Focal Adhesion Kinase (FAK) 與Integrin β4之交互作用於腫瘤細胞內之分析

Abstract

Integrin是一群位於細胞表面的膜蛋白家族，能夠調控與細胞外間質 (extracellular matrix, ECM) 的交互作用，進而影響各種細胞功能，例如細胞的生長 (cell growth)、分化 (differentiation) 與移動 (migration)，進而也牽涉到疾病的發生。許多先前的研究顯示，integrin β4會參與腫瘤的進程 (tumor progression)。當integrin β4和細胞外間質結合後，會在細胞膜上的聚集 (cluster)，並導致FAK (Focal adhesion kinase) 被活化且進一步影響下游的訊息傳遞。這些發現與先前FAK在腫瘤細胞發展過程中所扮演的角色相當一致，例如: 細胞複製 (cell proliferation)、細胞移動以及腫瘤發育 (tumor development)。然而我們發現，在一些較具侵略性 (aggressive) 細胞株，如: 乳癌細胞 (MDA-MB-231) 以及大腸癌細胞 (HCT-116) 中可以經由共免疫沉澱 (co-immuoprecipitate) 的方法，觀察到內源性 (endogenous) 的integrin β4與 FAK之間的直接交互作用，但在一些較不侵略性的細胞株中，則不具有這種直接的交互作用存在，由此可知此種交互作用極可能與腫瘤發育的過程有關。進一步利用FAK的deletion mutants的實驗以及利用表現短片段的25個可能進行交互作的FAK胺基酸發現，在FAK與integrin β4的交互作用中，FAK的11個胺基酸扮演了關鍵性的角色，接下來利用點突變的方式進一步找到此integrinβ4-FAK交互作用中最重要的兩個胺基酸，並加以分析其下游訊息傳導途徑。經由這些結果，我們首次證明了integrin β4與FAK之間的直接交互作用，顯示先前在integrin β4對於腫瘤進程的影響，極有可能是經由integrin β4-FAK的訊息傳遞路徑 (integrin β4-FAK dependent signaling pathway) 所導致，因此針對integrin β4-FAK交互作用所影響的訊息傳遞途徑以及生物功能加以釐清，對於將來在癌症的治療上將能夠提供新的策略與方向。

A numerous studies have shown that integrin β4 involved in tumor progression and clustering of integrin β4 might lead to the activation of focal adhesion kinase (FAK) and its downstream signaling. These findings are consistent with the roles of FAK in cell proliferation, migration and tumor development. Hence, we were further pursuing the possibility of direct interaction between integrin β4 and FAK. Indeed, we demonstrated that in several tumor cell lines, such as MDA-MB-231 and HCT116 but not HeLa, MCF7 and A549, integrin β4 could co-immuoprecipitate FAK protein endogenously. Besides, this integrin β4-FAK interaction is independent of FAK activity. It implicated the participation of this interaction in tumorigenesis. Moreover, the interaction required an 11-amino-acids motif within FAK’s amino-terminus. By using the in vitro binding assay and competition approach, this association is further confirmed. Among them, two out of 11 amino acids exhibited a critical role in interaction with integrin β4. And the further observations showed that this interaction existed only as cells are adherent.Our data resolved for the first time a physical interaction between integrins and FAK, suggesting a strong link regarding integrin-FAK signaling events. Future work will decipher the signaling pathway(s) and biological significance through the integrin β4-FAK interaction, which will shed a light on better strategies for cancer therapies.