Functional proteomics analysis of components of signaling pathways triggered by Epstein-Barr virus-encoded oncoprotein latent membrane protein 1 via novel phosphoprotein enrichment

5475 Nasopharyngeal carcinoma (NPC) is a common in Southeast Asia and is closely associated with infection of Epstein-Barr virus (EBV). The EBV encoded latent membrane protein 1(LMP1) is frequently detected in NPC and plays an important role in its pathogenesis. Previous studies have showed that LMP1 could trigger NFκB, AP-1, JAK/STAT pathways. However, many signaling molecules and downstream target proteins mediated by LMP1 in epithelial cells, particularly the NPC cells are largely unknown. To identify some functional components of signal transduction pathways triggered by LMP1, we combined the novel strategy of phosphoprotein enrichment with the proteomics technology to elucidate the signaling cascade activated by EBV-encoded oncoprotein LMP1 in NPC cell. First, we enriched phosphoproteins with phosphatase inhibitor to inhibit dephosphorylation of protein in vivo. Next, phosphoproteins were purified from cellular extract by phosphate metal affinity chromatography (PMAC). Finally, total phosphoproteins were separated by two-dimension gel electrophoresis, and different phosphoproteins were identified by MALDI-TOF-MS. We found that oncoprotein LMP1 could raise 18.03% of quantification of total cellular phosphoproteins and forty-three proteins showed significant changes in the degree of phosphorylation when LMP1 was expressed.Twenty-four signaling molecules or downstream targets of signal transduction pathways activated by LMP1 were identified, several of which had previously been implicated in LMP1 signal pathways such as putative NFκB activating protein, and remaining proteins including Annexin A2, HSP27, stathmin, Annexin I, basic transcription factor 3(BTF3) and porin were novel signaling molecules or targets with no previously known function in LMP1 signal transduction, and among them, some phosphorylated isoforms of protein were also found. The method used here have proven to be suitable for the identification signaling molecules involved in signal transduction pathways, and we obtained comprehensive understanding in the signaling pathways activated by LMP1.