Rkat cells to compare the activity of LYPR and LYPW in different promoters relevant for T cells, such as NF-AT/AP1 sites of the IL-2 promoter, Gal4-ELK or NF-kB (Figure S3). We observed in these assays that LYPW had a similar activity than LYPR. Next, we observed that LYPW-P695A and LYP-F620A/F700A, which are mutated in both P1 and P2 motifs and do not interact with CSK, inhibited the activation of the IL-2 minimal promoter as did LYPR and LYPW (Figure 4A). We also determined the activation of Erk in the presence of LYPR, LYPW or LYPW-P695A (Figure 4B) and the activation of p38 in the presence of LYPR or LYPW (Figure 4C), obtaining a similar inhibition, in agreement with the data obtained in the luciferase assays. Although the inhibitory role of LYPW in these assays could have been explained by the binding to endogenous CSK through the P2 motif, LYPWP695A and LYP-F620A/F700A do not bind to CSK at all, precluding any effect of the interaction of LYP with endogenous CSK in these assays. In addition, to address the cooperation of LYP and CSK in TCR signaling, we expressed CSK along with LYPR and LYPW to measure the induction of a promoter with NF-AT/AP1 sites of IL-2 (data not shown). In both cases CSK cooperated with LYPW as well as with LYPR, and in both cases co-expression of CSK and LYP produced a greater inhibition than any of the proteins alone. Likewise, when CSK-W47A, which AZ-876 showed no binding to LYP, was co-expressed with LYPR and LYPW there was an increase of the inhibition of the NF-AT/AP1 and IL-2 minimal promoter (Figure 4D and E). We also tested whether LYP and CSK versions that do not interact to one another could reduce the expression of CD25 in 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 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 Nobiletin site mobility (Figure 1A), we speculated that this shift could be due to 26001275 Tyr phosphorylation. To address this issue, a PBLs were stimulated through CD3 and CD28 r.Rkat cells to compare the activity of LYPR and LYPW in different promoters relevant for T cells, such as NF-AT/AP1 sites of the IL-2 promoter, Gal4-ELK or NF-kB (Figure S3). We observed in these assays that LYPW had a similar activity than LYPR. Next, we observed that LYPW-P695A and LYP-F620A/F700A, which are mutated in both P1 and P2 motifs and do not interact with CSK, inhibited the activation of the IL-2 minimal promoter as did LYPR and LYPW (Figure 4A). We also determined the activation of Erk in the presence of LYPR, LYPW or LYPW-P695A (Figure 4B) and the activation of p38 in the presence of LYPR or LYPW (Figure 4C), obtaining a similar inhibition, in agreement with the data obtained in the luciferase assays. Although the inhibitory role of LYPW in these assays could have been explained by the binding to endogenous CSK through the P2 motif, LYPWP695A and LYP-F620A/F700A do not bind to CSK at all, precluding any effect of the interaction of LYP with endogenous CSK in these assays. In addition, to address the cooperation of LYP and CSK in TCR signaling, we expressed CSK along with LYPR and LYPW to measure the induction of a promoter with NF-AT/AP1 sites of IL-2 (data not shown). In both cases CSK cooperated with LYPW as well as with LYPR, and in both cases co-expression of CSK and LYP produced a greater inhibition than any of the proteins alone. Likewise, when CSK-W47A, which showed no binding to LYP, was co-expressed with LYPR and LYPW there was an increase of the inhibition of the NF-AT/AP1 and IL-2 minimal promoter (Figure 4D and E). We also tested whether LYP and CSK versions that do not interact to one another could reduce the expression of CD25 in 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 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 26001275 Tyr phosphorylation. To address this issue, a PBLs were stimulated through CD3 and CD28 r.
