Phosphorylation of EPS8 Mediates Its Downstream Signaling and Biological Functions

Abstract

Epidermal growth factor receptor pathway substrate 8 (EPS8) is a scaffolding protein involved in regulating cell proliferation, actin dynamics and receptor trafficking in human cells. EPS8 expression is increased in a range of human cancers including head and neck squamous cell carcinoma (HNSCC). Previous studies have indicated that overexpression of EPS8 enhances mitogenesis and migration of tumor cells and is sufficient to convert non-tumorigenic cells to a tumorigenic phenotype. The non-receptor tyrosine kinase Src is reported to phosphorylate EPS8 at four tyrosine residues, although the impact of this on EPS8 function is unknown. The purpose of this study was to investigate the role of tyrosine phosphorylation of EPS8 at Src target sites in modulating biochemical functions, cell growth, motility, actin dynamics and tumorigenesis in HNSCC. Expression plasmids encoding EPS8 with amino acid substitutions to phenylalanine (F) at the four putative Src phosphorylation sites (Y485F, Y525F, Y602F, Y774F), and all four combined (FFFF), were prepared by site-directed mutagenesis. To evaluate the effect of unphosphorylated EPS8 on downstream signals and biological behavior, mutants were transfected into a model cell line, HN4, which expresses a low endogenous level of EPS8 similar to normal keratinocytes. Additionally, cells were treated with dasatinib, a Src inhibitor, to block phosphorylation of Src substrates. Expression of downstream targets of EPS8 was evaluated by western blotting. Wound closure, proliferation, immunofluorescence and tumorgenicity assays were used to investigate the impact of phenylalanine mutations on the biological functions of EPS8. Forkhead box (FOX) M1 transcription factor (FOXM1), aurora kinase A (AURKA) and aurora kinase B (AURKB) levels were decreased in cells expressing the non-phosphorylatable FFFF- and Y602F-EPS8 mutants, while cells harboring the Y485F-, Y525F- and Y774F-EPS8 mutants showed no differences in the expression of these proteins compared to controls. Consistent with this, dasatinib treatment resulted in a significant decrease in the expression of EPS8 downstream targets. In addition, both Y602F- and FFFF-EPS8 mutants elicited a significant reduction in tumor cell proliferation, motility and actin remodeling. However, blocking Src-mediated EPS8 phosphorylation showed increased tumorigenicity in vivo compared with non-transfected control. Conversely, blocking all putative Src phosphorylation sites except Y602 revealed an increase in the cell cycle mediators, tumor cell proliferation, motility, and a more prominent cortical actin cytoskeleton. These data suggest a crucial role for phosphorylation of EPS8 at Y602 in mediating pro-tumorigenic signal.