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標題: | 核分裂蛋白之結合蛋白(NuSAP)的構造與功能性分析 Structural and Functional Analysis of NuMA-s Associated Protein (NuSAP) |
作者: | 蔡坤穎 |
出版年 : | 1998 |
學位: | 碩士 |
摘要: | 核分裂蛋白NuMA(Nuclear Mitotic Apparatus)在細胞內的分佈與細胞週期有密切的關係。NuMA在細胞間期時位於細胞核內;分裂期(M phase)時則移動至紡綞極(spindle pole)附近。近來研究顯示,NuMA是核骨架蛋白(nuclear matrix)的成分之一,可能與RNA裁接(splicing)及穩定核骨架有關;有絲分裂時NuMA會和微小管(microtubule)上的動力蛋白(motor protein)dynein形成complex,穩定紡綞極的構造。 本實驗室藉由cDNA library篩檢定序及RT-PCR的方法,發現人類NuMA至少有NuMA-l、NuMA-m及NuMA-s等三種異型體(isoform)。NuMA-l為主要異型體,間期時位於細胞核內;NuMA-m及NuMA-s因RNA替代裁接(alternative splicing)而移除NuMA-l擁有的nuclear localization signal。這兩種異型體於間期時不分佈於細胞核內而分佈於中心體(centrosome)附近,當細胞進入分裂期則與NuMA-l同樣位於紡綞極附近。 為了瞭解NuMA-s的功能,本實驗室利用酵母菌雙雜合系統(yeast two hybrid)分離出與NuMA-s C端331個氨基酸相結合的蛋白。經由cDNA library的篩檢定序發現這是一個新發現的蛋白,並將其命名為NuSAP(NuMA-s Associated Protein)。NuSAP蛋白cDNA全長1757個鹽基對,可轉譯出390個氨基酸,其mRNA在人類十六種組織中均有表現,而其蛋白C端氨基酸序列則具有部分kinase保守序列。 為了證實NuMA-s的確是存在於細胞中,且比較其表現與NuMA-l的差異,我利用NuMA-l與NuMA-s特有序列做探針,偵測人類十六種組織中這兩種mRNA的表現。NuMA-l探針可辨認到一個明顯的8.0?8.5kb band,而NuMA-s探針則能辨認到微弱的9.5?10kb band。此外,比較NuMA-l及NuMA-s在十六種組織間表現的模式顯示,大部分組織有類似的表現模式,僅有骨骼肌、肝?及胰臟有較大的差異存在。這些結果使我們確信,NuMA-s異型體存在於正常組織細胞中,且其表現與NuMA-l略有不同。 確認NuMA-s與NuSAP蛋白間結合的實驗,包括colony-lifted assay,以及免疫沉澱法(immunoprecipitation)。以不同的NuMA-s C端片段與NuSAP蛋白進行colony-lifted assay發現,NuMA-s僅須要其特有序列的26個氨基酸便可與NuSAP蛋白結合,而包含NuMA-l特有序列的片段卻無法與NuSAP蛋白結合,顯示這個NuMA與NuSAP蛋白的結合可能是NuMA-s所特有的。此外,利用免疫沉澱法也證實了NuMA與NuSAP蛋白在哺乳類子宮頸癌細胞株SiHa中確實能結合。 在朝著NuSAP蛋白功能性角色研究的同時,本實驗室利用酵母菌雙雜合系統以NuSAP蛋白分離出與其結合之蛋白,這個蛋白居然是NuSAP本身。利用in vitro binding證實了NuSAP的dimerization特性,而酵母菌雙雜合系統則顯示NuSAP蛋白的dimerization區域位於氨基酸78至137之間。 將純化後的NuSAP-pET融合蛋白immunize紐西蘭白兔並獲得多源性抗體(polyclonal antibody),這個抗體可辨認大腸桿菌表現的NuSAP-pET, NuSAPN-pET(NuSAP蛋白N端)及pET蛋白,卻無法辨認哺乳類細胞表現的NuSAP蛋白。由於無法以NuSAP多源性抗體偵測哺乳類細胞中的NuSAP蛋白,因此短暫轉染(transient transfection)NuSAP-GFP或NuSAP-FLAG,使其於SiHa細胞中表現。免疫螢光染色顯示NuSAP-GFP或NuSAP-FLAG融合蛋白均分佈於細胞核與細胞質,且活體追蹤觀察過量表現NuSAP-GFP融合蛋白的SiHa細胞均能正常分裂。 一般的kinase活性測試是以抗體將kinase免疫沉澱下來,再以受質測試其被磷酸化的程度。由於NuSAP抗體無法辨認哺乳類細胞表現的NuSAP蛋白,因此我利用大腸桿菌表現並純化之NuSAP-pET(包含C端kinase-like domain)、NuSAPN-pET(去除C端kinase-like domain)以及pET蛋白於極稀釋的SiHa cell lysate下進行kinase活性測試。NuSAP-pET及NuSAPN-pET蛋白在SiHa cell lysate下可提高其32P標定,而pET蛋白則無32P標定的情形,顯示NuSAP及NuSAPN蛋白會被磷酸化。進一步將NuSAP-pET及NuSAPN-pET蛋白於cell lysate下同時進行kinase活性測試可發現NuSAP-pET是較NuSAPN-pET好的磷酸化受質。若將NuSAP-pET以65℃加熱處理不同時間使蛋白失去活性,可發現NuSAP-pET的32P標定隨著加熱時間的增加而逐漸降低。這些kinase活性測試的結果使我們認為NuSAP蛋白是一個被磷酸化調控的蛋白,但也不排除它具有kinase活性的可能性,然而進一步的研究將有助於釐清NuSAP蛋白與磷酸化的關係,進而瞭解NuMA-s在細胞中扮演的角色。 The nuclear mitotic protein (NuMA) changes its subcellular localization in a cell cycle specific manner. In interphase NuMA is presented in the nucleus; in mitosis it relocates to the spindle poles. Recent studies revealed that NuMA is a component of nuclear matrix with functions related to RNA splicing and nucleoskeleton structure stabilization. Other evidences show that NuMA associates with motor protein dynein and stablizes spindle pole structures. At least three different isoforms of NuMA, namely NuMA-1, NuMA-m, and NuMA-s were identified by DNA sequencing and RT-PCR in our lab. The NuMA-1 isoform that carries a nuclear localization signal (NLS) at its tail region is a predominant isoform presents in interphase nuclei, while NuMA-m and NuMA-s isoforms which remove the NLS by alternative RNA splicing, are excluded from the nucleus. In interphase, NuMA-m and NuMA-s isoforms are presented mainly at centrosomal region, but relocated to the mitotic spindle poles during mitosis. In order to clarify the functional role of NuMA-s isoform, our lab has isolated a protein associated with the C-terminal domain (331 amino acids) of NuMA-s by yeast two hybrid system. This novel protein, named NuSAP (NuMA-s Associated Protein), has a full-length cDNA of 1757 base pairs