Eceptors, and LYP was shown to be phosphorylated in tyrosine by

Eceptors, and LYP was shown to be phosphorylated in tyrosine by Western blot (Figure 5A). Next, to determine the kinase(s) responsible for this phosphorylation, we coexpressed in Jurkat cells LYPR-DA with several kinases relevantRegulation of TCR Signaling by LYP/CSK ComplexFigure 2. P1 and P2 LYP motifs bind to CSK. A, Lysates of HEK293 cells transfected with LYP and a deletion mutant of LYP tagged with the myc Title Loaded From File epitope that lacks the CTH PRM, tagged with the myc epitope, along with HA-CSK plasmids were immunoprecipitated and immunoblotted with theRegulation of TCR 11967625 Signaling by LYP/CSK Complexindicated Abs. Expression of these proteins was verified by IB in TL. B, Lysates from 36108 Jurkat cells were divided equally into five tubes and were subjected to pull-down assays with the indicated PRMs fused to GST. The presence of CSK in the precipitates was detected by IB with CSK Ab and GST-peptides were detected using a GST Ab. TL shows the presence of CSK in the lysate. C, Lysates of HEK293 cells transfected with Panels show duration of scratching response and right panels show total different LYP mutants in the P1 and P2 motifs, tagged with the myc epitope, along with HA-CSK were subjected to IP and IB as indicated. D, Interaction of HA-CSK with a myc-LYP mutant in the C-terminus of the P1 motif, IV, (Ile626Ala,Val627 Ala) was verified by IB after LYP IP in transiently transfected HEK293 cells. E, Lysates of HEK293 cells transfected with HA-CSK and different mutants of LYP in the P1, the P2, or in both motifs, tagged with the myc epitope, were immunoprecipitated and immunoblotted with the indicated Abs. F, Arg to Phe LYP mutants in P1 and P2 PRM tagged with myc were expressed in HEK293 cells and interaction with HA-CSK was detected by immunoblot after LYP immunoprecipitation. CSK blots in panels B, C, D, E and F were scanned and the values obtained were expressed as arbitrary units under the blot. doi:10.1371/journal.pone.0054569.gFigure 3. CSK SH3 and SH2 domains are involved in the association with LYP. A, Schematic representation of the NMR structure of the Pro rich motif P1 of Pep (orange) bound to the SH3 domain of CSK (blue) (PDB code 1JEG). P1 resid ues are numbered according to 1655472 the LYP sequence (613IPPPLPVRTPESFIVVEE630). Arg620 is involved in intermolecular polar contacts with Asp27 and Gln26 of CSK; hydrogen bonds are shown by dashed lines. In addition, Arg620 could establish an intramolecular hydrogen bond with Ser624. B, Jurkat cells were electroporated with HA-CSK wild type and several mutants of CSK SH3 domain along myc-LYPR as indicated. Interaction was detected by IB after LYP IP. C, Several HA-CSK mutants in the SH3 and SH2 domains were tested for interaction with myc-LYP-RDA. Jurkat cells were left untreated or treated with PV and LYP was immunoprecipitated from cell lysates. CSK interaction was detected by IB with specific anti-HA antibody. D, Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells cotransfected with different CSK plasmids, as indicated. The insert shows the IB of the CSK proteins expressed. doi:10.1371/journal.pone.0054569.gRegulation of TCR Signaling by LYP/CSK ComplexFigure 4. LYP/CSK interaction is not required to regulate TCR signaling. A, Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells co-transfected with different myc-LYP plasmids, as indicated. The insert shows the expression of the LYP proteins as detected by IB. B, Erk activation was assayed in Jurkat cells transfected with different.Eceptors, and LYP was shown to be phosphorylated in tyrosine by Western blot (Figure 5A). Next, to determine the kinase(s) responsible for this phosphorylation, we coexpressed in Jurkat cells LYPR-DA with several kinases relevantRegulation of TCR Signaling by LYP/CSK ComplexFigure 2. P1 and P2 LYP motifs bind to CSK. A, Lysates of HEK293 cells transfected with LYP and a deletion mutant of LYP tagged with the myc epitope that lacks the CTH PRM, tagged with the myc epitope, along with HA-CSK plasmids were immunoprecipitated and immunoblotted with theRegulation of TCR 11967625 Signaling by LYP/CSK Complexindicated Abs. Expression of these proteins was verified by IB in TL. B, Lysates from 36108 Jurkat cells were divided equally into five tubes and were subjected to pull-down assays with the indicated PRMs fused to GST. The presence of CSK in the precipitates was detected by IB with CSK Ab and GST-peptides were detected using a GST Ab. TL shows the presence of CSK in the lysate. C, Lysates of HEK293 cells transfected with different LYP mutants in the P1 and P2 motifs, tagged with the myc epitope, along with HA-CSK were subjected to IP and IB as indicated. D, Interaction of HA-CSK with a myc-LYP mutant in the C-terminus of the P1 motif, IV, (Ile626Ala,Val627 Ala) was verified by IB after LYP IP in transiently transfected HEK293 cells. E, Lysates of HEK293 cells transfected with HA-CSK and different mutants of LYP in the P1, the P2, or in both motifs, tagged with the myc epitope, were immunoprecipitated and immunoblotted with the indicated Abs. F, Arg to Phe LYP mutants in P1 and P2 PRM tagged with myc were expressed in HEK293 cells and interaction with HA-CSK was detected by immunoblot after LYP immunoprecipitation. CSK blots in panels B, C, D, E and F were scanned and the values obtained were expressed as arbitrary units under the blot. doi:10.1371/journal.pone.0054569.gFigure 3. CSK SH3 and SH2 domains are involved in the association with LYP. A, Schematic representation of the NMR structure of the Pro rich motif P1 of Pep (orange) bound to the SH3 domain of CSK (blue) (PDB code 1JEG). P1 resid ues are numbered according to 1655472 the LYP sequence (613IPPPLPVRTPESFIVVEE630). Arg620 is involved in intermolecular polar contacts with Asp27 and Gln26 of CSK; hydrogen bonds are shown by dashed lines. In addition, Arg620 could establish an intramolecular hydrogen bond with Ser624. B, Jurkat cells were electroporated with HA-CSK wild type and several mutants of CSK SH3 domain along myc-LYPR as indicated. Interaction was detected by IB after LYP IP. C, Several HA-CSK mutants in the SH3 and SH2 domains were tested for interaction with myc-LYP-RDA. Jurkat cells were left untreated or treated with PV and LYP was immunoprecipitated from cell lysates. CSK interaction was detected by IB with specific anti-HA antibody. D, Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells cotransfected with different CSK plasmids, as indicated. The insert shows the IB of the CSK proteins expressed. doi:10.1371/journal.pone.0054569.gRegulation of TCR Signaling by LYP/CSK ComplexFigure 4. LYP/CSK interaction is not required to regulate TCR signaling. A, Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells co-transfected with different myc-LYP plasmids, as indicated. The insert shows the expression of the LYP proteins as detected by IB. B, Erk activation was assayed in Jurkat cells transfected with different.