RESEARCH FOR MOLECULAR EVOLUTION OF PROTEIN KINASE C GENES OF PROTISTA EUGLENA GRACILIS Z AND ROLES OF THE KINASES.

蛋白激酶在细胞信号传导过程中起着重要作用。根据氨基酸序列的聚类分析，将这些激酶分为5类。蛋白激酶A（Protein Kinase A，PKA）、蛋白激酶G（Protein Kinase G，PKG）和蛋白激酶C（Protein Kinase C，PKC）相互归类为一组，称为AGC家族。在脊椎动物中，每个AGC家族蛋白激酶在信号转导过程中具有不同的功能，其起源可能是单细胞真核生物，如原生生物。然而，我们对激酶的分子进化知之甚少。针对这些问题，本研究从一种原生生物纤细裸藻中筛选了新的AGC家族蛋白激酶。单细胞真核生物可能是动物、植物和真菌等多细胞生物的祖先。根据纤细眼虫蛋白激酶基因沿着保守序列设计引物，采用RT-PCR方法扩增了该蛋白激酶基因。 关于我们 主要是各种AGC激酶。共鉴定出12个与PKA、PKC、S6-激酶和RACK基因同源的蛋白激酶基因片段。从制备的纤细眼虫cDNA文库中筛选出一个与AGC激酶基因具有广泛相似性的基因片段，并以此为依据筛选出该激酶的完整编码区。获得了具有不同激酶基因但彼此具有高相似性的四个克隆。对激酶基因（EPK-3、EPK-4、EPK-16、EPK-19）进行了测序。北方分析显示在1.6Kb位置有一条带。这些新的激酶具有典型的AGC激酶的几个共有序列，由激酶结构域组成，没有调节区。根据氨基酸序列推测的系统发育分析表明，EPK家族激酶由PKA、PKC和其他相关激酶组成。这些结果支持了这样一种观点，即这种新型的蛋白激酶（EPK家族）可能是各种高度进化的生物体中已知的AGC家族激酶的起源。少

Protein kinases play important roles in the process of cell signaling. The kinases have been classified into 5 groups on the claster analysis inferred from their amino acid sequences. The protein kinase A (PKA), protein kinase G (PKG) and protein kinase C (PKC) are clastered each other in a group, AGC-family. It is well-known especially on vertebrate that each AGC-family protein kinse shows various function in signaling processes.The origin of the AGC-family kinase seems to be to single cell eucalyote such as protists. However, we have been much less informed about the molecular evolution of the kinases. Focussing these problems, new AGC-family protein kinases of a protist Euglena gracilus were screened. The single cell eucalyotic organism is a possible ancestor of all the multicelluler organisms, such as animal, plant and fungi. The protein kinase gene from Euglena gracilus were amplified by RT-PCR using several designed primers according to the consensus sequences along the kinase do … More main of the various AGC-kinase. Twelve fragments of protein kinase genes homologous to the PKA,PKC,S6-kinase and RACK genes were identified. A gene fragment showing widely similarity to AGC-kinase genes was used as a prove for selecting overall coding region of the kinase from the prepared cDNA libraries of Euglena gracilus z. Four clones having different kinase genes, but with high simirality each other, were obtained. The kinase genes (EPK-3, EPK-4, EPK-16, EPK-19) were sequenced. Northern analysis showed the single band corresponding 1.6Kb position. These new kinases havinf the several consensus sequences og typical AGC-kinases consist of a kinase domain without regulatory regions. Phyrogenetic analysis inferred from the amino acid sequences indicates that EPK family kinases make an claster out of PKA,PKC and other related kinases clasters. These results suppot the idea that the new type of protein kinase (EPK-family) is a possible origin of the well-known AGC-family kinases from various highly evolved organisms. Less