The interplay between Eps8 and IRSp53 contributes to Src-mediated transformation

Abstract

As an oncoprotein, Eps8 participates in v-Src-induced cellular transformation. To delineate the underlying mechanism, we conducted a yeast two-hybrid screening and identified IRSp53S, a protein critical in cell mobilization, as one of the Eps8-binding partners from a human brain cDNA library. The association was mediated by the multiple proline-rich regions of Eps8 and the C-terminal SH3-WWB containing domains of IRSp53S. In this study, we observed that Eps8 modulated the expression of IRSp53 in v-Src-transformed cells (IV5), raising the question of whether Eps8/IRSp53 interaction was crucial in carcinogenesis. To address this issue, we generated IV5-expressing irsp53 siRNA cells. Attenuation of IRSp53 reduced cell proliferation of IV5 in culture dish and tumor formation in mice, which could be partly rescued by ectopically expressed human IRSp53S. In addition, IRSp53 knockdown impaired activity of phosphatidylinositol 3-kinase (as reflected by Pi-Ser473 AKT) and Stat3 (as reflected by Pi-Tyr705 Stat3), and reduced cyclin D1 expression that culminated to impede G 1 -phase cell-cycle progression. Ectopically expressed human IRSp53S, but not its Eps8-binding defective mutants (that is, D363 and PPPDA), rescued these defects and partly restored cell proliferation. Remarkably, through activation of Src, EGF increased the formation of Eps8/ IRSp53 complex and Stat3 activation in HeLa cells. With these results, we show for the first time that IRSp53, through its interaction with Eps8, not only affects cell migration but also dictates cellular growth in cancer cells.

Eps8 Protein Facilitates Phagocytosis by Increasing TLR4-MyD88 Protein Interaction in Lipopolysaccharide-stimulated Macrophages

“…In addition to EGF receptor, Eps8 is also a substrate of Src tyrosine kinase (20) and is involved in Src-mediated transformation (21,22). The enzymatic activity of Src positively regulates not only the expression of Eps8 (20,21) but also the interaction between Eps8 and IRSp53, leading to the activation of Stat3 in Src-transformed cells (23).…”

EPS8, an Adaptor Protein Acts as an Oncoprotein in Human Cancer

“…Its interaction with Eps8 was originally observed from a GST-IRSp53 SH3 pull-down experiment [22] and confirmed by a yeast two-hybrid screening utilizing N-terminal Eps8 sequences as a bait to search for Eps8 binding partners [23]. IRSp53 contains an N-terminal I-BAR (inverse-Bin-Amphiphysin-Rev) domain, followed by a CRIB, an SH3, a WW-binding sequence (WWB), and a PDZ (post synaptic density 95, disc large, zonula occludens-1) domain.…”

“…Interaction between IRSp53 SH3 domain and Eps8 N-terminal proline-rich sequences (aa207-aa221) activates Rac and results in cell motility and invasion of cancer cells [22]. Interestingly, in addition to the SH3 domain, PPPDY (aa468-aa471; GeneBank accession number NP_001138360) within the IRSp53 C-terminal WWB domain also participates in the association with Eps8 SH3 [23]. The interplay between Eps8 and IRSp53 is important for Srcmediated STAT3 activation that leads to cell proliferation in cancer cells [23].…”

“…Interestingly, in addition to the SH3 domain, PPPDY (aa468-aa471; GeneBank accession number NP_001138360) within the IRSp53 C-terminal WWB domain also participates in the association with Eps8 SH3 [23]. The interplay between Eps8 and IRSp53 is important for Srcmediated STAT3 activation that leads to cell proliferation in cancer cells [23].…”

“…Furthermore, two Eps8 proteins were demonstrated to form an interwound dimer with their SH3 domains being located at the interface [19]. In addition to the aforementioned Eps8-interacting proteins, Eps8 was also shown to bind directly to EGFR [20], Sos1 [21], and IRSp53 [22,23]. Strikingly, Eps8 is an actin capper whose barbed-end capping activity resides in its Cterminal effector domain and is regulated by protein-protein interaction with Abi1 [24,25].…”