探討細胞核內的 βII-tubulin 蛋白對於 Notch 訊號傳遞路徑之調控

1. 探討細胞核內的βII-tubulin蛋白對於Notch訊號傳遞路徑之調控探討細胞核內的βII-tubulin蛋白對於Notch訊號傳遞路徑之調控 • Notch接受子對於生物的發育過程扮演很重要的調控角色。Notch-1接受子的活化是透過Notch-1接受子細胞外區域與鄰近細胞的ligand接觸，造成Notch-1接受子經過蛋白水解反應而釋放出Notch-1接受子細胞內區域( N1IC, Notch1 intracellular domain)。Notch-1接受子細胞內區域移動至細胞核中與RBP-Jκ結合 ( RBP-Jκ是結合在DNA上的蛋白)，形成N1IC/RBP-Jκ複合體，並且取代RBP-Jκ/histone deacetylase複合體結合在DNA上，活化Notch訊息傳導的下游基因。之前本實驗室曾經利用yeast two-hybrid 方法尋找與Notch-1接受子結合的蛋白質，βII-tubulin在此實驗被證實與N1IC有結合關係。βII-tubulin在之前研究被發現存在C6 glioma cell、MCF-7 breast carcinoma cell、HeLa cervix carcinoma cell等細胞株的細胞核中(Keliang Xu, 2002)。在此，本論文欲證實βII-tubulin與N1IC的結合關係，並且探討βII-tubulin在Notch訊息傳導所扮演的角色。首先，本論文在K562以及HeLa細胞株的細胞核偵測到βII-tubulin蛋白，並且進一步證實在HeLa細胞株的細胞核中，細胞核內的βII-tubulin會與Notch-1接受子有結合關係。以taxol處理K562以及HeLa細胞株後，經由CBF-1媒介的Notch-1訊息傳導會受到活化；以colchicine處理細胞則不影響經由CBF-1媒介的Notch-1訊息傳導。此外，以taxol處理K562以及HeLa細胞株，會造成細胞核內βII-tubulin、α-tubulin蛋白量的上升，以colchicine處理細胞則不影響細胞核中的βII-tubulin、α-tubulin蛋白含量。經由不同時間點的實驗證實經由taxol處理而進入細胞核的βII-tubulin、α-tubulin可以穩定存在細胞核中。以上結果顯示細胞核內βII-tubulin與Notch-1接受子在癌細胞細胞核內有結合關係，並且可以調控Notch-1訊息傳導。

2. Roles of the nuclear βII-tubulin in the modulation of Notch signaling • The Notch transmembrane receptors play important roles in cell fate decisions during development. Ligand binding initiates the signal through the proteolysis of Notch-1 receptor to generate the Notch-1 intracellular domain (N1IC). The N1IC translocates into the nucleus to target the DNA-binding protein RBP-Jκ. The N1IC/RBP-Jκ complex overcomes the transcriptional repression by replacement of the RBP-Jκ/histone deacetylase complex to activate the target genes. Previously, we used yeast two-hybrid assay to identify proteins which associate with Notch-1 receptor, and βII-tubulin was identified to associate with N1IC. βII-tubulin was detected in the nuclei of C6 glioma cell, MCF-7 breast carcinoma cells, HeLa cervix carcinoma cells, etc (Keliang Xu, 2002). The aim of this paper was to characterize the roles of the nuclear bII-tubulin in the modulation of Notch signaling. Herein, I found that βII-tubulin was localized in the nuclei of HeLa and K562 cells. Furthermore, βII-tubulin associated with N1IC in the nucleus of HeLa cells. The CBF1-mediated transactivation activity of N1IC was modulated by taxol in K562 and HeLa cells, but not by colchicine. The contents of both nuclear βII- and a-tubulins were elevated by taxol in K562 and HeLa cells but not by colchicine. In time course experiment, the induction of nuclear βII-tubulin by taxol was stable in the nucleus of HeLa cell. These results suggested that nuclear βII-tubulin associated with the activated Notch-1 receptor to modulate Notch signaling.