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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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Structure and subcellular localization of ROZA.(A) Scheme of the ROZA sequence including the N-terminal residues from p56 Lck, the linker sequence and residues 171-178 of LAT (underlined) surrounding Y175 (in bold). (B) Model showing the schematic structure of ROZA (without its membrane anchor), and illustrating that, in the absence of ZAP-70 activity, ROZA may adopt several conformations, some of them allowing FRET. Following phosphorylation of the LAT based sequence by ZAP-70, ROZA adopts a constrained conformation incompatible with FRET. (C) In Jurkat T-cells transiently transfected with ROZA, the probe is mainly located at the plasma membrane as visualized by CFP fluorescence.
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pone-0001521-g001: Structure and subcellular localization of ROZA.(A) Scheme of the ROZA sequence including the N-terminal residues from p56 Lck, the linker sequence and residues 171-178 of LAT (underlined) surrounding Y175 (in bold). (B) Model showing the schematic structure of ROZA (without its membrane anchor), and illustrating that, in the absence of ZAP-70 activity, ROZA may adopt several conformations, some of them allowing FRET. Following phosphorylation of the LAT based sequence by ZAP-70, ROZA adopts a constrained conformation incompatible with FRET. (C) In Jurkat T-cells transiently transfected with ROZA, the probe is mainly located at the plasma membrane as visualized by CFP fluorescence.

Mentions: As previously described [13], [14], a ratiometric FRET based probe for a tyrosine kinase activity can be prepared by fusing a peptide sequence encoding a known substrate sequence for the targeted kinase with its cognate phospho-tyrosine binding domain (e.g. a Src homology (SH) 2 domain). When assembled with the appropriate linker sequences and flanked by compatible donor and acceptor autofluorescent proteins, such an engineered protein can undergo a phosphorylation dependent change in its FRET signal, presumably due to a phosphorylation dependent intramolecular binding event which changes the relative distance and/or orientation of the autofluorescent protein domains (See Figure 1B). In order to create a ZAP-70 specific probe, we targeted peptide sequences derived from mouse LAT encompassing tyrosine residues Y132, Y175 or Y195, known to be substrates for ZAP-70 [15]. The peptides were fused to their respective SH2 domain binding partners such as the N-terminal SH2 domain of mouse PLCγ1 for Y132 and the SH2 domain of mouse Grb2 for Y175and Y195 [16]. ROZA variants were produced in which the substrate peptide, the SH2 binding partner as well as the length and composition of the interdomain linkers were varied. Constructs were screened for their expression levels in Jurkat J-Tag cells and their ability to generate a phosphorylation dependent change in FRET signal upon pervanadate (PV) and anti-CD3 stimulation. The sequence resulting in the most significant phosphorylation dependent FRET change (see Fig. 1A) was used for all subsequent experiments. In order to facilitate the encounter of active ZAP-70 with the substrate probe, ROZA was targeted to the plasma membrane by incorporating the 13 N-terminal residues of mouse Lck. As shown in Fig. 1C, the protein is indeed largely restricted to the membrane of Jurkat T-cells. A variant was also prepared incorporating the N-terminal 35 residues of LAT, encompassing the transmembrane domain and palmitoylation sites, however this anchor led to a largely vesicular probe expression (data not shown).


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)

Structure and subcellular localization of ROZA.(A) Scheme of the ROZA sequence including the N-terminal residues from p56 Lck, the linker sequence and residues 171-178 of LAT (underlined) surrounding Y175 (in bold). (B) Model showing the schematic structure of ROZA (without its membrane anchor), and illustrating that, in the absence of ZAP-70 activity, ROZA may adopt several conformations, some of them allowing FRET. Following phosphorylation of the LAT based sequence by ZAP-70, ROZA adopts a constrained conformation incompatible with FRET. (C) In Jurkat T-cells transiently transfected with ROZA, the probe is mainly located at the plasma membrane as visualized by CFP fluorescence.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211399&req=5

pone-0001521-g001: Structure and subcellular localization of ROZA.(A) Scheme of the ROZA sequence including the N-terminal residues from p56 Lck, the linker sequence and residues 171-178 of LAT (underlined) surrounding Y175 (in bold). (B) Model showing the schematic structure of ROZA (without its membrane anchor), and illustrating that, in the absence of ZAP-70 activity, ROZA may adopt several conformations, some of them allowing FRET. Following phosphorylation of the LAT based sequence by ZAP-70, ROZA adopts a constrained conformation incompatible with FRET. (C) In Jurkat T-cells transiently transfected with ROZA, the probe is mainly located at the plasma membrane as visualized by CFP fluorescence.
Mentions: As previously described [13], [14], a ratiometric FRET based probe for a tyrosine kinase activity can be prepared by fusing a peptide sequence encoding a known substrate sequence for the targeted kinase with its cognate phospho-tyrosine binding domain (e.g. a Src homology (SH) 2 domain). When assembled with the appropriate linker sequences and flanked by compatible donor and acceptor autofluorescent proteins, such an engineered protein can undergo a phosphorylation dependent change in its FRET signal, presumably due to a phosphorylation dependent intramolecular binding event which changes the relative distance and/or orientation of the autofluorescent protein domains (See Figure 1B). In order to create a ZAP-70 specific probe, we targeted peptide sequences derived from mouse LAT encompassing tyrosine residues Y132, Y175 or Y195, known to be substrates for ZAP-70 [15]. The peptides were fused to their respective SH2 domain binding partners such as the N-terminal SH2 domain of mouse PLCγ1 for Y132 and the SH2 domain of mouse Grb2 for Y175and Y195 [16]. ROZA variants were produced in which the substrate peptide, the SH2 binding partner as well as the length and composition of the interdomain linkers were varied. Constructs were screened for their expression levels in Jurkat J-Tag cells and their ability to generate a phosphorylation dependent change in FRET signal upon pervanadate (PV) and anti-CD3 stimulation. The sequence resulting in the most significant phosphorylation dependent FRET change (see Fig. 1A) was used for all subsequent experiments. In order to facilitate the encounter of active ZAP-70 with the substrate probe, ROZA was targeted to the plasma membrane by incorporating the 13 N-terminal residues of mouse Lck. As shown in Fig. 1C, the protein is indeed largely restricted to the membrane of Jurkat T-cells. A variant was also prepared incorporating the N-terminal 35 residues of LAT, encompassing the transmembrane domain and palmitoylation sites, however this anchor led to a largely vesicular probe expression (data not shown).

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
Related in: MedlinePlus