Activation of FAK by integrin β4 in an EGFR/Src dependent manner in tumor progression

Focal adhesion kinase(FAK)是一種酪胺酸(tyrosine)的磷酸脢，是integrin下游的重要訊息傳遞者。Integrin是由α、β二種次單元經由不同組合而成的雙複合(heterodimer)受體，位於細胞表面，會與細胞外的基質(extracellular matrix)進行交互作用，負責細胞的黏著、移動、增生等重要的功能。先前研究發現integrin和FAK在癌細胞發展中皆扮演重要角色，其中報導顯示integrin β4會透過調節FAK的活性而影響癌細胞的轉移；故此研究針對integrin β4是否可直接與FAK有交互作用進行研究，並進一步探討其對癌細胞功能的影響。實驗首先利用免疫螢光染色法(immuno-fluorescent stain)，發現在比較具侵略性的MDA-MB231乳癌細胞中，integrin β4與FAK皆位於細胞邊緣的相同位置，而在較不具侵略性的MCF7乳癌細胞中則無此現象出現；另外，利用免疫組織染色(immunohistochemistry)和西方墨點轉漬法(Western blotting)分析人類結腸癌組織，結果顯示integrin β4和FAK在結腸癌細組織中皆有大量表現，且透過co-immunoprecipitation實驗發現此二蛋白質具有交互作用的現象。更進一步經由co-immunoprecipitation、pull down及far Western分析，發現FAK的N端25個胺基酸會與integrin β4的cytodomain有直接的交互作用；同時我們利用不同位置點突變的FAK分別與integrin β4進行co-immunoprecipitation實驗，發現FAK的N端25個胺基酸中，有三個胺基酸是參與此一交互作用的主要位置。我們觀察到這樣的交互作用是在細胞呈現貼附的狀態時所產生，且此交互作用會促進FAK第397個酪胺酸自體磷酸化的能力；此外，在Src蛋白質活性的刺激下，此特殊且具有專一性的交互作用將更緊密。另外，發現在表皮生長因子(Epidermal Growth Factor, EGF)刺激下，發現此二蛋白質的交互作用，會進一步影響到下游signal transducer and activator of transcription 3 (STAT3)與AKT蛋白質的活性。更進一步經由Boyden chamber細胞移動、BrdU細胞增生和matrigel細胞侵入的實驗分析顯示，在HCT116結腸癌細胞和MDAMB231乳癌細胞中，大量加入FAK的N端25個胺基酸，會干擾FAK和integrin β4的交互作用，並減少細胞移動、增生和入侵的能力。故推測FAK和integrin β4的交互作用，可能會影響癌細胞生成。此次研究首次發現integrin β4與FAK有直接的交互作用，且此交互作用會影響到細胞的功能，未來期望依據生物化學、生物學和醫學臨床等研究結果，能進一步研發治療癌細胞的藥物。Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, is an important downstream mediator of integrins which belong to one family of cell adhesion receptors consisted of α and β subunits. Both integrin β4 and FAK play important roles in the tumor progression according to numerous previous reports, one report had shown integrin β4 can stimulate FAK’s activity, furthermore, it can interfere the integrin β4-mediated metastasis of invasive tumor. Here, we demonstrated a physical and functional association between these two molecules in vivo and in vitro. In the immunofluorescence staining assay, integrin β4 and FAK were co-localized at cell peripheral plasma membrane in MDA-MB 231 cell line, but not in non-aggressive MCF-7 tumor cell line. We also investigate that integrin β4 and FAK were over expression in human colon cancer by immunohistochemistry and Western blotting assay. According to co-immunoprecipitation, pull down and far Western assays, we conclude that a 25 amino-acid motif within FAK’s N terminus is responsible for interacting with the cytoplasmic tail of integrin β4. Furthermore, three of the 25 amino acids within the amino-terminus of FAK act as crucial sites in interaction with integrin β4. The interaction resulted in phosphorylation and activation of FAK in an adhesion-dependent manner. Furthermore, the particular interaction between integrin β4 and FAK is promoted by Src which subsequently affects STAT3 and AKT as the downstream signaling mediators upon this interaction. In addition, in both of HCT116 and MDA-MB-231 cells, disruption of this interaction reduced cell proliferation, motility and invasion, suggesting the involvement of this interaction in tumorigenesis and perhaps metastasis in these tumor cells. Our data resolved for the first time a novel pathway of integrin β4-mediated cellular functions via direct interaction with FAK in the regulation of tumor progression, thereby strengthening a link between integrin β4 and FAK biochemically and functionally.CONTENTS………………………………………………………………….………..IIST of FIGURES……………………………………………………………..……III文摘要……………………………………………………………………….…….IVBSTRACT…………………………………………………..…………….….……VINTRODUCTION……………………………………………………………………1ATERIALS and METHODS………………………… ………………………....12eagents…………………………………………………………..……….…12ell culture and transfection………………………………………………….13mmunoprecipitation and Western blotting analysis..…………….………….13NA constructs...…………………………………………………..………...14recombinant adenoviruses………………………………………...………..15reparation of fusion protein..……………………………………...………...16mmunofluorescent staining..……………………………………...…………17mmunohistochemistry……………………………………………...………..17ar Western assay…………………………………………………..………...18ell migration assay………………………………………………..………...19romodeoxyuridine incorporation assay..……………………………………20atrigel invasion assay……………………………………………..………..21ESULTS……………………………………………………………………………22ntegrin β4 associates with FAK in tumor cells……………………………...22he amino-terminus of FAK enables direct interaction with integrin β4 ...….24 AK’s 25 amino acids enables interact with the cytodomain of integrin β4...25ntegrin β4 interacting with FAK is in a cell adhesion-dependent manner…..27he interaction between integrin β4 and FAK is promoted by activated Src..28TAT3 and AKT are the downstream signal proteins of this interaction…….28ffects of integrin β4-FAK complexes in tumor cell proliferation, migration and invasion…………………………………………………………………..29ISCUSSION ……………………………………………...……………………….31IGURES……………………………………………………………………….…...36EFERENCES…………………………………………………………………..….4