探討 Cten 與 β-catenin 和 α-actinin4 之間的交互作用及 Cten 於細胞質和細胞核間穿梭的機制

Abstract

C-terminal tensin-like (Cten) 蛋白質位於集中附著點並參與細胞附著和移動。CTEN 在各個時期的大腸癌中皆有過量表現的情形，於細胞核中也能偵測到高含量的 Cten，顯示細胞核中的 Cten 可能參與大腸癌細胞癌化的過程。本論文發現在大腸癌細胞株 SW480 中，Cten 與 β-catenin、α-actinin4 在細胞核中有交互作用，Cten 和β-catenin 無直接的交互作用，而 Cten 和α-actinin4 則是會直接結合，α-actinin4 同時利用其 N 端和 C端與 Cten 的 C 端 (327~715) 結合。Cten、β-catenin 和 α-actinin4 之間的交互作用，並不影響彼此於細胞質和細胞核的分佈。本論文由 qPCR 的結果發現 Cten不影響 α-actinin4 所參與調控的 NFκB 下游基因表現。在探討 Cten 於細胞質和細胞核間穿梭的機制方面，實驗結果發現 SH2-PTB domain 是 Cten 進核的重要區塊。Cten 出細胞核的機制是透過 CRM1 所運送，利用線上軟體預測出可能的 NES 序列位於 102~118 胺基酸，將 Cten 的 102~118 胺基酸剔除後，發現 Cten 會於細胞核中累積。說明 Cten於細胞質與細胞核間可能的穿梭機制。

C-terminal tensin-like protein (Cten) locates in focal adhesion complex and participates in regulating cell adhesion and migration. Elevated Cten level has been detected in all stage of colon cancers. Furthermore, a high population of Cten is located in the nucleus. Therefore, nuclear Cten may play an important role in colon cancer cell progression. In this study, we demonstrated that Cten interacts with β-catenin and α-actinin4 in the nucleus. Cten indirectly interacts with β-catenin. Whereas α-actinin4 associates with C-terminal half of Cten (327~715) through its N- and C-terminal domain. The interactions of Cten, β-catenin and α-actinin4 may not be required for the nuclear targeting of each protein. Using the qPCR, we showed that nuclear Cten may not participate in regulating α-actinin4 -mediated NFκB downstream genes expression. In elucidating the molecular mechanism underlying nucleocytoplasmic shuttling of Cten, we found that Cten may translocate to the nucleus through its SH2-PTB domain and is exported by the chromosome region maintenance-1 (CRM-1) dependent pathway. In silico analysis by two NES database shows that Cten contains nuclear export signal (NES) between 102 to 118 amino acid residues. Deletion in putative NES of Cten results in its nucleus retention. These findings provide possible molecular mechanisms for nucleocytoplasm shuttling of Cten.