M2 Phosphorylation and Latency Figure 2. Role of the phosphotyrosine M2 motif in B-lymphocytes. (A) TCLs from A20 cells expressing the indicated proteins were incubated with anti-Fyn antibodies and subjected to western blot analysis using the indicated antibodies. As control, aliquots of the same TCLs were analyzed with anti-Vav1 and anti-Myc antibodies to reveal the levels of endogenous Vav1 and ectopic M2 proteins present in these lysates. (B) M2-transfected A20 cells were lysed and TCLs analyzed either by immunoprecipitation with anti-M2 antibodies followed by immunoblot with the indicated antibodies (two upper panels) or by direct immunoblot with the indicated antibodies (rest of panels). The mobility of M2, Fyn and Vav1 proteins is indicated by arrowheads. The mobility of M2-dependent phosphorylated cellular proteins is indicated by arrows (fifth panel from top). a, anti; IP, immunoprecipitation; p, phosphorylated; WB, western blot. doi:10.1371/journal.pone.0001654.g002 Phosphorylated M2 binds directly to Vav1 and Fyn SH2 domains The observation that tyrosine phosphorylation regulates the binding of Fyn and Vav1 to M2 was quite unexpected, since we had shown before that the interaction of these proteins was mediated by the M2 PRR [22]. We surmised that the involvement of the phosphotyrosine M2 motif in this heteromolecular interaction could be due to two different causes. On the one hand, it was possible that tyrosine phosphorylation could induce a conformational change in M2 that resulted in the exposure of the hidden PRR, thereby facilitating the interaction of this second motif with the M2 target proteins. On the other hand, it was also feasible that the phosphorylation sites of M2 could serve as additional docking sites for Vav1 and Fyn via interactions with the SH2 domains of these two signalling proteins. To verify the former possibility, we assessed the binding of several M2 mutants containing Y to D mutations at positions 120 and 129 to Fyn and Vav1. These mutations mimic the negative charge created by the incorporation of the phosphate group on the tyrosine residue so, if the hypothesis of the conformational change were correct, we expected that some of these mutants could enhance the binding of Fyn and Vav1 to M2. However, we found that the Y to D mutations induced effects identical to those elicited by the Y to F missense mutations, indicating that the role of this phosphorylation site was not due through conformation-dependent effects (Fig. 3A,B, top panels). To assess the second possibility, we first tested the hypothesis that the SH2 domains of Fyn and Vav1 could bind M2 sequences containing the phosphorylated Y120 residue. To this end, COS1 cell lysates containing either the wild type or the SH2 mutant versions of Fyn and Vav1 were subjected to pull-down experiments with phosphopeptides (pY) containing either the phosphorylated Y120 or Y129 residues. After binding, the association of Vav1 and Fyn to these peptides was determined by immunoblot analysis. As a control for the specificity of the possible interactions, we included parallel pull-downs in which peptides were dephosphorylated by the YOP phosphatase prior to the incubation with cell extracts. As shown in Figure 4A (two top panels), the pY120 peptide, but not the pY129 peptide, could bind to both Fyn and Vav1. However, this interaction was lost in the case of the SH2 mutants of Fyn and Vav1 (Fig. 4A, two top panels) or when the dephosphorylated peptide was used in the experiments (Fig. 4A, two top panels). These results indicate that pY120 residue can act as an effective docking site for Vav1 and Fyn. In order to assess if this interaction required other chaperone proteins, we repeated these experiments using the isolated SH2 domains of Vav1 and Fyn purified from Escherichia coli as glutathione S-transferase (GST) fusion proteins. As a negative control, we performed similar pull-down experiments with the non-chimeric GST protein. We found that the pY120 peptide could bind directly both SH2 domains, but that the SH2 domain of Fyn bound more efficiently than that of Vav1 (Fig. 4B). The phosphorylated peptide failed to bind to GST (Fig. 4B), confirming the specificity of the interactions. These differences could not be attributed to loading artefacts, given that equivalent amounts of peptide and input GST proteins were used in each pull down experiment. PLoS ONE | www.plosone.org 4 February 2008 | Volume 3 | Issue 2 | e1654