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標題: 14-3-3 sigma蛋白的胺基酸H180對調控Src活性及抑制肺癌進程的重要性
Amino acid H180 of 14-3-3sigma protein is important for regulating Src activity and inhibiting lung cancer progression
作者: 許峻豪
Hsu, Chun-Hao
關鍵字: lung cancer progression;14-3-3sigma;Src;14-3-3sigma;肺癌;Src
出版社: 生物醫學研究所
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The family of 14-3-3 proteins is implicated with many biological activities by binding to other proteins. It consists of seven known highly conserved isoforms (β, γ, ε, ζ, η, σ, and τ) and most of them are identified as oncogenic functions except for 14-3-3sigma, a well-known tumor suppressor. To investigate the suppressor characteristic of 14-3-3sigma divergent from others, we analyzed the protein sequences of 14-3-3 family and found that the major difference between 14-3-3sigma and its oncogenic members was due to loss of the phospho-site at His180. Thus, H180Y mutation was generated by site-direct mutagenesis to determine the role of this amino acid in the suppressive characteristic of 14-3-3sigma. First, we showed that overexpression of 14-3-3sigma could decrease the abilities of cancer cell invasion, migration and proliferation, as well as repress cell scattering. On the contrary, H180Y not only induced cell surface protrusions and branchings but also switched 14-3-3sigma from suppressor to oncogene. Meanwhile, H180Y mutant also generates a SH2-binding motif (YYEI) for Src binding. The results of co-immunoprecipitation demonstrated that the association between H180Y mutant and Src was higher than that of wild type, indicating the importance of H180 site for preventing 14-3-3sigma from binding to Src. We also observed that overexpression of H180Y mutant enhanced cell invasion capability and MMP2 secretion. induced the formation of podosome rings and led to matrix degradation. Finally, we demonstrated that H180Y overexpression enhanced cell migration, colony formation, proliferation in vitro, and tumorigenesis in vivo. Taken together, our findings suggest that amino acid H180 of 14-3-3sigma is an essential site to disrupt the 14-3-3sigma-Src interaction and plays important roles in the regulation of Src activity and the tumor suppression of 14-3-3sigma.

14-3-3家族成員利用與相關蛋白質進行交互作用來調控生物活性。在哺乳動物當中,14-3-3家族被發現有七個家族成員,分別為β、γ、ε、ζ、 η、σ和τ,而這些家族成員大部分被視為致癌基因,除了14-3-3sigma (YWHAS)被定義為是一個腫瘤抑制基因。為了瞭解14-3-3sigma與其他家族成員的差異,我們進行所有家族成員的蛋白質序列分析,發現14-3-3sigma與其他致癌基因的家族成員最主要的差異是在第180個胺基酸,由可被磷酸化的酪胺酸取代為組胺酸。因此,我們利用單點突變方法構築H180Y突變株,以探討在14-3-3sigma腫瘤抑制特性中此胺基酸的角色。研究發現,大量表現14-3-3sigma會抑制細胞的移動、侵襲與生長能力。然而,突變的H180Y蛋白質不只會增加細胞的突出及分支情形,也會將原有的腫瘤抑制基因功能轉變成致癌性。此外,突變的H180Y會獲得與其他家族成員同樣擁有的SH2結合區域(YYEI)。我們利用免疫沉澱實驗發現,突變的H180Y與野生性的YWHAS相比,的確會提升與Src之間的交互作用,顯示H180的位置對於14-3-3sigma與Src結合是重要的。我們觀察到大量表現帶有H180Y的構築,會促進細胞的侵犯能力和導致MMP2的分泌;突變的H180Y也會誘導podosome環的形成,造成瓦解細胞基質能力的提升。最後,研究也證實大量表現突變的H180Y會促進細胞的移動、聚落形成、生長以及腫瘤形成。總而言之,由我們的結果推測14-3-3sigma的第180個胺基酸對於其與Src的交互作用是必要的,且在其調控Src活性及抑制肺癌進程中扮演重要的角色。
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