EBP50參與調控β-catenin/TCF-1訊息傳遞之探討

Abstract

β-連鎖蛋白(β-catenin)在Wnt 訊息傳遞路徑中扮演極重要的角色，Wnt 訊息傳遞路徑調控生物發育的許多階段，例如生物體的形成，細胞型態發生(morphogenesis)過程中的移動，以及細胞增殖。在細胞核中，β-連鎖蛋白與T cell factors (TCFs) 轉錄因子結合並負責調控Wnt 誘導基因的表現。磷酸化蛋白50 能和β-catenin 結合，過度表現磷酸化蛋白50 促進β-連鎖蛋白轉錄活性，然而其作用機制仍未知。我們的研究發現磷酸化蛋白50 會進入細胞核，並且進核行為是受到細胞密度調控。經由ChIP-on-chip 的分析，磷酸化蛋白50 主要結合於具有TCF/LEF 共同序列的啟動子區域。藉著資料庫搜尋，我們發現TCF 轉錄因子isoform TCF-1B 的蛋白質序列符合PDZ 結合motif 的特徵。我們進一步證明磷酸化蛋白50 藉由PDZIdomain 和TCF-1 有交互作用。經由和磷酸化蛋白50 結合，不僅可以加強β-連鎖蛋白和TCF-1 複合物的穩定性，並且能回復β-連鎖蛋白和缺乏β-連鎖蛋白結合區域的TCF 轉錄因子，稱為顯性負向TCF 轉錄因子的結合，進而使顯性負向TCF轉錄因子由抑制轉變成促進生長的角色。同時我們證明磷酸化蛋白50 作用於Wnt 誘導基因啟動子上，並且參與β-連鎖蛋白的轉錄功能。利用siRNA 干擾技術抑制大腸癌細胞中磷酸化蛋白50 蛋白表現，可以減少細胞增殖，細胞的非貼附性生長及裸鼠移植腫瘤的生長。此外，免疫組織染色研究亦發現腫瘤侵入的前緣會異常表現核內磷酸化蛋白50，且此現象與大腸直腸癌病人的預後不佳有顯著相關性。

β-Catenin plays a pivotal role in Wnt signaling pathway which controls a variety of cellular processes during development, such as body patterning, migration during morphogenesis and cell proliferation. T-cell factor (TCF) family transcriptional factors cooperate with β-catenin in the nucleus to regulate the expression of Wnt-responsive genes. EBP50 is a β-catenin-associated protein and overexpression of EBP50 has been known to enhance β-catenin transcriptional activity. However, the underlying mechanism has remained elusive. According to our data, EBP50 displayed a nuclear translocation modulated by cell density. Through ChIP-on-chip assay, we disclosed that nuclear EBP50 predominantly associates with the promoter regions containing TCF/LEF consensus binding sequence. By database search, we found TCF-1B has a PDZ binding motif in its C-terminal end. Thus, we further revealed a novel interaction between nuclear EBP50 and TCF-1 through PDZ1 domain. In spite of stabilizing the interaction between β-catenin and full-length TCF-1, binding of nuclear EBP50 also restores the dnTCF-1/β-catenin binding and converts the dominantly negative dnTCF-1 which lacks a β-catenin binding domain into a tumor facilitator. Also, we demonstrated that nuclear EBP50 was recruited to the promoter regions of β-catenin target genes and participated in β-catenin mediated transcriptional activity. EBP50 knockdown in colonic cancer cells led to reduced cell proliferation, anchorage independent growth, and tumorigenesis in nude mice. Moreover, the immunohistochemistry study identified abnormal EBP50 localization in the nucleus at the tumor invasive fronts the colorectal carcinoma specimens that manifested a poor clinical outcome.