N Jurkat cells. This experiment showed that a combination of plasmids like LYPWP695A and CSK-W47A, are still able to reduce the induction of this activation marker (Figure 4F). Collectively, we conclude from these data that the cooperation of LYP and CSK 370-86-5 web proteins to regulate TCR signaling does not require a direct interaction between them.CSK SH2 and SH3 25033180 Domains are Involved in Binding to LYPThe LYP residues that contribute to CSK binding have been studied largely. However, less attention has been paid to the CSK aa critical for this interaction. To address this issue, we generated D27A and W47A CSK mutants, which interact with Arg620 and Pro618 in LYP, respectively [23]. In addition, a careful examination of the NMR models of Ghose et al. suggested that Gln26 could form a hydrogen bond with Arg620 in LYP (Figure 3A), which prompted us to mutate Gln26 to Ala and test its binding to LYP. We observed that while D27A and W47A (-)-Indolactam V chemical information mutants blocked the association with LYP, the Q26A mutant seems less critical for this association (Figure 3B). Given that LYP/CSK interaction was increased by PV treatment (Figure 1A), we asked whether the SH2 domain of CSK was involved in the interaction with LYP. To prove this, we mutated CSK Arg107 to Met, because this residue, conserved 
in SH2 domains, is critical for binding to phospho-Y in protein ligands [24]. The R107M mutation decreased the association of CSK with LYP in cells treated with PV, but also in resting cells (Figure 3D). Whereas W47A mutation abolished the interaction with LYP, as did the triple mutant D27A/W47A/LYP is Phosphorylated in TyrosineAs PV treatment produced a shift in LYP SDS-PAGE mobility (Figure 1A), we speculated that this shift could be due to Tyr phosphorylation. To address this issue, a PBLs were stimulated through CD3 and CD28 receptors, 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 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 detec.N Jurkat cells. This experiment showed that a combination of plasmids like LYPWP695A and CSK-W47A, are still able to reduce the induction of this activation marker (Figure 4F). Collectively, we conclude from these data that the cooperation of LYP and CSK proteins to regulate TCR signaling does not require a direct interaction between them.CSK SH2 and SH3 25033180 Domains are Involved in Binding to LYPThe LYP residues that contribute to CSK binding have been studied largely. However, less attention has been paid to the CSK aa critical for this interaction. To address this issue, we generated D27A and W47A CSK mutants, which interact with Arg620 and Pro618 in LYP, respectively [23]. In addition, a careful examination of the NMR models of Ghose et al. suggested that Gln26 could form a hydrogen bond with Arg620 in LYP (Figure 3A), which prompted us to mutate Gln26 to Ala and test its binding to LYP. We observed that while D27A and W47A mutants blocked the association with LYP, the Q26A mutant seems less critical for this association (Figure 3B). Given that LYP/CSK interaction was increased by PV treatment (Figure 1A), we asked whether the SH2 domain of CSK was involved in the interaction with LYP. To prove this, we mutated CSK Arg107 to Met, because this residue, conserved in SH2 domains, is critical for binding to phospho-Y in protein ligands [24]. The R107M mutation decreased the association of CSK with LYP in cells treated with PV, but also in resting cells (Figure 3D). Whereas W47A mutation abolished the interaction with LYP, as did the triple mutant D27A/W47A/LYP is Phosphorylated in TyrosineAs PV treatment produced a shift in LYP SDS-PAGE mobility (Figure 1A), we speculated that this shift could be due to Tyr phosphorylation. To address this issue, a PBLs were stimulated through CD3 and CD28 receptors, 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 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 detec.