The Src family tyrosine kinase, Lyn, negatively regulates Akt activation in LMP2A-expressing B lymphocytes




Brandon, Jillian

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The Epstein-Barr virus (EBV) protein, Latent Membrane Protein 2A (LMP2A), is critical for maintaining viral latency and provides pro-survival and pro-migratory signals to EBV-positive B and epithelial cell malignancies. The N-terminus of LMP2A contains several protein-protein interaction motifs involved in the recruitment of cellular signalling proteins and it is through the recruitment of these proteins that LMP2A is able to initiate signalling. In B lymphocytes, LMP2A's ability to initiate signalling was originally proposed to proceed via a two step mechanism. Firstly, recruitment of the Lyn tyrosine kinase to the tyrosine phosphorylated YEEA site in LMP2A allows for tyrosine phosphorylation of the LMP2A immunoreceptor tyrosine-based activation motif (ITAM). This, in turn, facilitates the recruitment and activation of Syk tyrosine kinase which then initiates downstream signalling events. However, recent findings suggest this model may not be correct and argue that Syk recruitment to LMP2A is independent of the YEEA site. Therefore, we undertook a series of experiments to better understand the role of the YEEA motif and Lyn in the initiation of LMP2A signalling in B lymphocytes. We found that the YEEA site was not absolutely required for tyrosine phosphorylation of the LMP2A ITAM, or for LMP2A to activate Syk. Using siRNA to silence Lyn expression in LCLs. we found that reducing Lyn expression inhibited the ability of LMP2A to promote Syk tyrosine phosphorylation. In contrast, DG75 B cells or Lyn-deficient DT40 B cells transiently expressing higher levels of LMP2A did not require Lyn for LMP2A-mediated Syk phosphorylation. Furthermore, Lyn was not required for LMP2A-mediated Akt activation in DG75 B cells, but rather Akt activation was significantly enhanced in LMP2A-expressing cells where Lyn was reduced by siRNA. We propose that Lyn negatively regulates LMP2A-mediated Akt activation by phosphorylating Syk on Y323, which serves to recruit the c-Cbl E3 ubiquitin ligase to Syk and targets Syk for ubiquitin-mediated degradation. In sum, this work provides novel insight into how LMP2A uses Lyn to initiate and titre signalling in B cells and brings to light an unappreciated role for Lyn as a negative regulator of LMP2A-mediated Akt activation.



Epstein-Barr virus, Protein-tyrosine kinase