Phosphoproteomics Analysis Of EGFR Signaling Pathway In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China, its incidence and mortality occupies the first place of the world, and is a great threat to people's health and lives.Although several groups had conducted proteomics of NPC, little study was invovled in phosphoproteomics, oncogenic signaling and cancer research about NPC. Analysis of phosphotyrosine proteins from transforming growth factor alpha (TGF-a) triggered phosphotyrosine proteome permitted the identification of novel downstream substrates of the epidermal growth factor receptor (EGFR). Using functional proteomics technology based on 2-DE,2-D Western blotting, and mass spectrometry, we identified and quantified the tyrosine phosphorylation levels of 16 proteins between control and TGF-a treated CNE2 human NPC cells. These proteins were involved in cell apoptosis, proliferation and metabolism. In addition, among these identified proteins, ANXA3, KRT8 and KRT18 were validated to be novel tyrosine-phosphorylation targets of EGFR signaling by IP-western blotting and part of a complex EGFR phosphotyrosine signaling network.To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the activation of EGFR signaling, nasopharyngeal carcinoma CNE2 cell line was treated with TGF-a and/or PD153035. After phosphoprotein enrichment and 2-D DIGE analysis,38 protein spots were quantitated and showed a 1.5-fold or greater change in all three groups and 34 differential phosphoproteins were identified.35 (92.1%) from 38 differential protein spots were found to be phosphorylated in poststaining of 2D DIGE gels with Pro-Q Diamond phosphospecific fluorescent stain. Only spot 4 (BAG5) from all differential phosphoproteins was no reports to phosphophorylation modification in two online phosphophorylation modification sites analysis resources (PhosphoSitePlusTM on-line system biology resourse and the Phospho.ELM database). Among them, we detected 19 (55.9%) from 34 identified differential phosphoproteins were signal pathway proteins with DAVID Bioinformatics Resources. Hierarchical clustering analysis showed expression patterns for the differential phosphoproteins. Pathway network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in EGFR signalling. In this study, five previously known EGFR-regulated phosphoproteins (KRT8, hnRNPK, KRT18, GRB2, STMN1) and twenty-seven novel EGFR signalling phospho-target proteins were identified. Interestingly, two different spots (spot 22 and spot 33) were uniformly identified as GST family member Glutathione S-transferase P (GSTP1) in 2-D DIGE analyses. Subsequently, Western blotting showed that GSTP1 undergoes phosphorylation and nonphosphorylation following with EGFR activation and inactivation in enrichment phosphoprotein samples. IP-western blotting analysis showed that GSTP1 formed a complex with EGFR and had high levels of tyrosine-phosphorylated GSTP1 only in EGFR-activated cells in total protein lysates.These novel finding will provide new insights into the complex EGFR phosphorylation signaling and may have implications in carcinogenesis of NPC. Our systematic profiling of phosphorylation changes responding to TGF-a stimulation and/or PD153035 inhibition not only presented a comprehensive phosphorylation network regulated by canonical EGFR signaling but also found novel molecules and phosphorylation involved in EGFR signaling in NPC cells. Our results indicated that GSTP1 might serve as a novel phospho-target protein of EGFR signalling pathway and play an important role in pathogenesis of NPC.