透過您的圖書館登入
IP:3.91.203.238
  • 學位論文

CTEN 磷酸化與其於核質間穿梭之調控機制

Regulatory mechanisms of phosphorylation and nucleocytoplasmic shuttling of CTEN

指導教授 : 廖憶純
若您是本文的作者,可授權文章由華藝線上圖書館中協助推廣。

摘要


C-terminal tensin-like (CTEN) 為集中附著點 (focal adhesion) 蛋白質 tensin 家族中的一員,它在細胞附著、遷移和癌症發生過程中扮演重要角色。CTEN 在大腸癌中有表現量上升的現象,並且會促進癌症的轉移及侵略等特性;此外,在腫瘤細胞的細胞核中也能偵測到高含量的 CTEN。本實驗室過去研究中證實了與非惡性細胞相比,CTEN 在癌細胞的細胞核中有明顯的累積。我們藉由比對 CTEN 蛋白質序列後發現,CTEN 並未帶有已知的 nuclear localization signal (NLS),而在第102~118 個胺基酸上則帶有 nuclear export signal (NES)。本研究藉由篩選出穩定表現 NES deletion (ΔNES) CTEN 的細胞株,分析其細胞形態、增生、貼附等能力,發現 CTEN 蛋白質序列上 NES 的缺失並未如預期的使 CTEN 在細胞核中有明顯的累積,而在大腸癌細胞株 SW480 細胞增生以及貼附能力上,與控制組相比亦無明顯差異。此外,本研究也探討 CTEN 磷酸化是否影響其於核質間穿梭,我們藉由質譜的方式發現 CTEN 的磷酸化位點位於 Thr347、Ser350 和 Ser386,而細胞核中的 CTEN 與細胞質中 CTEN 有相同的磷酸化位點,推測此磷酸化修飾可能不影響 CTEN 於細胞中的分佈情形。實驗進一步以 Kinasephos 2.0 探討 CTEN 上游可能的蛋白質激酶,並建構帶有磷酸化位點突變的 CTEN 質體,以探討 CTEN 磷酸化的調控機制與其對 CTEN 功能的影響。

關鍵字

核質分佈 磷酸化

並列摘要


C-terminal tensin-like (CTEN) molecule, a member of the tensin family, is a focal adhesion protein that plays an important role in cell adhesion, cell migration and the development of tumorigenesis. Elevated CTEN level has been detected in colon cancer and that promotes the cancer metastasis and invasiveness. Furthermore, high CTEN expression is detected in the nucleus of cancer cell. We have confirmed that more CTEN accumulates in the nuclei of colon cancer cells than in non-malignant cells in our previous studies. We have also found that CTEN contains nuclear export signal (NES) between 102 to 118 amino acid residues by in silico analysis but no classical nuclear localization signal (NLS) on its sequence. In this study, by selection of cells stably expressing NES-deletion (ΔNES) CTEN, we found that CTEN does not remarkably accumulate in the nucleus when the NES was deleted and that the expression of ΔNES-CTEN does not significantly alter cell proliferation and cell adhesion in SW480 colon cancer cells. Moreover, we explore whether the phosphorylation of CTEN regulates the mechanisms underlying the subcellular distribution of CTEN. CTEN is phosphorylated on Thr347, Ser350, and Ser386. The mass spectrometry results showed that the phosphorylation residues identified on nuclear CTEN are the same as those on cytoplasmic CTEN, which suggested that phosphorylation of CTEN might not contribute to its localization. However, in silico analysis was carried out to identify kinases that might be responsible for phosphorylation of CTEN. We also constructed the CTEN plasmids with phosphorylation site mutations to elucidate the regulatory mechanisms underlying CTEN phosphorylation and its effects on CTEN’s function.

參考文獻


Albasri A, Aleskandarany M, Benhasouna A, Powe DG, Ellis IO, Ilyas M, et al. (2011). CTEN (C-terminal tensin-like), a novel oncogene overexpressed in invasive breast carcinoma of poor prognosis. Breast Cancer Res. Tr. 126:47–54.
Calderwood DA, Fujioka Y, de Pereda JM, Garcia-Alvarez B, Nakamoto T, Margolis B, et al. (2003). Integrin β cytoplasmic domain interactions with phosphotyrosine-binding domains: A structural prototype for diversity in integrin signaling. Proc. Natl. Acad. Sci. U.S.A. 100: 2272-2277
Cui Y, Liao YC, Lo SH. (2004). Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3. Mol Cancer Res 2: 225-232.
Davis S, Lu ML, Lo SH, Lin S, Butler JA, Druker BJ, et al. (1991). Presence of an SH2 domain in the actin-binding protein tensin. Science 252: 712-715
Derrick Lee. (2014). Studies on the interactions of Cten with β-catenin and α-actinin4 and the molecular mechanism underlying nucleocytoplasmic shuttling of Cten. Department of Biochemical Science and Technology College of Life Science, National Taiwan University, Master Thesis.

延伸閱讀