergoes a phosphorylation dependent decrease in FRET. The authors postulate that the close positioning of the N and C termini of the SH2 domain would allow a relative proximity of the linked autofluorescent protein domains and thus FRET in the probe's basal state. This relative proximity would be disrupted during the phosphorylation dependent intramolecular binding event and the FRET ratio would decrease. The N and C termini of the SH2 domain of Grb2 are also found on the same face of the domain in its crystal structure which is consistent with the FRET decrease observed in ROZA [19]. Fig 1B illustrates such a model, taking into account the possibility that in the basal state, the probe may adopt several conformations. In the following figures, ZAP-70 activity is arbitrarily expressed as the inverse of FRET ratio (Fig. 2B inset). Using this representation a FRET decrease corresponds to an increase in net ZAP-70 dependent activity.

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".

Show MeSH