Dergai O. Identification and characterization of interactions of Latent membrane protein 2A (LMP2A) of Epstein-Barr virus with endocytic proteins.

Latent Membrane Protein 2A (LMP2A) is an Epstein-Barr virus-encoded protein that is important for the maintenance of latent infection. Its activity affects cellular differentiation, migration, proliferation and B cell survival. LMP2A resembles a constitutively activated B cell antigen receptor and exploits host kinases to activate a set of downstream signaling pathways. In the current study we demonstrate the interaction of LMP2A with intersectin 1 (ITSN1), a key endocytic adaptor protein. This interaction occurs via both the N- and C-tails of LMP2A and is mediated by the SH3 domains of ITSN1. Additionally, we identified the Shb adaptor and the Syk kinase as novel binding ligands of ITSN1. The Shb adaptor interacts simultaneously with the phosphorylated tyrosines of LMP2A and the SH3 domains of ITSN1 and mediates indirect interaction of ITSN1 to LMP2A. Syk kinase promotes phosphorylation of both ITSN1 and Shb adaptors in LMP2A-expressing cells. In contrast to ITSN1, Shb phosphorylation depends additionally on Lyn kinase activity. Considering that Shb and ITSN1 are implicated in various receptor tyrosine kinase signaling, our results indicate that LMP2A can affect a number of signaling pathways by regulating the phosphorylation of the ITSN1 and Shb adaptors.Using bioinformatic analysis SGIP1 (SH3 domain GRB2-like (endophilin) interacting protein 1) was predicted to bind SH3 domains of ITSN1 and AMPH1. The interaction of ITSN1 with SGIP1 was confirmed with coimmunoprecipitation approach. We have found that SH3A and SH3E domains of ITSN1 mediated its binding to SGIP1. Confocal imaging of ITSN1 and SGIP1 at the plasma membrane evidenced for significant overlapping of ITSN1 and SGIP1 signals. Moreover, we found that SH3 domain of AMPH1 could precipitate SGIP1, evidencing for interaction between these proteins. Latent membrane protein 2A of Epstein-Barr virus regulates phosphorylation of several adaptor proteins by mean of associated with it tyrosine kinases. Interactions with some endocytic proteins can impact on exosomal secretion and/or intracellular traffic of LMP2A.