Analysis of Latent Membrane Protein (LMP) 2A Signaling Through Src Family Kinases and the Role of LMP2B in Modulating LMP2A SignalingPublic Deposited
Latent membrane protein 2A (LMP2A) and LMP2B are viral proteins expressed during Epstein-Barr virus (EBV) latency in EBV-infected B cells both in cell culture and in vivo. Although Lyn has been shown to be important in mediating LMP2A signaling, it is still unclear if Lyn is used preferentially or if LMP2A associates promiscuously with other Src family kinase (SFK) members. To investigate the role of various SFKs in LMP2A signaling, I crossed LMP2A transgenic mice (TgE) to Lyn -/-, Fyn -/- or Blk -/- mice. TgE Lyn -/- mice had a larger IgM-positive population in comparison to TgE mice, suggesting that the absence of Lyn prevents LMP2A from delivering survival and developmental signals to the B cells. LMP2A was also transiently transfected into the human EBV-negative B cell line BJAB to determine which SFK members associate with LMP2A. Lyn was detected in LMP2A immunoprecipitates whereas Fyn was not. Both Lyn and Fyn were able to bind to an LMP2A mutant which contained a sequence shown previously to bind tightly to the SH2 domain of multiple SFK members. The similarity in structure between LMP2A and LMP2B suggests that they may localize to the same cellular compartments. To investigate the function of LMP2B, B cell lines expressing LMP2A, LMP2B, LMP2A/LMP2B and the relevant vector controls were analyzed. As previously shown, cells expressing LMP2A had a dramatic block in normal BCR signal transduction as measured by calcium mobilization and tyrosine phosphorylation. Interestingly, when LMP2B was expressed in conjunction with LMP2A, there was a restoration of normal BCR signal transduction upon BCR crosslinking. The expression of LMP2B did not alter the cellular localization of LMP2A, but did bind to and prevent the phosphorylation of LMP2A. A restoration in Lyn levels, but not a change in LMP2A levels was also observed in cells co-expressing LMP2B with LMP2A. Taken together, these results show that LMP2A signals preferentially through Lyn and that LMP2B is able to modulate LMP2A signaling. This preferential association and signaling through Lyn is due in part to the SH2 domain of Lyn associating with the YEEA motif of LMP2A.