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。众所周知，尤其是在脊椎动物中，每个AGC家族的蛋白激酶在信号转导过程中表现出不同的功能。PKA、PKG和PKC基因在高等植物中尚未发现，从一种高等植物芸苔中克隆到了PKA、PKG和PKC基因，根据PKC激酶结构域沿着的保守序列设计引物，采用RT-PCR方法扩增了20株人源性大肠杆菌的cDNA，并对扩增的基因进行了亚克隆和测序。鉴定出8个蛋白激酶基因片段。PKC同源物（bcpk 1）与里氏木霉（Trichoderma reesei）PKC激酶结构域的氨基酸序列具有高度的相似性（92.4%），并含有几个PKC特异的保守序列，如GGDLM（位于亚结构域V）、FYAAE（位于亚结构域VIa）和TFCGT（位于亚结构域VIII）。这些结果支持了高等植物芸苔具有与PKC样蛋白激酶有关的信号转导系统。通过DEAE-memsep柱层析，部分纯化了PKC样激酶。允许PKC样激酶级分与部分纯化的硝酸还原酶（NR）一起重结晶。只有在磷脂酰丝氨酸（PS）、甘油二酯（DG）和Ca^2+存在的情况下，NR蛋白才能被磷酸化，表明NR磷酸化可能与PKC样蛋白激酶有关。

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.