之前研究已證實Eps8為一種致癌蛋白並且參與在癌症形成和v-Src誘導細胞轉型中。為了進一步分析Eps8是如何參與在癌化的作用機轉，利用yeast two-hybrid的系統從腦 cDNA資料庫找出和Eps8 N端的結合蛋白質IRSp53/BAIAP2。IRSp53是一種和肌動蛋白絲形成的相關蛋白並且參與在細胞移動過程中。這兩種蛋白相互作用是經由透過 Eps8 proline-rich區域和IRSp53的SH3-WWB domain結合。並且也觀察到在IV5細胞 (v-Src transformed cells)中，Eps8可以調控IRSp53的表達。因此本研究中，我們進一步探討Eps8-IRSp53的結合是如何參與在v-Src所誘導細胞轉型作用中。首先觀察到當IV5細胞中降低IRSp53的蛋白表現量時，不論在培養皿，或在軟瓊脂和老鼠皮下，都會明顯抑制細胞的生長。並且，當IRSp53S再送入IRSp53低表現量的IV5 細胞中，細胞生長缺失會回復過來；但外送進缺乏和Eps8結合的△363或PPPDA mutant於IRSp53 低表現量的 IV5細胞中，細胞生長缺失不會回復過來。進一步分析Eps8-IRSp53參與的分子機轉，也觀察到降低IRSp53表達會造成v-Src調控Stat3酪胺酸705的磷酸化有下降的情形以及PI3K的不活化(反應在下游Akt絲胺酸473的磷酸化下降) 。同時，cyclin D1和FAK蛋白的表現量降低，導致G1期細胞週期的積累，最終造成v-Src促進細胞生長能力降低。並且，當細胞大量表達IRSp53S 於Eps8低表現量的IV5 細胞中，也會使得細胞生長回復過來；但外送進△363或PPDA mutant，細胞生長缺失並不會回復過來。進一步觀察在HeLa細胞中，當EGF刺激之下促進Src活化時可以增加Eps8-IRSp53的結合和Stat3活化。因此我們的實驗結果顯示IRSp53和Eps8不僅能影響細胞的移動，而且IRSp53會和Eps8結合共同參與在v-Src所誘導的細胞轉型中。

As an oncoprotein, Eps8 participates in v-Src-induced cellular transformation. To delineate the underlying mechanism, we perform a yeast two-hybrid screening from human brain cDNA library and IRSp53S, a protein critical in cell mobilization, was identified. The interaction domains of was identified at the multiple proline-rich regions of Eps8 and the C-terminal SH3-WWB containing sequence of IRSp53S. In addition, Eps8 knockdown decreases the expression of IRSp53 in v-Src-transformed cells (IV5), raising the question of whether Eps8-IRSp53 interaction was crucial in Src-mediated transformation. Indeed, attenuation of IRSp53 reduced anchorage dependent and independent growth in IV5 cells and tumor formation in mice, which could be partly rescued by siRNA-resistant human IRSp53S but not by the Eps8-binding defective mutants (i.e. 363 and PPPDA). Accompanying with decreased cell proliferation, Src-mediated activation of PI3K (as reflected by Pi-Ser473 AKT), Stat3 (as reflected by Pi-Tyr705 Stat3), and expression of cyclin D1 and FAK was abolished in IRSp53-attenuated IV5 cells. Consistently, G1-phase cell cycle progression was retarded in these cells. Furthermore, the interaction between Eps8 and IRSp53 was abolished by Src family kinase inhibitor SU6656. Remarkably, through activation of Src, EGF increased the formation of Eps8/IRSp53 complex and Stat3 activation in HeLa cells. With these results, we demonstrate for the first time that IRSp53, through its interaction with Eps8, not only affects cell migration but also dictates cellular growth in cancer cells.

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