RESEARCH FOR MOLECULAR EVOLUTION OF PROTEIN KINASE C GENES OF PROTISTA EUGLENA GRACILIS ZAND 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，每种agc家族蛋白激酶在信号传导过程中具有不同的功能。编码PKA、PKG和PKC的基因在高等植物中未发现。采用RT-PCR方法，对高等植物芸苔（Brassica campestris L.）的蛋白激酶基因进行了扩增。对扩增的基因进行亚克隆和测序。鉴定出8个蛋白激酶基因片段。它们与PKC、PVPK、s6激酶或其他类型的蛋白激酶基因（如MAPKK）相似。PKC同源物（bcpk 1）与里氏木霉PKC激酶结构域的氨基酸序列（92.4%）具有高度的相似性，并包含PKC序列特有的GGDLM（位于子结构域V）、FYAAE（位于子结构域VIa）和TFCGT（位于子结构域VIII）等保守序列。这些结果支持了高等植物油菜具有pkc样蛋白激酶的信号转导系统。目前正在进行进一步的研究。采用DEAE-memsep层析纯化了pkc样激酶。pkc样激酶片段允许用部分纯化的硝酸还原酶（NR）重新读取。只有在磷脂酰丝氨酸（PS）、二酰基甘油（DG）和Ca^<2+>离子存在的情况下，磷基团才会被强烈地转移到NR蛋白上，这表明pkc样蛋白激酶可能参与了NR磷酸化。

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 kinase shows various function in signaling processes. The genes coding PKA,PKG and PKC were not found in higher plants.The protein kinase gene from a higher plant, Brassica campestris L., were amplified by RT-PCR using several designed primers according to the consensus sequences along the kinase domain of the PKC.The amplified genes were subcloned and sequenced. Eight protein kinaes genes fragment were identified. They showed similarity to PKC,PVPK,S6-kinase or other type of orotein kinase genes such as MAPKK.The PKC homologue (bcpk 1) shows high simirality to the amino acid sequences of Trichoderma reesei PKC kinase domain (92.4%) and contains several conserved sequences specific in PKC sequence such as GGDLM (on subdomain V), FYAAE (on subdomain VIa) and TFCGT (on subdomain VIII). These results support that a higher plant, Brassica campestlis posseses signal transduction system concerning a PKC-like protein kinase. Further studies have been under progress.A PKC-like kinase was partially purified by DEAE-memsep chromatography. A PKC-like kinase fraction allowed to readt with partially purified nitrate recuctase (NR). Phosphorus group was transferred to NR protein strongly only when the reaction was performed in the presence of phosphatidylserine (PS), diacylglycerol (DG) and Ca^<2+> ion, indicative that NR-phosphorylation is likely to be involved by PKC-like protein kinase.