utilises different structural motifs present in its primary structure. For example, it has been shown that the activation of the Vav pathway requires the interaction of the M2 PRR with the C- terminal SH3 domains of Vav1 and Vav2 proteins [22]. Likewise, the M2-dependent down-modulation of the STAT pathway requires a scattered motif distributed between the central basic region and N-terminal amino acids of M2 [35,36]. Whether other regions of M2 contribute to these or other functions remained to be determined up to now. Here, we show that a tyrosine- phosphorylated region of M2 constitutes a third M2 regulatory motif that contributes to the establishment of MHV-68 latency in B cells. This region contains two tyrosine residues (Tyr120 and 129) that are inducibly phosphorylated in ectopic systems by Fyn and constitutively phosphorylated in the A20 B-cell line. By using mutant proteins targeted in these two residues, we have demonstrated that Tyr120 is the primary phosphorylation site of M2 while Tyr129 contributes much less to the overall phosphor- ylation of the protein. Interestingly, we have observed that this new region and the M2 PRR need to work together in order to activate optimally the Fyn/Vav1 pathway. The structural dissection of these two motifs indicates that the cooperative action of the phosphotyrosine residues and the PRR is not mediated by major structural changes that expose putative cryptic binding sites of the M2 molecule. Instead, we have demonstrated that these two motifs work as independent docking sites for the SH3 and SH2 domains of both Vav1 and Fyn. In agreement with this view, we have observed that the optimal binding of Fyn to M2 requires the presence of both the phosphotyrosine and PRR motifs. Furthermore, we have also shown that Vav1 proteins with non-functional C-terminal SH3 or SH2 domains can still bind to M2, although at much lower levels than those observed with the wild type protein. Supporting this dual docking site model, we have also shown that the defective interaction of the Vav1 SH3 mutant with M2 is rescued by the overexpression of Fyn and the subsequent phosphorylation of M2. The requirement of two independent motifs for the optimal binding of Vav proteins to other partners is not unprecedented. Thus, previous studies have shown that binding of Vav1 to Cbl-b requires the SH2 and SH3 regions [37] and, of particular relevance to the current study, it has been reported that the binding of Vav2 (and presumably the rest of Vav family proteins) to mDia/interacting protein requires the simultaneous engage- ment of a tyrosine phosphorylation motif and a PRR [38]. The study of the signalling properties of M2 and its mutants in B-cells has revealed that this protein may also use other B-cell targets. Thus, we have shown that the enforced expression of M2 in A20 cells results in the tyrosine phosphorylation of five additional proteins whose molecular weights range between 50 and 150 kDa. Consistent with our observations with Vav1 and Fyn, we have observed that the mutation of M2 in either the phosphotyrosine motif or the PRR disrupt these phosphorylation events. Likewise, we have shown that the Y120 phosphorylation residue has a much relevant biological role than the Y129 site in this cellular response. These results underscore the concept that the phosphotyrosine motif and the PRR of M2 exert intertwined actions for the optimal manipulation of the B-cell signalling machinery. At this moment, we do not know whether these additional M2-dependent phosphoproteins are direct M2 binding partners or, alternatively, downstream elements activated by either Fyn or Vav1. Preliminary experiments favour the former possibility, since we have observed by pull-down experiments that M2 phosphopeptides can bring down some of those phosphopro- teins. Thus, it is likely that, as in the case of the KSHV K1 protein [9,11], M2 could mediate the formation of a complex signalosome M2 Phosphorylation and Latency with a subset of B-cell signalling proteins. We are conducting currently proteomic experiments to identify these new putative M2 targets. The presence of two interacting motifs in the M2 structure raises interesting regulatory possibilities. According to the previous model of interactions of Vav1 and Fyn with the M2 PRR, it was difficult to understand how the M2/Fyn/Vav1 complex could be assembled, since Fyn and Vav1 will be binding to the same M2 PRR. The presence of two docking sites involved in this interaction solves this problem, since it is possible that the trimeric complex will be formed through the independent binding of Vav1 and Fyn to either the phosphotyrosine motif or the PRR. Given that both proteins can bind to these two regions of M2, this trimeric complex may form in different ways. For example, Vav1 could associate with the PRR or the phosphotyrosine motif when Fyn is bound to the phosphotyrosine or PRR, respectively. In favour of this hypothesis, we have observed that Fyn can be detected in an M2-dependent manner in the anti-Vav1 immuno- precipitates regardless of whether this GEF has inactivating mutations in either the SH2 (that disrupts its association with the M2 phosphotyrosine motif) or the C-terminal SH3 domain (which blocks its association to the M2 PRR). However, we cannot exclude the possibility that the trimeric complex could be also formed with those two proteins binding simultaneously to the two M2 docking sites. This alternative mechanism of assembly is only feasible if M2 forms homodimers within the host cell. Preliminary experiments conducted in our lab indicate that this is not the case (data not shown). An issue that is still problematic according to this model of interaction is the understanding of how Fyn becomes associated with M2 in the first place. According to our present results, Fyn only binds to M2 when both the PRR and the phosphotyrosine domains are functional. If so, how is this complex formed when M2 is non-phosphorylated? To solve this signalling conundrum, we favour a model in which the association of Fyn and M2 will entail two independent, and mechanistically separable steps. In the first step, the initial binding of Fyn to M2 would be triggered by the prior trans-phosphorylation of M2 by Fyn in the absence of complex formation or, alternatively, by the binding of Fyn to M2 molecules previously phosphorylated by other protein tyrosine kinases. This trans-phosphorylation could occur in the absence of direct physical interactions since the constitutive localization of M2 in the plasma membrane makes it possible the presence of other membrane-bound kinases in the neighbour- hood [22]. This model is mechanistically similar to the binding of Syk to the tyrosine-phosphorylated sequences of the Iga and Igb ITAM regions. This initial phosphorylation step would lead to the subsequent physical interaction of Fyn to either uncomplexed M2 proteins or to the M2/Vav1 complex, a result that would trigger the subsequent phosphorylation of Vav1 by the associated kinase. In any case, it is worth mentioning that M2 can be detected in binary complexes with Vav1 and Fyn alone, indicating that M2 will display different forms of engagement during the establish- ment of viral latency such as free forms, binary complexes with either Vav1 or Fyn, and the trimeric M2/Vav1/Fyn complex. This spectrum of states may be even larger if the additional phosphoproteins triggered by M2 in B-cells also associate with M2 following Vav1- or Fyn-like mechanisms. Our results with the Y to F M2 mutants also suggest that the phosphorylation levels of the wild type M2 protein could be used to induce different signalling outputs within the host cells. For instance, we have observed that the phosphorylation pattern induced by M2 in B-cells follows a gradient response, with a total absence of signal in the case of the M2Y mutant, a low but detectable signal elicited by the M2Y120F mutant, and the robust PLoS ONE | www.plosone.org 9 February 2008 | Volume 3 | Issue 2 | e1654