M2 Phosphorylation and Latency Figure 1. Y120 is the predominant tyrosine required for the formation of the M2/Vav1/Fyn complex and for M2-dependent phosphorylation of Vav1. COS1 cells were transiently transfected with plasmids encoding the indicated proteins. (A,B) Cells were lysed and clarified extracts incubated with either anti-M2 (A) or anti-Vav1 antibodies (B). Immunoprecipitates were analysed by western blot with the indicated antibodies (right side of panels). Western blot analysis of a representative aliquot of total cell lysates (TCL) confirmed expression of proteins (bottom panels). (C) Cell lysates were incubated with an anti-Vav1 antibody and the resulting immunoprecipitates subjected to an immunocomplex kinase assay exactly as described previously [22]. Lower panel is an overexposure showing phosphorylation of M2 and its mutant forms. The position of molecular weight markers is indicated on the right. 2, without; +, with; a, anti; IP, immunoprecipitation; p, phosphorylated; WB, western blot. doi:10.1371/journal.pone.0001654.g001 primary phosphorylation residue involved in the formation of the M2/Vav1/Fyn complex and in Vav1 phosphorylation. Constitutive phosphorylation of M2 in B cells In order to address the role of M2 phosphorylation in a more physiological context, we next monitored the coassembly with Fyn and Vav1 and the phosphorylation of M2 in A20 cells, a mouse B cell lymphoma line expressing surface IgGs. To this end, M2 and its phosphotyrosine mutants were ectopically expressed in these cells. For comparative purposes, we also analyzed in these experiments the binding properties and phosphorylation of M2P2, a previously described M2 mutant protein that lacks the PRR involved in the recognition of both Vav1 and Fyn [22]. The formation of heteromolecular complexes was assessed by immunoblot analysis of Fyn immunoprecipitates for the presence of Vav1 and M2 proteins. These results indicated that Y120 is the primary phosphorylation residue involved in the formation of the M2/ Vav1/Fyn complex in B cells (Fig. 2A). Next, we examined the phosphorylation levels of M2 proteins by anti-PTyr immunobloting of M2 immunoprecipitates. Under these conditions, we observed that wild type M2 was constitutively phosphorylated on tyrosine residues even in the absence of BCR stimulation (Fig. 2B, upper panel; data not shown). This constitutive phosphorylation could be due to the relatively high basal levels of activation of this cell line [26]. This phosphorylation was maintained upon mutation of the Y129 residue but abolished in the case of the replacement of the Y120 M2 residue (Fig. 2B, upper panel). As expected [22], the elimination of the proline rich region eliminated the phosphorylation of M2, as assessed by the lack of detectable phosphorylation levels in the M2P2 mutant protein (Fig. 2B, upper panel). These results indicate that the main phosphorylation site of M2 is Y120 independently of the cell type used. To analyze the impact of these mutations in the activation of Vav1 in B-cells, we monitored by immunoblot analysis the levels of phosphorylation of endogenous Vav1 on tyrosine 174 in the transfected A20 cells. Phosphorylation of this Vav1 site is a good reflection of its activation status, since it has been shown before that this posttranslational modification induces a conformational change in the Vav1 molecule that stimulates its GDP/GTP exchange activity towards Rho/Rac proteins [27–29]. Furthermore, it has been previously shown that M2 induces Vav1 phosphorylation on this site and the concomitant activation of the downstream Rac1 GTPase [22]. As shown in Fig. 2B, the phosphorylation of Vav1 induced by the overexpression of M2 in B-cells did not occur in the case of M2Y120F-transfected cells. As previously described, Vav1 phosphorylation was also impaired when the M2P2 mutant protein was expressed in A20 cells. In contrast, the Y129F mutation did not affect the phosphorylation of the endogenous Vav1 protein at the Y174 position (Fig. 2B). To verify whether M2 could affect other intracellular proteins in addition to Vav1, we analyzed total cell extracts from A20 cells by anti-PTyr immunoblot to monitor the possible phosphorylation of other B-cell proteins on tyrosine residues upon overexpression of M2 and the indicated mutants. We observed that M2 and the M2Y129F mutant induced the phosphorylation of several proteins of approximately 52, 54, 56, 75, 95 (likely Vav1 itself) and 150 kDa (Fig. 2B, fifth panel from top). Interestingly, the phosphorylation of these proteins was not observed in A20 cells expressing either M2Y and/or M2P2 and significantly reduced in the case of cells expressing the M2Y120F mutations (Fig. 2, fifth panel from top). Taken together, these results show that Y120 is the predominant phosphorylated tyrosine of M2 both in lymphoid and non-hematopoietic cells. Moreover, they indicate that M2 is also able to promote the BCR-independent phosphorylation of Vav1 and an additional subset of cellular proteins. This effect is totally dependent on the presence of both the Y120 residue and the PRR of M2. PLoS ONE | www.plosone.org 3 February 2008 | Volume 3 | Issue 2 | e1654