2008-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/679043摘要：在我們近年的研究已發現Grb7在與phosphoinosides結合後，可進一步被FAK磷酸化；此一過程對於FAK/Grb7調控細胞移動扮演關鍵的角色。為了進一步瞭解FAK/Grb7可促進細胞移動的分子機制及其訊息傳導路徑，我們將首先利用定點突變及質譜儀分析Grb7被FAK磷酸化的麩氨酸位置。而所確認可被FAK磷酸化的Grb7突變株將可被用來探討此一磷酸化在細胞移動的角色和功能，以及其所誘發、調控的訊息傳導路徑為何。同時，我們也正在建立以lentiviruses為基礎的基因knockdown的系統。利用此knockdown的技術，也可提供探討Grb7在細胞內生化和功能的分子工具。另外，Grb7常被報導與乳癌的轉移有高度的正相關性，然而，其分子機轉則是缺乏未知，因此對於其是否成為抗癌藥物的標的分子無法有有效的認知。我們已建立起一套標準有效的免疫組織染色，可清晰偵測Grb7蛋白質大量表現於胃癌及直腸癌病人檢體。同時，以人類癌症細胞株進行西方點漬分析，也發現SkBr3和A431乳癌細胞及HCT116直腸癌細胞有Grb7的表現。因此，我們將可利用此一標準流程提供於臨床上的診斷參考。而本研究所獲得到的Grb7在細胞移動及癌症轉移上的分子機制，將可提供未來抗癌藥物研發的標的。<br> Abstract: Recently, we have found that the promotion of Grb7 in cell migration requires phosphoinositide binding and subsequently tyrosine phosphorylated by FAK in an adhesion dependent manner. To further elucidate the molecular mechanism and downstream signaling of FAK-Grb7 mediated cell migration, we first map the phosphorylation site(s) of Grb7 by FAK using the site-directed mutagenesis analysis and MALDI-TOF approach. The identified site(s) will be further investigated the role in cell migration and its downstream signaling by performing cell migration assay, western blots, and mutagenesis study. Consistent with the role in cell migration, Grb7 has also been reported crucial in metastasis in breast cancer. To understand the significance of Grb7 in human cancer, we also performed the immunohistochemical staining of Grb7 on various human cancers. Interestingly, we found that over-expression of Grb7 is prominent in gastric and colon cancers, thereby prompting us to further sought for the importance and involvement of Grb7 in tumor progression. Therefore, we are establishing Grb7 and varied mutant stable cell lines in HCT116 (colon cancer), A431 (breast cancer), and SkBr3 (breast cancer) tumor cells. In addition, lentivirus-based Grb7 knockdown technique is employed to proceed the loss of function studies in Grb7 in vitro and in vivo. These studies will not only provide the molecular and cellular mechanism of FAK-Grb7 mediated cell migration, also shed a light on the understanding the pathological role of Grb7, an emerging adaptor protein, in metastasis as well as potential diagnostic and therapeutic applications.FAK (focal adhesion kinase)Grb7 (Growth factor receptor bound protein 7)細胞移動癌症轉移訊息傳導FAK (focal adhesion kinase)Grb7 (Growth factor receptor bound protein 7)cell migrationmetastasissignal transduction