Abstract
BackgroundEPS8 is a scaffolding protein that regulates proliferation, actin dynamics and receptor trafficking. Its expression is increased in cancer, enhancing mitogenesis, migration and tumorigenesis. Src phosphorylates EPS8 at four tyrosine residues, although the function is unknown. Here we investigated the pro-oncogenic role of EPS8 tyrosine phosphorylation at Src target sites in HNSCC.MethodsPlasmids expressing EPS8 Src-mediated phosphorylation site mutants (Y485F, Y525F, Y602F, Y774F and all four combined [FFFF]) were expressed in cells containing a normal endogenous level of EPS8. In addition, cells were treated with dasatinib to inhibit Src activity. EPS8 downstream targets were evaluated by western blotting. Wound closure, proliferation, immunofluorescence and tumorgenicity assays were used to investigate the impact of phenylalanine mutations on EPS8 biological functions.ResultsFOXM1, AURKA, and AURKB were decreased in cells expressing FFFF- and Y602F-EPS8 mutants, while cells harbouring the Y485F-, Y525F- and Y774F-EPS8 mutants showed no differences compared to controls. Consistent with this, dasatinib decreased the expression of EPS8 targets. Moreover, Y602F- and FFFF-EPS8 mutants reduced mitogenesis and motility. Strikingly though, FFFF- or Y602F-EPS8 mutants actually promoted tumorigenicity compared with control cells.ConclusionsPhosphorylation of EPS8 at Y602 is crucial for signalling to the cell cycle and may provide insight to explain reduced efficacy of dasatinib treatment.
Highlights
epidermal growth factor receptor (EGFR) pathway substrate 8 (EPS8) is a scaffolding protein that regulates proliferation, actin dynamics and receptor trafficking
HN4 cells were first stably transfected with FFFF-EPS8 mutants, total cell lysates were prepared and the expression of FOXM1 and two of its targets, AURKA and AURKB, was determined by western blotting
To determine whether blocking Src-mediated EPS8 phosphorylation is responsible for the reduced expression of cell cycle regulators, HN4 cells were treated with 400 nM of the Src inhibitor, dasatinib, for 24 h
Summary
EPS8 is a scaffolding protein that regulates proliferation, actin dynamics and receptor trafficking. We investigated the pro-oncogenic role of EPS8 tyrosine phosphorylation at Src target sites in HNSCC. EGFR is a receptor tyrosine kinase that has been found to be overexpressed in 80–100% of advanced-stage HNSCC.[11,12] This kinase transduces signals through multiple pathways to promote cell proliferation, motility and survival.[13,14,15,16,17,18] EGFR pathway substrate 8 (EPS8) is one of the mediators involved in postreceptor EGFR signalling.[19,20] EPS8 is a scaffolding protein that exists as a dimer[21] and shows several structural features: a Src homology 3 (SH3) domain, a split pleckstrin homology (PH)
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