Que X, Samuelson J, Reed S
Department of Pathology and Medicine, University of California, San Diego.
Mol Biochem Parasitol. 1993 Aug;60(2):161-70. doi: 10.1016/0166-6851(93)90128-k.
Eleven Entamoeba histolytica protein-serine/threonine-kinase gene segments were identified using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers to conserved amino acids in subdomains VI and VIII of the catalytic domain of protein-serine/threonine kinases. These ameba gene segments were homologous to myosin light chain kinases, protein kinase C, phosphorylase b kinase, and kinases that regulate glucose repression in yeast and cell growth in mammalian cells. One of these PCR products, which was homologous to the Dictyostelium discoideum protein kinase 2, was used to identify a full-length protein-serine/threonine-kinase gene (Eh rac1) from an E. histolytica genomic library. The open reading frame of Eh rac1 was 409 amino acids long (encoding a 47-kDa protein) and included an amino terminal segment containing 87 mostly charged and polar amino acids and a 322-amino acid carboxyl terminal segment containing the catalytic domain. The catalytic domain of Eh rac1 was homologous to the rac family of protein-serine/threonine-kinases, which are related to cAMP-dependent protein kinases and protein kinase Cs. Southern blots of ameba DNA showed that the Eh rac1 gene was present as a single copy in all strains tested, however pathogenic amebae expressed four times more Eh rac1 mRNAs than did nonpathogenic amebae. These studies suggest that E. histolytica, a primitive unicellular eukaryote, has a complex protein kinase family.
利用聚合酶链反应（PCR）和针对蛋白质丝氨酸/苏氨酸激酶催化结构域亚结构域VI和VIII中保守氨基酸的简并寡核苷酸引物，鉴定出11个溶组织内阿米巴蛋白质丝氨酸/苏氨酸激酶基因片段。这些阿米巴基因片段与肌球蛋白轻链激酶、蛋白激酶C、磷酸化酶b激酶以及调节酵母中葡萄糖阻遏和哺乳动物细胞中细胞生长的激酶同源。其中一个与盘基网柄菌蛋白激酶2同源的PCR产物，被用于从溶组织内阿米巴基因组文库中鉴定出一个全长蛋白质丝氨酸/苏氨酸激酶基因（Eh rac1）。Eh rac1的开放阅读框长409个氨基酸（编码一个47 kDa的蛋白质），包括一个氨基末端片段，含有87个大多带电荷和极性的氨基酸，以及一个322个氨基酸的羧基末端片段，含有催化结构域。Eh rac1的催化结构域与蛋白质丝氨酸/苏氨酸激酶的rac家族同源，它们与环磷酸腺苷依赖性蛋白激酶和蛋白激酶C相关。阿米巴DNA的Southern印迹显示，Eh rac1基因在所有测试菌株中均以单拷贝形式存在，然而致病性阿米巴表达的Eh rac1 mRNA是非致病性阿米巴的四倍。这些研究表明，作为一种原始单细胞真核生物的溶组织内阿米巴拥有一个复杂的蛋白激酶家族。
