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转导过程中发挥着重要作用。根据它们的氨基酸序列，通过Claster分析将它们分为5个类群。蛋白激酶A(PKA)、蛋白激酶G(PKG)和蛋白激酶C(PKC)在AGC家族中相互分离。众所周知，特别是在脊椎动物中，每个AGC家族蛋白家族在信号传递过程中都发挥着不同的功能，AGC家族蛋白激酶的起源似乎是单细胞桉树，如原生生物。然而，我们对这些激酶的分子进化知之甚少。针对这些问题，我们筛选出了一种新的纤细裸藻AGC家族蛋白激酶。单细胞桉树生物可能是所有多细胞生物的祖先，如动物、植物和真菌。根据纤细眼虫蛋白激酶基因DO-…的同源序列，设计了多对引物，通过RT-PCR法扩增了纤细眼虫蛋白激酶基因更主要的是各种AGC-激酶。鉴定出12个与PKA、PKC、S6-K和RACK基因同源的蛋白激酶基因片段。以一段与AGC-KEK基因高度相似的基因片段为依据，从已构建的纤细眼虫(Euglena Gracilus Z)的cDNA文库中筛选出AGC-KK的全编码区，获得了4个具有不同KK基因但高度相似的克隆。对其蛋白激酶基因(EPK-3、EPK-4、EPK-16、EPK-19)进行了序列测定。Northern分析表明，该单条带对应1.6Kb的位置。这些新的激酶具有几个典型的AGC-激酶的共同序列，由一个没有调节区的激动域组成。氨基酸序列的系统发育分析表明，EPK家族的蛋白激酶是由PKA、PKC和其他相关的蛋白激酶组成的。这些结果支持一种观点，即新类型的蛋白激酶(EPK-家族)可能是各种高度进化的生物体中众所周知的AGC-家族的一个起源。较少

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