which can translate into a 390-amino acids polypeptide. Northern blot analysis demonstrated that NuSAP mRNA were expressed in 16 human tissues examined, and amino acid sequence analysis revealed that the carboxyl terminal of NuSAP contains a kinase-like domain. In order to prove the true existence of NuMA-s and compare the expression pattern between NuMA-1 and NuMA-s, we used NuMA-1 and NuMA-s specific probes to detect the mRNAs expressed in multiple tissues by Northern blot analysis. NuMA-1 showed an obvious 8.0?8.5kb band, while NuMA-s showed a faint 9.5?10kb band. Both NuMA-1 and NuMA-s revealed a similar expression pattern in most tissues examined, but some differences were observed in skeletal muscle, liver, and pancreas. According to these findings, it is believed that NuMA-s isoform truly exists, and exhibits some differential expression patterns in comparison with NuMA-1. Colony-lifted assay and immunoprecipitation have been performed to confirm the association between NuMA-s and NuSAP. Colony-lifted assay shows that a 26-amino acids polypeptide domain of NuMA-s is necessary and sufficient for interaction with NuSAP. In combination with the finding that NuMA-1 specific domain (235 amino acids) cannot interact with NuSAP, we concluded that NuSAP is specifically associated with NuMA-s, not NuMA-1. Furthermore, the direct interaction between NuMA and NuSAP in SiHa cells (mammalian cervical carcinoma cell line) was confirmed by immunoprecipitation. In order to search for the functional roles of NuSAP, our lab has isolated a protein associated with NuSAP. Surprisingly, this protein is NuSAP itself. Dimerization property of NuSAP has been proved by in vitro binding assay, and the dimerization domain was narrowed down to amino acid 78?137 by yeast two-hybrid system. A polyclonal antibody was raised by immunizing New Zealand white rabbits with purified E. coli-expressed fusion protein NuSAP-pET. The antibody can recognize E. coli expressed NuSAP-pET, NuSAPN-pET, and pET proteins, however, it cannot recognize mammalian cell-expressed NuSAP protein. As a result, we transiently transfected NuSAP-GFP or NuSAP-FLAG and allowed it to overexpress in SiHa cells. Immunofluorescence analysis was performed using GFP and M5 monoclonal antibodies which indicated that NuSAP fusion proteins were expressed in both nucleus and cytosol. Moreover, NuSAP-GFP overexpressing cells can subdivide into daughter cells normally. A general kinase assay was performed by immunoprecipitation followed by quantitating 32P labelling of a specific substrate. Because NuSAP antibody cannot immunoprecipitate mammalian expressed NuSAP protein, we could only perform kinase assay using E.coli-expressed NuSAP-pET (with kinase-like domain), NuSAPN-pET (without kinase-like domain), and pET proteins in the presence of diluted SiHa cell lysate. The phosphorylated intensity of NuSAP-pET and NuSAPN-pET can be enhanced in diluted SiHa cell lysate, while pET protein cannnot. This implies that NuSAP and NuSAPN might be phosphorylated. When we put NuSAP-pET and NuSAPN-pET in the same kinase reaction, it was found that NuSAP-pET is a better phosphorylation substrate than NuSAPN-pET. The phosphorylation of NuSAP-pET can be heat (65℃) inactivated, and the phosphorylation ability was decreased with the increasing inactivation time. Taken together, our data implied that NuSAP might retain some kinase activities, or NuSAP may be phosphorylated by other unknown kinases. Nevertheless, more experiments are required to unravel the functional role of NuSAP and NuMA-s. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76388 |
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顯示於系所單位: | 動物學研究所 |
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