Role of Src homology domain binding in signaling complexes assembled by the murid γ-herpesvirus M2 protein

γ-Herpesviruses express proteins that modulate B lymphocyte signaling to achieve persistent latent infections. One such protein is the M2 latency-associated protein encoded by the murid herpesvirus-4. M2 has two closely spaced tyrosine residues, Tyr120 and Tyr129, which are phosphorylated by Src family tyrosine kinases. Here we used mass spectrometry to identify the binding partners of tyrosine-phosphorylated M2. Each M2 phosphomotif is shown to bind directly and selectively to SH2-containing signaling molecules. Specifically, Src family kinases, NCK1 and Vavl, bound to the Tyr(P)120 site, PLCγ2 and the SHP2 phosphatase bound to the Tyr(P)129 motif, and the p85a subunit of PI3K associated with either motif. Consistent with these data, we show that M2 coordinates the formation of mul-tiprotein complexes with these proteins. The effect of those interactions is functionally bivalent, because it can result in either the phosphorylation of a subset of binding proteins (Vavl and PLCγ2) or in the inactivation of downstream targets (AKT). Finally, we show that translocation to the plasma membrane and subsequent M2 tyrosine phosphorylation relies on the integrity of a C-terminal proline-rich SH3 binding region of M2 and its interaction with Src family kinases. Unlike other γ-herpesvi-ruses, that encode transmembrane proteins that mimic the activation of ITAMs, murid herpesvirus-4 perturbs B cell signaling using a cytoplasmic/membrane shuttling factor that nucleates the assembly of signaling complexes using a bilayered mechanism of phosphotyrosine and proline-rich anchoring motifs.