MOLECULAR-CLONING AND CHARACTERIZATION OF A NOVEL PUTATIVE PROTEIN-SERINE KINASE RELATED TO THE CAMP-DEPENDENT AND PROTEIN-KINASE-C FAMILIES
MOLECULAR-CLONING AND CHARACTERIZATION OF A NOVEL PUTATIVE PROTEIN-SERINE KINASE RELATED TO THE CAMP-DEPENDENT AND PROTEIN-KINASE-C FAMILIES
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DOI：
10.1111/j.1432-1033.1991.tb16305.x
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发表时间：
1991-10-15
期刊：
影响因子：
--
通讯作者：
WOODGETT, JR
中科院分区：
文献类型：
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作者：
COFFER, PJ;WOODGETT, JR
根据蛋白-丝氨酸激酶之间的保守区域设计的高度简并寡核苷酸引物已专门用于通过聚合酶链式反应 (PCR) 扩增人上皮 (HeLa) cDNA。在如此分离的编码推定激酶的几个新的cDNA片段中，其中一个被进一步表征。用 PCR 片段筛选人成纤维细胞和牛脑 cDNA 文库，产生了几个具有 479 个氨基酸开放阅读框的克隆，其中包含蛋白丝氨酸激酶的所有保守序列基序。预测的蛋白质与蛋白激酶 C (PKC)/cAMP 依赖性蛋白激酶 (PKA) 家族最相似，因此其基因被称为 pkb。 pkb 基因表达普遍，但在脑、心脏和肺中表达最高。 pkb RNA 的体外翻译产生了 57-kDa 蛋白 (PKB)，可被细菌表达的 PKB/TrpE 融合蛋白的抗血清识别。用激酶 cDNA 转染 COS 细胞，合成了 60 kDa 的蛋白质，该蛋白质通过 Mono Q 阴离子交换层析进行了部分纯化。与对照细胞的类似组分相比，含有 PKB 免疫反应蛋白的柱组分表现出升高的组蛋白 H1 激酶活性，证明了该蛋白激酶的酶活性。
Highly degenerate oligonucleotide primers designed from regions conserved between protein-serine kinases have been used specifically to amplify human epithelial (HeLa) cDNA by the polymerase chain reaction (PCR). Of several novel cDNA fragments encoding putative kinases thus isolated, one was further characterised. Screening of human fibroblast and bovine brain cDNA libraries with the PCR fragment yielded several clones with an open reading frame of 479 amino acids containing all of the conserved sequence motifs of protein-serine kinases. The predicted protein was most similar to the protein kinase C (PKC)/cAMP-dependent protein kinase (PKA) families and its gene has thus been termed pkb. Expression of the pkb gene is general but highest in brain, heart and lung. Translation of pkb RNA in vitro generated a 57-kDa protein (PKB) recognised by antisera raised to a bacterially expressed PKB/TrpE fusion protein. Transfection of COS cells with the kinase cDNA resulted in the synthesis of a 60-kDa protein which was partially purified by Mono Q anion-exchange chromatography. Column fractions containing PKB-immunoreactive protein exhibited elevated histone H1 kinase activity compared with similar fractions from control cells, demonstrating the enzymatic activity of this protein kinase.