2014-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/708791摘要：Growth factor receptor bound protein-7 (Grb7) 為一不具有酵素活性的銜接蛋白 (adaptor), 屬 Grb family 的一員,其具有數個功能性區域可與許多訊息分子產生交互作用而活化多重之訊息傳遞路徑以調控細胞功能。由目前研究中指出 Grb7 基因 在某些乳癌細胞中會與 Her2/erbB2 ( human epidermal growth factor 2 )有共同大量 表現 (co-amplification) 的現象, 並可能參與癌症的轉移(metastasis)、侵襲(invasive) 及腫瘤生成 (tumorigenesis) 的過程。為了進一步探討 Grb7 所參與之訊息調控,先 前實驗室由酵母菌雙雜合分析 (yeast two-hybrid assay) 中篩選會與 Grb7 進行交互作用之下游蛋白,發現 Grb7 與 Pin1 蛋白 ( peptidyl-prolyl cis/trans isomerase ) 間具有大量結合之現象。Pin1 為一可針對磷酸化受質蛋白之特定 pSer/Thr motif 進行 cis/trans 異構化以調控蛋白的穩定、活化及構形。藉由共免疫沉降和定點突變分析證實 Pin1 和 Grb7 具有直接之交互作用,且確認 Grb7 上 Pin1 可能之主要結合位置為 pSer194-Pro motif。另外,藉由 lentiviral-based gene silencing 技術,發現在 Pin1 基因表現抑制的情況下,Grb7 的蛋白表現量有提高之趨勢,進一步利用即時定量 RT-PCR 及 cycloheximide pulse-chase 分析中顯示 Pin1 可能在磷酸化蛋白之後修飾機制中透過與Grb7之pSer194-Pro motif結合而負向調控Grb7蛋白的穩定性。事實上，我們發現 Pin1 可運用其異構化活性 (isoermase) 造成 Grb7 易於被 ubiquitinated 進而進入 proteasome proteolysis 的系統中被降解。綜合上述，我們提出了 Pin1 與 Grb7 結合對癌症進程 (progression) 和惡化（malignancy) 具有負調控的角色。為了證明此一假說，本&#30799;究計劃將試圖進一步以不同的 in vitro 以及 in vivo 的癌症模式來探討 Grb7 和 Pin1 的交互作用的分子機制、調控的訊息傳遞以及在癌症病理意義。藉此,希望對 Grb7 和 Pin1 蛋白間之研究可協助我們對癌症生成的過程及訊息路徑有更進一步的了解並提供相關疾病未來治療策略的研究和應用。<br> Abstract: Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways to regulate various cellular functions. The Grb7 gene is often co-amplified with EGF or HER2/erbB2 receptors to potentiate metastasis, invasion and tumorigenesis of breast cancers. Thus, Grb7 is an adverse prognostic marker in breast cancers. Previously, in a yeast two-hybrid screening, we found a novel interaction between Pin1 and Grb7. Pin1 is a peptidyl-prolyl cis/trans isomerase capable of isomerizing the specific pSer/Thr-Pro bonds and such conformational change leading to the alterations in protein functionality, stability, and/or intracellular localization. By co-immunoprecipitation and mutational analysis, we found that Pin1 enables directly interacting with Grb7 through the phospho-Ser 194-Pro motif on Grb7 and the WW domain of Pin1. Moreover, knockdown of Pin1 expression through lentiviral-mediated gene silencing could retain elevated Grb7 expression. Furthermore, utilizing cycloheximide pulse-assay and quantitative RT-PCR analysis, we conclude that Pin1 could negatively regulate Grb7 protein stability at the post-translational level. Indeed, we found that Pin1 could exert its isomerase activity to modulate Grb7 protein expression in an ubiquitin/proteasome-dependent proteolysis pathway. Thus, we hypothesized that the Pin1-Grb7 complex formation enables negatively regulating tumor progression and/or malignant processes due to the influence of Grb7 protein expression. To prove this idea, in this proposed study, we attempt to further investigate the molecular and cellular mechanisms as well as pathologic significance of the Pin1-modulated Grb7 expression using various in vitro and in vivo cancer models.Grb7Pin1ubiquitinisoermase惡化Grb7Pin1ubiquitinisoermasemalignancy