Intrinsic oxidative stress regulates enzymatic activity of protein tyrosine phosphatases and FAK-mediated signal transduction in Ras-transformed fibroblasts

摘要 目前有越來越多的證據指出，活性氧以訊息傳遞分子的角色參與在許多細胞生理現象，包括了細胞生長、細胞凋零與細胞老化等。活性氧參與在訊息傳導路徑主要是透過可逆氧化抑制細胞中蛋白質酪氨酸去磷酸酶(protein tyrosine phosphatases)來調控蛋白質酪氨酸磷酸化程度。過去有許多研究認為細胞轉型和癌症的發生與細胞持續不斷製造活性氧有關。本篇論文主旨是針對在轉型細胞中，大量的活性氧在細胞訊息傳遞上所扮演的角色功能探討。 在正常細胞與致癌基因Ras與Src引發之轉型細胞的比較上，我們觀察到轉型細胞製造高量活性氧的同時，細胞中酪氨酸磷酸化蛋白程度也隨之上升。當以細胞中負責產生活性氧之酵素Nox的抑制劑DPI來抑制轉型細胞所製造的活性氧時，實驗結果顯示細胞蛋白中酪氨酸磷酸化有減少的現象發生。在此同時，轉型細胞在有DPI的處理下發生了細胞型態的變化。從我們的實驗結果發現，對於細胞遷移具有重要功能的focal adhesion kinase (FAK)第397酪氨酸的磷酸化在DPI處理後會有減弱的情形。而在Ras轉型細胞中另一個重要的蛋白激酶Src，其活性在長時間DPI的處理後也會減低。同時在FAK上屬於Src磷酸化位置的酪氨酸也會隨著Src活性的減弱有磷酸化下降的現象。在本實驗亦證實，經過DPI處理抑制活性氧，會造成細胞中受到可逆氧化調控的蛋白質酪氨酸去磷酸酶酵素活性的恢復。因此我們認為轉型細胞製造高量的活性氧，進而氧化抑制細胞中蛋白質酪氨酸去磷酸酶，以促進細胞中蛋白質酪氨酸磷酸化。這個機制所造成的影響，包括了對於FAK與Src主導的訊息傳遞路徑。Reactive oxygen species (ROS) have emerged as intracellular signaling molecules in multiple cellular processes such as proliferation, apoptosis, and senescence. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, particularly protein tyrosine phosphatases (PTPs). Recent studies suggested that the abnormally high levels of cellular ROS production are concurrent with the development of human diseases such as cancers. We examined the role of ROS in the control of signal transduction in Ras- and Src-transformed NIH-3T3 cells which constitutively produce more ROS and possess more active tyrosine phosphorylation signals than parental NIH-3T3 cell. Treatment of transformed cells with diphenyleneiodonium (DPI), a NADPH oxidase (Nox) inhibitor, led to the suppression of cellular ROS production. Under this condition, we found that the activity of endogenous PTPs was elevated, whereas the tyrosine phosphorylation level of cellular proteins was decreased. Such an effect was more pronounced in Ras-transformed cells, compared with that in Src-transformed cells, suggesting that Ras-mediated signal transduction is essentially ROS-dependent. This hypothesis was further investigated. In Ras-transformed cells treated with DPI, a 120 kDa protein, which showed a significant decrease of tyrosine phosphorylation, was subsequently identified as focal adhesion kinase (FAK). Upon the inhibition of cellular ROS production, FAK was dephosphorylated at tyrosine 397 (Tyr397), an integrin-induced auto-phosphorylation site whose phosphorylation activates FAK and enhances cell migration. Treatment of DPI also led to the decrease of phosphorylation levels of Tyr576, Tyr577, Tyr861 and Tyr925 of FAK, concurrent with the decreased kinase activity of Src that recognizes and phosphorylates those Tyr residues in FAK. We also observed that the overexpression of dominant negative Rac1N17, which has been shown to block Nox activity, resulted in the dephosphorylation of FAK and Src. Furthermore, we showed that Ras-transformed cells lost their membrane protrusions in a time-dependent manner in response to DPI treatment, concomitant with the reactivation of cellular PTPs that were originally undergoing reversible oxidation. Our data thus suggested that the Ras-mediated migration signal is regulated by the ROS-dependent activation of FAK and Src through constitutive oxidation and inactivation of endogenous PTPs.目錄 中文摘要 …………………………………………………………………1 英文摘要 …………………………………………………………………2 緒論 ………………………………………………………………………3 實驗材料…………………………………………………………………16 實驗方法…………………………………………………………………21 實驗結果…………………………………………………………………26 討論………………………………………………………………………36 圖表………………………………………………………………………43 附圖………………………………………………………………………64 參考文獻…………………………………………………………………6