In an attempt to study the role of Eps8 in human carcinogenesis, we observe that ectopic overexpression of Eps8 in SW480 cells (low Eps8 expression) increases cell proliferation. By contrast, expressing eps8 small interference RNA in SW620 and WiDr cells (high Eps8 expression) reduces their proliferation rate. Interestingly, attenuation of Eps8 decreases Src Pi-Tyr-416, Shc Pi-Tyr-317, and serum-induced FAK Pi-Tyr-397 and Pi-Tyr-861. Remarkably, by virtue of mammalian target of rapamycin/STAT3 Pi-Ser-727, Eps8 modulates FAK expression required for cell proliferation. Within 62% of colorectal tumor specimens examined, >2-fold enhancement of Eps8 as compared with their normal counterparts is observed, especially for those from the advanced stage. In agreement with the modulation of FAK by Eps8, the concomitant expression of these two proteins in tumor specimens is observed. Notably, Eps8 attenuation also impedes the motility of SW620 and WiDr cells, which can be rescued by ectopically expressed FAK. This finding discloses the indispensability of Eps8 and FAK in cell locomotion. These results provide a novel mechanism for Eps8-mediated FAK expression and activation in colon cancer cells.The signal transduction of the epidermal growth factor receptor (EGFR) 2 is important for normal cell physiology (1, 2). During the search for novel EGFR substrates, Eps8 (EGFR pathway substrate number 8) as suggested by its designation was originally identified as a putative EGFR target devoid of phosphotyrosine-binding SH2 domain (3). Among its 97-and 68-kDa isoforms, only the former is well characterized and thus referred to as Eps8. Later, Eps8 also is the substrate for Src tyrosine kinase (4). In addition to tyrosyl phosphorylation, expression of Eps8 is also affected by Src activity (4, 5). Remarkably, its aberrant overexpression in murine fibroblasts can lead to cellular transformation (6), and of note, Eps8 overexpression contributes to Src-mediated transformation (7).As an adaptor protein, Eps8 contains several structural features such as a split pleckstrin homology, a putative nuclear targeting sequence, a central SH3 domain, and several prolinerich regions. Although the split pleckstrin homology confers the ability of Eps8 to associate with plasma membrane in response to serum stimulation and conveys signals to ERK activation (6), Eps8 can also complex with Abi-1/E3b1 and RN-tre separately through its SH3 domain (8, 9). By interacting with Abi-1 or RN-tre, Eps8 integrates signals leading to actin cytoskeleton via Rac and receptor endocytosis via Rab5, respectively (10, 11). Recently, IRSp53 has been demonstrated as an Eps8-binding protein whose complex with Eps8 reinforces Rac activation and cell migration in fibrosarcoma cells (12). Besides, Eps8 is also identified as an actin capper, which is capable of regulating actin-based motility (13).Focal adhesion kinase (FAK) is an intracellular tyrosine kinase localized prominently within focal adhesion (14, 15) and participates in a variety of integrin-elicited biological ac...