Affinity purification of mammalian RNA polymerase I - Identification of an associated kinase
Affinity purification of mammalian RNA polymerase I - Identification of an associated kinase
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DOI：
10.1074/jbc.273.2.1257
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发表时间：
1998-01-09
影响因子：
4.8
通讯作者：
Rothblum, L
中科院分区：
文献类型：
--
作者：
Hannan, RD;Hempel, WM;Rothblum, L
从 Reuber 肝癌 cDNA 文库中分离出编码大鼠 RNA 聚合酶 I 两个最大亚基的重叠 cDNA 克隆，命名为 A194 和 A127。对推导的氨基酸序列的分析表明，A194 和 A127 是酵母 A190 和 A135 的同源物，并且与大肠杆菌 RNA 聚合酶 I 的 β' 和 β 亚基具有同源性。针对重组 A194 和 A127 蛋白产生的抗体在总蛋白印迹上识别大约 190 和 120 kDa 的单个蛋白。 哺乳动物来源的细胞蛋白。分离表达重组 His 标记的 A194 和 FLAG 标记的 A127 蛋白的 N1S1 细胞系，这些蛋白被整合到功能性 RNA 聚合酶 I 复合物中，并且含有 FLAG 标记的 A127 的活性酶可以在抗 FLAG 亲和柱上的单个色谱步骤中免疫纯化至约 80% 均质性，A194 的免疫沉淀 用抗 A194 抗血清从 P-32 代谢标记的细胞中提取，证明该亚基是磷蛋白。将 FLAG 亲和纯化的 RNA 聚合酶 I 复合物与 [gamma-P-32]ATP 一起孵育，导致 RPI 的 A194 亚基自磷酸化，表明相关激酶的存在。其中一种激酶被证明是 CK2，一种参与细胞生长和增殖调节的丝氨酸/苏氨酸蛋白激酶。
Overlapping cDNA clones encoding the two largest subunits of rat RNA polymerase I, designated A194 and A127, were isolated from a Reuber hepatoma cDNA library. Analyses of the deduced amino acid Sequences revealed that A194 and A127 are the homologues of yeast A190 and A135 and have homology to the beta' and beta subunits of Escherichia coli RNA polymerase I. Antibodies raised against the recombinant A194 and A127 proteins recognized single proteins of approximately 190 and 120 kDa on Western blots of total cellular proteins of mammalian origin. N1S1 cell lines expressing recombinant His tagged A194 and FLAG-tagged A127 proteins were isolated, These proteins were incorporated into functional RNA polymerase I complexes, and active enzyme, containing FLAG-tagged A127, could be immunopurified to approximately 80% homogeneity in a single chromatographic step over an anti-FLAG affinity column, Immunoprecipitation of A194 from P-32 metabolically labeled cells with anti-A194 antiserum demonstrated that this subunit is a phosphoprotein. Incubation of the FLAG affinity-purified RNA polymerase I complex with [gamma-P-32]ATP resulted in autophosphorylation of the A194 subunit of RPI, indicating the presence of associated kinase(s). One of these kinases was demonstrated to be CK2, a serine/threonine protein kinase implicated in the regulation of cell growth and proliferation.