Ed Jurkat derived cell lines deficient in LCK, JCam1.6, and in Zap70, P116 (Figure 5C). PV treatment of Jurkat and P116 cells lead to similar levels of LYP Tyr phosphorylation, while in JCam1.6 cells there was a residual phosphorylation that can be explained by the presence of FYN or CSK in these cells. We also detected in vitro LYP phosphorylation by LCK (Figure 5D), further supporting LCK as a key kinase in LYP tyrosine phosphorylation. Our 69-25-0 analysis on LYP phosphorylation was followed by the identification of the tyrosines phosphorylated. To this end, we transfected several Tyr to Phe mutants, in a LYPR-DA inactive version, chosen based on the phosphorylation sites predicted by Netphos [25] or Scansite [26], and on the degree of evolutionary conservation. Co-transfection of LCK with 
the LYP Tyr to Phe mutants showed that the main sites phosphorylated by LCK were Tyr526 and Tyr536 (Figure 5E). We also tested whether there was any difference in the phosphorylation of LYPR and LYPW in Jurkat cells by LCK, which in fact was similar (Figure 5F). Then, we evaluated whether LYP phosphorylation on these residues, Tyr526 and Tyr536, was involved in the regulation of TCR signaling. Expression of LYP Y526F and Y536F mutants showed no effect with respect of LYPR on the activation of the IL-2 promoter in luciferase assays (Figure 5G), in disagreement with data published recently [14]. We also tested whether these mutants affected the interaction of LYP with CSK, but our results showed that they are not involved in this interaction (Figure S4),These results indicate that TCR stimulation leads to Y-phosphorylation of LYP, and that phosphorylation of Tyr526 and Tyr536 does not affect LYP function during TCR signaling.DiscussionThe C1858T polymorphism of LYP plays a critical role in the pathogenesis of several autoimmune diseases. This polymorphism changes to a Trp the Arg620 that is essential for LYP SIS 3 site binding to CSK, suggesting that a change in the association between these proteins will contribute to the generation of autoimmunity. Here, we analyzed the association between LYP and CSK, and its relevance to TCR signaling. Our work suggests that LYP/CSK interaction is more complex than expected from the data reported on Pep/Csk binding. Whereas Pep/Csk interaction is constitutive [6], LYP binding to CSK is dynamic 
and is increased by cellular activation, involving CSK SH3 and SH2 domains along with LYP P1 and P2 motifs. Extensive mutation of critical residues in the P1 motif: Pro615, Pro618, Arg620, Ser624, Ile626, Val626 did not abolished CSK binding to LYP, differing from the data reported on Pep [8]. Our data also show that CSK does bind to LYPW. Although this observation is not entirely novel, it is novel the demonstration that this binding is mediated by the P2 motif of LYP. Again, this is another controversial fact regarding LYP. The study of Pep/Csk interaction showed initially that a weak interaction exists on a yeast two-hybrid assay when the P1 motif is deleted [12]. Another work reported that mutations in the P1 motif of Pep abrogated Csk binding [8]. Later on, it was found that Pep binds to Csk after deletion of the P1motif in stable cell lines generated with different constructs of Pep [6]. Other reports have not detected LYPW interaction with CSK [27,28]. However, in agreement with our data, other studies have reported that LYPW interacts with CSK [10,14]. Particularly, the work of Fiorillo et al. showed this interaction in vitro between.Ed Jurkat derived cell lines deficient in LCK, JCam1.6, and in Zap70, P116 (Figure 5C). PV treatment of Jurkat and P116 cells lead to similar levels of LYP Tyr phosphorylation, while in JCam1.6 cells there was a residual phosphorylation that can be explained by the presence of FYN or CSK in these cells. We also detected in vitro LYP phosphorylation by LCK (Figure 5D), further supporting LCK as a key kinase in LYP tyrosine phosphorylation. Our analysis on LYP phosphorylation was followed by the identification of the tyrosines phosphorylated. To this end, we transfected several Tyr to Phe mutants, in a LYPR-DA inactive version, chosen based on the phosphorylation sites predicted by Netphos [25] or Scansite [26], and on the degree of evolutionary conservation. Co-transfection of LCK with the LYP Tyr to Phe mutants showed that the main sites phosphorylated by LCK were Tyr526 and Tyr536 (Figure 5E). We also tested whether there was any difference in the phosphorylation of LYPR and LYPW in Jurkat cells by LCK, which in fact was similar (Figure 5F). Then, we evaluated whether LYP phosphorylation on these residues, Tyr526 and Tyr536, was involved in the regulation of TCR signaling. Expression of LYP Y526F and Y536F mutants showed no effect with respect of LYPR on the activation of the IL-2 promoter in luciferase assays (Figure 5G), in disagreement with data published recently [14]. We also tested whether these mutants affected the interaction of LYP with CSK, but our results showed that they are not involved in this interaction (Figure S4),These results indicate that TCR stimulation leads to Y-phosphorylation of LYP, and that phosphorylation of Tyr526 and Tyr536 does not affect LYP function during TCR signaling.DiscussionThe C1858T polymorphism of LYP plays a critical role in the pathogenesis of several autoimmune diseases. This polymorphism changes to a Trp the Arg620 that is essential for LYP binding to CSK, suggesting that a change in the association between these proteins will contribute to the generation of autoimmunity. Here, we analyzed the association between LYP and CSK, and its relevance to TCR signaling. Our work suggests that LYP/CSK interaction is more complex than expected from the data reported on Pep/Csk binding. Whereas Pep/Csk interaction is constitutive [6], LYP binding to CSK is dynamic and is increased by cellular activation, involving CSK SH3 and SH2 domains along with LYP P1 and P2 motifs. Extensive mutation of critical residues in the P1 motif: Pro615, Pro618, Arg620, Ser624, Ile626, Val626 did not abolished CSK binding to LYP, differing from the data reported on Pep [8]. Our data also show that CSK does bind to LYPW. Although this observation is not entirely novel, it is novel the demonstration that this binding is mediated by the P2 motif of LYP. Again, this is another controversial fact regarding LYP. The study of Pep/Csk interaction showed initially that a weak interaction exists on a yeast two-hybrid assay when the P1 motif is deleted [12]. Another work reported that mutations in the P1 motif of Pep abrogated Csk binding [8]. Later on, it was found that Pep binds to Csk after deletion of the P1motif in stable cell lines generated with different constructs of Pep [6]. Other reports have not detected LYPW interaction with CSK [27,28]. However, in agreement with our data, other studies have reported that LYPW interacts with CSK [10,14]. Particularly, the work of Fiorillo et al. showed this interaction in vitro between.