Src Kinase and Syk Activation Initiate PI3K Signaling by a Chimeric Latent Membrane Protein 1 in Epstein-Barr Virus (EBV)+ B Cell Lymphomas

Abstract

The B lymphotrophic γ-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.

LMP1 signaling pathway activates IRF4 in latent EBV infection and a positive circuit between PI3K and Src is required

“…40–42 A previous report has also shown that the levels of LMP1 and Src activity positively correlate in EBV latency, and a chimeric NGFP-LMP1 was able to activate the Src family member Fyn. 43 As we show here that LMP1 interacts with c-Src via P85 (Figures 2 and 3), we were interested in examining the possibility that c-Src mediates LMP1 activation of Akt. First, we show that transient expression of wild-type Flag-c-Src or the constitutively active mutant Flag-c-Src(Y527F), but not the kinase-dead mutant Flag-c-Src(Y418F), triggers Akt activation in DG75 B cells (Figure 5a).…”

The interplay between Epstein–Barr virus and B lymphocytes: implications for infection, immunity, and disease

“…It is likely that IL-10 produced by EBV infected B cells also acts to negatively regulate the immune response, akin to regulatory IL-10 producing B cells, that have been more recently described to play a role in peripheral tolerance in autoimmunity, cancer, and organ transplantation (40). In the case of EBV-infected B cells, we demonstrated that LMP1 signaling is sufficient to elicit IL-10 production through p38 MAPK and PI3K-dependent pathways (41) and that activation of PI3K depends upon the Syk tyrosine kinase and the Src family kinase Fyn (42) (Fig 2B). Moreover, LMP1-induced PI3K activation drives B cell survival by preventing loss of XIAP induced by the mitochondrial protease HtrA2 (43).…”

The Latent Membrane Protein 1 (LMP1)

“…However, CTAR2 alone was as efficient as CTAR1 in inducing AKT phosphorylation in C666-1 cells, indicating the existence of a CTAR2-dependent PI3-K/AKT pathway (Shair et al 2008). The precise mechanism of PI3-K/AKT activation by LMP1 has not been fully elucidated but may involve the Src family tyrosine kinases Fyn and Syk (Hatton et al 2012). …”