利用酵母菌雙雜合系統分離核分裂蛋白之結合蛋白並分析其構造與功能

Abstract

核分裂蛋白NuMA(nuclear mitotic apparatus protein)在細胞內的分佈與細胞週期(cell cycle)有密切的關係。NuMA在間期(interphase)時位於細胞核內；而在有絲分裂(mitosis)時則移動至紡綞極(spindle pole)附近。近來的實驗結果發現，NuMA是核骨架蛋白(nuclear matrix)的成分之一，可能與RNA裁接(splicing)有關，並且在有絲分裂時負責紡綞絲的形成與穩定。 本實驗室籍由cDNA library的篩檢定序及反轉錄?－聚合?鏈反應，發現人類NuMA至少有T33、U4及U6三種異型體(isoform), 其中T33為主要的異型體，間期時位於細胞核內。U4及U6異型體因RNA替代裁接(alternative splicing)的關係而缺乏T33異型體所擁有的NLS (nuclear localization signal)，因此不分佈在細胞核內。細胞於間期時，這兩種異型體主要分佈於中心體(centrosome)附近，在有絲分裂時則與T33異型體同樣位於紡綞極附近。 為了探討U6異型體的功能，我們利用酵母菌雙雜合系統（yeast two hybrid system）尋找與U6異型體結合的蛋白質，並由人類淋巴球的cDNA library中分離到與U6蛋白質C端有強烈結合能力的clone K2C，並暫將此一基因稱為K2。 由進一步的雙雜合系統實驗發現，U6異型體是以它尾端特有胺基酸序列與K2C結合。而由北方點墨法（Northern blot）分析的結果發現，K2基因轉錄的mRNA略小於人類的18S rRNA，大約是1700個鹼基對，而經由對人類不同組織的北方點墨法分析結果發現，K2 mRNA在所測試的十六種人類組織中均有表現。 對人類脾臟cDNA library的篩檢及DNA定序結果，K2基因的cDNA全長共1757個鹼基對，轉譯的蛋白質可能含有 390個胺基酸。經由DNA及胺基酸序列比對的結果發現，K2是一個新發現的基因。將K2蛋白質的胺基酸序列和已知的蛋白質激?(protein kinase)胺基酸保守序列比較發現，K2蛋白質含有部分保守序列。因此，K2可能是一個激?。 將K2 C短暫轉染至細胞中發現，K2 C分佈在細胞質和細胞核除了核仁以外的所有區域。而當K2 C大量在細胞中表現時，隨著時間的推進，被轉染的細胞出現多核的比例有逐漸升高的趨勢，且大部分的細胞會在30小時之內死亡。由此推論，K2蛋白質可能與有絲分裂時染色體分離或紡綞絲的功能有關。

The nuclear mitotic apparatus 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 and are essential for mitotic spindle pole assembly and stabilization. At least three different isoforms of NuMA, namely T33, U4 and U6 were identified by DNA sequencing and rt-PCR in our lab. The T33 isoform that carries a NLS (nuclear localization signal) at its tail region is a predominant isoform mainly present in interphase nuclei, while U4 and U6 isoforms which lack the NLS by alternative RNA splicing, are excluded from the nucleus. In interphase, U4 and U6 isoforms are presented mainly at centrosomal region, but relocated to the mitotic spindle poles during mitosis. In order to clarify the functional role of U6 isoform, we have utilized the yeast two hybrid system to screen for cellular proteins which interact with the U6 isoform. One clone K2C, isolated from human lymphocyte cDNA library, showed a significant binding ability with the C terminal portion of U6 isoform, and we have temporarily named this gene K2. Using a series of U6 deletion mutants, the K2 specific binding domain of U6 isoform was further narrowed down to the tail specific region of U6 isoform. Northern blot analysis of mRNAs from two human cell lines (Molt-4 and SiHa) revealed a transcript slightly smaller than human 18S rRNA (?l.8 Kb). Tissue specific Northern blot demonstrated that K2 mRNA were expressed in all human tissues examined. A human spleen cNDA library was screened to obtain a full-length cDNA clone of K2. The resulting 1757 bp sequence of K2 has a single open reading frame which may encode a 390 amino acid polypeptide. The nucleotide and amino acid analysis revealed no sequence homology with that of any reported gene presented in the known databases. Therefore K2 could be a novel gene. However, by comparing the amino acid sequence of K2 protein with the conserved sub-domain peptide sequences of most protein kinases, it revealed that K2 contains several of these conserved peptide sequences which suggest that K2 protein might belong to a novel protein kinase family. When transiently transfected the C terminal end of K2 clone (K2C) into SiHa cells, the K2C protein dispersed throughout the cytoplasm and nucleus, but was excluded from nucleolus. As time progressed, the transfected cells tended to become multinuclei and most of the transfected cells appeared to die out within 30 hours after transfection, which suggests a possible role of K2 protein involved in chromosome segregation or spindle formation.