Eps8 controls Src- and FAK-dependent phenotypes in squamous carcinoma cells.
Bottom Line: Eps8 is an actin regulatory scaffold protein whose expression is increased in squamous cell carcinoma (SCC) cells.Here, we describe two new roles for Eps8.Therefore, Eps8 is a crucial mediator of Src- and FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine the spatial localization of Src, and modulates the functions of Src and FAK during invasive migration.
Affiliation: Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, EH4 2XR Edinburgh, UK.Show MeSH
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Mentions: We next investigated whether Eps8 plays a key role in the trafficking of active Src to autophagosomes or whether it was co-trafficked as a ‘passenger’. We used two independent siRNAs to efficiently suppress expression of endogenous Eps8 as judged by immunofluorescence and immunoblotting (Fig. 1E; Fig. 2A; Fig. 5A). Knockdown of Eps8 in FAK WT SCC cells had no effect on the localization of active Src, which remained at focal adhesions (Fig. 5A, top panel, solid arrows). However, in FAK−/− SCC cells, Eps8 knockdown significantly reduced the number of Src-positive intracellular puncta (Fig. 5A, bottom panels, dashed arrows). Active Src was now predominantly re-localized to focal adhesions as indicated by co-staining with paxillin (Fig. 5A, bottom panel, solid arrows and supplementary material Fig. S3C). Similar results were obtained by shRNA-mediated stable knockdown of Eps8 (supplementary material Fig. S3D,E). Furthermore, we found that the biochemical complex between active Src and LC3B in SCC FAK−/− cells was reduced upon stable knockdown of Eps8, providing biochemical evidence that Eps8 function is required to induce the binding of Src to LC3B during autophagic targeting of Src. Eps8 did not affect general autophagic flux as judged by immunoblotting for LC3B (Fig. 5B), including after chloroquine treatment (supplementary material Fig. S4A,B). These results indicate that Eps8 is required for the selective trafficking of active Src to autophagosomes.
Affiliation: Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, EH4 2XR Edinburgh, UK.