以磷酸化蛋白質譜鑑定乳癌激酶之受質

Abstract

乳癌激酶是一種非受體型酪胺酸激酶，以經常在乳癌中過量表現而聞名。過去研究指出位於表皮生長激素（EGF）傳遞路徑下游的乳癌激酶能促進細胞的增殖和移動，而這些功能被認為來自於它對細胞中某些蛋白的磷酸化所造成，然而至今卻只發現它極少數的受質。因此在本篇論文中我們利用系統性蛋白質體學方法來比較過量表現和沒表現乳癌激酶的細胞裡有被酪胺酸磷酸化的蛋白之間的差異，並藉此找出一些可能是乳癌激酶受質的蛋白。當那些有被酪胺酸磷酸化的蛋白經免疫親和純化和胰蛋白酵素分解後，其中的磷酸化胜肽經質譜分析可發現有127種磷酸化蛋白特別出現於過量表現乳癌激酶的細胞中。大部分被發現的蛋白都會和核醣核酸作用且參與多種不同的核酸代謝途徑，例如核酸剪接與加工和去氧核醣核酸的轉錄。除此之外，當中還有某些蛋白會調控細胞移動和細胞骨架的變動。而且乳癌激酶的一些已知受質Sam68、SLM-2、PSF也在其列。接著我們從中選出GIT2和ARHGEF6這兩個蛋白來證實是否為此激酶的受質。結果發現乳癌激酶在細胞中不僅使這兩個蛋白的磷酸化程度上升，也與它們有著交互作用。另外我們也發現GIT2的Y286和Y592這兩個酪胺酸位點可被乳癌激酶給磷酸化。最後我們又另外研究一個名叫EPS8且早已被我們實驗室發現也是乳癌激酶受質的蛋白。我們以生物體外激脢活性試驗證實ESP8確實是乳癌激酶的受質，也連帶確定了此蛋白中的某五個酪胺酸位點可被此激酶給磷酸化。整體來說我們的研究發現了數個乳癌激酶下游的作用目標，並可做為未來研究乳癌激酶生物功能的基礎。

Breast tumor kinase (Brk), a non-receptor tyrosine kinase, is frequently overexpressed in breast cancers. Previous studies have shown that Brk acts downstream of epidermal growth factor to promote cell proliferation and migration. These biological functions of Brk are thought to be resulted from its phosphorylation of cellular proteins, but only a limited number of substrates have been discovered to date. In the thesis, we took use a systematic proteomic approach to identify putative Brk substrates by comparing the tyrosine phosphorylated proteins in Brk-overexpresing cells and control cells. Using immunoaffinity purification of tyrosine-phosphorylated proteins followed by enrichment of phosphopeptides, 127 phosphoproteins are observed only in Brk-overexpresing cells. Most of them belong to RNA-binding proteins involving in various RNA metabolic processes, such as splicing, transcription, and RNA processing. In addition, several of them are known to elicit effects on regulating actin cytoskeleton and/or cell migration. Importantly, several known Brk substrates, including Sam68, SLM-2, and PSF, are also recovered. We next validate the Brk-induced tyrosine phosphorylation of two identified proteins, GIT2 and ARHGEF6. Importantly, Brk not only promotes their phosphorylation in vivo but also physically interacts with both proteins. Moreover, we show that the tyrosine residues Y286 and Y592 of GIT2 are both involved in Brk-induced phosphorylation. Finally, we have extended our analysis to EPS8, another putative Brk substrate identified previously in our laboratory. Using in vitro kinase assay, we demonstrate EPS8 as a direct substrate of Brk and identify five Brk-targeting tyrosine residues. Together, our analysis has uncovered several Brk downstream targets, which would provide a basis for future analysis of Brk’s biological functions.