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。根据这些激酶的氨基酸序列进行分类分析，将其分为5类。蛋白激酶A (PKA)，蛋白激酶G （PKG）和蛋白激酶C （PKC）彼此聚在一个组中，称为agc家族。众所周知，特别是在脊椎动物中，每个agc家族蛋白激酶在信号传导过程中具有不同的功能。agc家族激酶的起源似乎是单细胞真核生物，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，筛选了一种原生草芥agc家族蛋白激酶。单细胞真核生物可能是所有多细胞生物（如动物、植物和真菌）的祖先。采用RT-PCR方法，根据agc -激酶的一致序列，设计了几种引物，扩增出了绿叶菊蛋白激酶基因。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。以一个与agc激酶基因具有广泛相似性的基因片段为依据，从制备的甘茅cDNA文库中选择了agc激酶的总编码区，得到了4个具有不同激酶基因的克隆，但彼此之间具有较高的相似性。对激酶基因（EPK-3、EPK-4、EPK-16、EPK-19）进行测序。Northern分析显示单波段对应1.6Kb位置。这些新激酶具有典型agc激酶的几个共识序列，由一个没有调节区域的激酶结构域组成。根据氨基酸序列的进化分析表明，EPK家族激酶由PKA、PKC和其他相关激酶分型组成一个分型。这些结果支持了一种观点，即新型蛋白激酶（epk家族）可能是各种高度进化生物中众所周知的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