酵母菌組蛋白激酶Ipl1 複合體之親和純化與功能性鑑定

Abstract

組蛋白H3第10號絲胺酸之磷酸化，為一具有兩種意義之基因外分子遺傳標誌，其首先被發現高度發生於進行有絲分裂的細胞中，推測可能與染色體濃縮相關；然而此磷酸化現象，也在經有絲分裂源刺激的細胞及轉錄活化的基因座上被觀察到。因此，組蛋白H3第10號絲胺酸之磷酸化似乎和兩種截然不同的染色質狀態有關，而專一進行此磷酸化反應的酵素也已被發現。證據顯示出芽酵母菌中，Ipl1在有絲分裂期間是該絲胺酸的主要修飾者。 Ipl1是出芽酵母菌中唯一的極光激酶，其主要在細胞分裂中期藉由磷酸化下游受質來調節紡錘絲的雙向性。除此之外，在分裂不同時期發生的染色體濃縮、紡錘體關卡的活化及胞質分裂等詎料也需要該酵素的活性參與。 Ipl1的完整功能性依賴與其結合的蛋白質，因此在本研究中，首先致力於Ipl1蛋白複合體的純化，此部份採取並列親和純化的技術。藉由在Ipl1的C端加入TAP摽記，使用硫酸銨初分劃加上兩步驟的親和純化，可以得到足夠純度的Ipl1複合體。結果顯示Ipl1在自然狀態下為一具有三個次單元的複合體，根據分子量預測及前人的研究，該複合體中的另兩個次單元應為Sli15及Bir1。 為了更進一步論證Ipl1和其交互作用者，特別是Sli15在功能上的重要性，因此製造出數個ipl1的突變種。溫度敏感突變株ipl1T260A及ipl1P340L並不影響其與Sli15的交互作用，然而它們均使激酶活性下降。胜肽刪除突變種ipl1ΔK166-V185，抑制了大部分和Sli15的交互作用卻僅造成相對較低的活性喪失，說明K166-V185可能是與Sli15結合的重要區域。在此和Sli15交互作用缺乏的突變株中，細胞週期遭受干擾而產生大規模細胞死亡及染色體不穩定的表現型。另外分析Ipl1及Sli15的表現量，發現有些許差異，且結果暗示在細胞週期期間Ipl1必然會與Sli15結合。最終發現若失去與Sli15之結合，會使有絲分裂時的組蛋白磷酸化狀態下降，此觀察亦說明Sli15可以幫助Ipl1正常執行組蛋白激酶的功能。

Histone H3S10 phosphorylation is an epigenetic mark with two faces. It was first described to occur at high levels in mitotic cells and to be correlated with chromosome condensation. However, phosphorylation on this residue was also found in mitogen-stimulated cells and transcriptionally activated loci. Thus, histone H3S10 phophorylation seems to be associated with two chromatin states. Enzymes that specifically catalyze this phosphorylation reaction were also studied. In budding yeast, it has been shown that Ipl1 is a major modifier on histone H3S10 during mitosis. Ipl1 is the only aurora kinase in S. cerevisiae and it regulates the spindle biorientation during metaphase by phosphorylating downstream substrates. Concerning other functions during cell division, it has been reported that cellular pathways like chromosome condensation, spindle checkpoint activation and cytokinesis also requires the enzyme activity. Fully functional Ipl1 relies on its binding partners. In this study, I devoted myself to purification of Ipl1 complex first. The technique of tandem affinity purification (TAP) was applied. By addition of a TAP tag into the C-terminal of Ipl1, ammonium sulfate pre-fractionation and two successive affinity purification steps yielded Ipl1 complex with sufficient purity. The results suggested Ipl1 forms a three-subunit complex in normal status. Based on molecular weight predictions and previous studies, the other two subunits should stand for Sli15 and Bir1. To further demonstrate the functional importance between Ipl1 and its interactors, especially Sli15, multiple ipl1 mutants have been generated. The temperature-sensitive mutants, ipl1T260A and ipl1P340L, did not affect the interactions between Ipl1 and Sli15. However, they reduced the kinase activity overall. Peptide truncation mutant ipl1ΔK166-V185 abolished most affinity with Sli15 but resulted in a relatively minor activity loss. This suggested K166-V185 might serve as the critical interaction region with Sli15. Phenotypes in this interaction-deficiency mutant showed that cell cycle was disturbed, resulted in large population of cell death and genome instability. By analyzing the expression levels of Ipl1 and Sli15, they showed differential patterns which implied a constitutively Sli15-bound Ipl1 during cell cycle. At last, loss of Sli15 binding also led to reduction of the mitotic histone H3 phosphorylation status. The observation suggested Sli15 also functions in guiding Ipl1 as a histone kinase.