2014-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647702摘要：磷酸化蛋白 50(Ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50))乃主要幫助蛋白與蛋白在細胞膜彼此互動的蛋白。我們最近的研究發現，此一高度磷酸化的蛋白會異常地表現於大腸直腸癌細胞的細胞核中，藉由恢復了β-catenin 和TCF-1 複合物的穩定性，進而促進細胞朝癌化生長的過程進展 (J Clin Invest. 2012;122(5):1881–1894.)。利用 siRNA 干擾技術，我們篩選到 RSK1 (ribosomal s6 kinase 1)可調控磷酸化蛋白 50 進入細胞核的過程。我們也發現 RSK1 蛋白羧基端的PDZ motif 可與磷酸化蛋白 50 第一 PDZ domain 結合，並且血清刺激會促使兩者分離。初步實驗結果顯示 RSK1 可能以磷酸化蛋白 50 為其受質，對其中一段含有RXRXXS/T 氨基酸序列的酥胺酸 (Threonine)進行磷酸化反應。為進一步研究對RSK1 影響 EBP50 在細胞核內表現之分子機轉及可能蘊含之細胞生物學重要性，我們將製備能夠鑑識磷酸化蛋白 50 該一特定磷酸化酥胺酸的專一性抗體，此外也製造能夠表現該一特定酥胺酸位點的擬磷酸化與抗磷酸化質體並穩定細胞株。由於 RSK1之活性已知是由 Ras-Raf-Erk 訊息傳遞途徑來調控，因此我們將收集大腸直腸癌細胞的病理檢體，進行 Ras 基因定序，RSK1 之活化的程度，以及磷酸化蛋白 50 異常地表現細胞核的程度，進行相關性的研究。我們假設會觀察到一些大腸直腸癌的病人，他們是有野生型 Ras 基因定序，但是磷酸化蛋白 50 卻也仍舊進入細胞核，我們將關注這類大腸直腸癌病人是否比具有野生型 Ras 基因定序，但磷酸化蛋白 50 卻不會進入細胞核的大腸直腸癌病人，有更為不良的預後。 <br> Abstract: Ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) is a scaffold protein that mediates protein–protein interactions at plasma membrane. We recently reported this highly phosphorylated protein is aberrantly expressed in the nucleus of colorectal cancer cells and facilitates colon tumorigenesis by modulating the interaction between β-catenin and TCF-1 (J Clin Invest. 2012;122(5):1881–1894.). Further experimental approaches including RNAi library screening indicate that RSK1 (ribosomal s6 kinase 1) regulate nucleocytoplasmic trafficking of EBP50. RSK1 binds to the first PDZ domain of EBP50 through its C-terminal PDZ binding motif. This interaction apparently decreased upon serum stimulation. Interestingly, our preliminary assay showed that EBP50 at the threonine 156 could be phosphorylated by RSK1 in an in vitro kinase assay. To study the regulatory molecular mechanism by RSK1 for EBP50 nuclear retention, we plan to generate phosphorylation specific antibody and construct phosphor-mimetic and phosphor-resistant EBP50 mutant plasmids at the potential RSK1 action site of EBP50 and corresponding stably transfect clones to study the effects in the interaction between EBP50 and RSK1 and the cell biological effects.. Furthermore, RSK1 is known to be regulated by Ras-Raf-Erk signaling pathway and we would examine the nuclear EBP50 expression pattern in colorectal cancer specimen, for both the Ras wild type and oncogenic mutated samples. We hypothesize that there exists a group of patients who might have bypassed the requirement of Ras oncogenic mutation along the carcinogenic progression by establishing nuclear EBP50 localization.ribosomal s6 kinase 1磷酸化蛋白 50ribosomal s6 kinase 1EBP50