熱休克蛋白70在三氧化二砷誘引有絲分裂停滯細胞為Plk1受質之 探討

Abstract

在過去的研究中發現HeLa S3細胞處理三氧化二砷 (ATO) 之後會停滯在有絲分裂期；當處理激酶 (kinase) 抑制劑staurosporine會迫使這些停滯的細胞離開有絲分裂期。這過程中的詳細機制至今仍未明，為了瞭解在這些被三氧化二砷處理下所停滯細胞中的分子機制，本論文中採取了高效率的蛋白質體學來分析staurosporine 處理三氧化二砷所停滯的細胞後所產生的磷酸蛋白質體的變化（意指大量或是一系列蛋白質上的磷酸根產生變化，增加或減少 ）。在這一系列的蛋白質中，發現有一些蛋白，例如：Hsp70, Rad23B和eIF4B等 的上游激酶是Plk1。這是一個全新的發現，更重要的是Plk1是一個在細胞分裂週期中扮演很重要角色的激酶。由於三氧化二砷會引起細胞大量表現Hsp70，所以我們後續探討Plk1和Hsp70之間的關係。為了進一步證實Hsp70上游的激酶是Plk1，而後利用了二氧化鈦純化和質譜儀來鑑定Plk1磷酸化Hsp70之氨基酸位置，結果顯示在Thr13, Ser362, Ser631和Ser633 這幾個位置都可以被Plk1磷酸化。進一步使用針對Hsp70磷酸化專一性的抗體和抑制Plk1的激酶活性（沒有活性的Plk1加上Plk1的化學專一抑制劑）證實了Plk1的確可以磷酸化Hsp70，至少在Ser631。另外，Plk1和Hsp70的確在三氧化二砷所導致有絲分裂期中是互相結合在一起的。因此，從這部分的研究可以得知在三氧化二砷所誘引的有絲分裂停滯細胞中，Plk1藉由大量磷酸化Hsp70的方法來延緩或避免細胞的死亡。 在有絲分裂期中，紡錘絲會形成雙軸分佈；在每一個特定的細胞種類裡紡錘絲的雙軸之間的距離一般而言是固定的。然而，過去研究發現在三氧化二砷所誘引的有絲分裂期的細胞中，紡錘絲的雙軸之間的距離特別長，為了要瞭解其間究竟是什麼調控了紡錘絲的長度。透過上面的研究，發現Plk1與Hs70之間的交互作用可以在三氧化二砷所導致有絲分裂期的細胞中調控紡錘絲的雙軸之間的距離。利用免疫沈澱以及螢光染色，首先發現了Plk1和Hsp70在細胞中的中心粒的位置互相結合。此外，也進一步利用質譜儀鑑定了在Hsp70上Ser631和Ser633 被Plk1磷酸化會使得Hsp70座落於中心粒上進而調整三氧化二砷所導致有絲分裂期的細胞中調控紡錘絲的雙軸之間得距離。除此之外，利用點突變的方法，將Hsp70上Ser631和Ser633 突變成Asp後，將此突變之Hsp70轉染進入細胞中會使的細胞的紡錘絲對於nocodzole對其降解的速度變慢。由此可推知，在三氧化二砷所導致有絲分裂期的細胞中，Plk1 磷酸化Hsp70上Ser631和Ser633使其座落於中心粒上去調控紡錘絲的雙軸之間得距離，另外可以推測是因為Hsp70具有保護或抑制紡錘絲降解的動力所造成的。 綜合上述，利用蛋白質體學來研究三氧化二砷所導致有絲分裂期的細胞中的分子機制變化，這樣的研究可以幫助未來在使用三氧化二砷於臨床治療的使用。更重要的是，發現了Hsp70為Plk1的全新受質，這不僅是提供了對於三氧化二砷如何影響或調控有絲分裂期間激酶的活性，並提供了Plk1在細胞壓力下的角色，尤其是和Hsp70之間的交互作用調控細胞中有絲分裂的紡錘絲以及死亡機制。

It has been previously demonstrated that when arsenic trioxide (ATO)-induced mitotically arrested HeLa S3 cells (AIMACs) were treated with staurosporine the cells rapidly exited mitosis. To better define the cellular targets and the underlying mechanisms of AIMACs, we applied 2-D DIGE followed by LC-MS/MS analysis and showed that staurosporine induced a significant change in the phosphoproteome of AIMACs. Among the proteins whose phosphorylation was modulated by staurosporine, we identified Hsp70, Rad 23B, and eIF4B as potentially new substrates of Plk1, an essential serine/threonine kinase with versatile mitotic functions. Since Hsp70 is a stress protein responsible for ATO treatment, we further identified Thr13, Ser362, Ser631 and Ser633 on Hsp70 intracellularly phosphorylated in AIMACs by combining TiO2 phospho-peptides enrichment and MS/MS analysis. Using antibody specifically against phosph-Ser631 Hsp70 and further aid by expression of kinase-dead Plk1 and pharmacological inhibition of Plk1, we concluded that Ser631 on Hsp70 is phosphorylated by Plk1 in AIMACs. By immnuofluorescent staining, we found the colocalization of Hsp70 and Plk1 in AIMACs but not in interphase cells. In addition, Plk1-mediated phosphorylation of Hsp70 prevented AIMACs from mitotic death. The results reveal that Hsp70 is a novel substrate of Plk1 and that its phosphorylation contributes to attenuation of ATO-induced mitotic abnormalities. It is known that the bipolar spindle formation is essential for faithful chromosome segregation mitosis. Moreover, during cell division, the mitotic spindles maintain constant length in given cell types, especially in metaphase. Recently, it has been reported that the abnormal mitotic spindle may result in cellular defects after cell division. However, the abnormal elongated spindle was observed in AIMACs, and how these processes are regulated is unknown. Here, this thesis showed that Plk1 interplayed with Hsp70 to regulate mitotic spindle length in AIMACs. First of all, using immunoprecipitation and immunofluorescence microscopy, Plk1 and Hsp70 were found to colocalize at the centrosome in mitotically arrested cells. Moreover, co-immunoprecipitation of Hsp70 by antibody against γ-tubulin, site-directed mutagenesis, the results showed that Hsp70 located at centrosome was likely phosphorylated by Plk1 at Ser631 and Ser633 in mitotically arrested cells and that these phosphorylations were required direct phosphorylated Hsp70 to the centrosome and thereby set the length of mitotic spindle. Cells with expression of Hsp70 in which Ser631 and Ser633 mutated to Asp were more resistant to nocodazole depolymerization. Therefore, this study concluded that Plk1 contributes to the targeting Hsp70 to mitotic spindle poles, suggesting a model in which Hsp70 regulates spindle length by inhibiting microtubule depolymerizing activities at spindle poles. Proteomic approach provides a systematic platform to consider insight into the molecular mechanism in AIMACs and thereby a rationale to evaluate the therapeutic effect of ATO, especially the mitotic catastrophe. More importantly, identification of Hsp70 as a novel substrate of Plk1 offers not only a more comprehensive understanding of how ATO can affect mitotic kinase but an important clues for the role of Plk1 under stress condition, especially in mitosis and the dynamics of mitotic spindle mediated by Hsp70.