Properties of the delta-, epsilon-, and zeta- Subspecies of Protein Kinase C

蛋白激酶C在信号转导中起着至关重要的作用，是一个多亚种的家族。本研究旨在探讨delta-， epsilon-和zeta-亚种的作用，这些亚种最初是通过cDNA克隆分析从脑文库中分离出来的。delta和epsilon亚种从大鼠脑中纯化到均匀性，并证明依赖于二酰基甘油和磷脂，但不需要钙离子来维持其激酶活性。此外，这两个亚种表现出不同的酶促特性，脂肪酸增强了epsilon亚种的活性，而抑制了delta亚种的活性。建立了过量产生蛋白激酶C各亚种的CHO细胞系，研究了该酶家族对细胞表型的影响。12-肉豆蔻酸13-乙酸佛波酯是蛋白激酶C家族的一种有效激活剂，对过量产生δ亚种的细胞生长有明显抑制作用，而表达其他亚种的细胞系则没有明显影响。这些结果表明，蛋白激酶C家族的每个亚种在生长调节中具有不同的作用。由于很难从大鼠组织中纯化zeta-亚种，因此该亚种用杆状病毒系统表达，并将从感染Sf9细胞的提取物中纯化。delta-和epsilon-亚种也在昆虫细胞中表达。我们正计划制备针对这些亚种的多克隆抗体，通过免疫学方法研究蛋白激酶C家族的作用。

Protein kinase C plays crucial roles in signal transduction, and is a family of multiple subspecies. This study aimed to investigate the roles of the delta-, epsilon-, and zeta-subspecies, that are initially isolated from brain libraries by the analysis of cDNA clones. The delta-and epsilon-subspecies were purified to homogeneity from rat brain and shown to be dependent on diacylglycerol and phospholipid, but do not require calcium ion for their kinase activities. In addition, these two subspecies showed distinct enzymatic properties, the activity of the epsilon-subspecies is enhanced by fatty acids, whereas the delta-subspecies is inhibited. CHO cell lines overproducing each protein kinase C subspecies were established to study the effects of this enzyme family on cell phenotypes. The growth of the cells overproducing the delta-subspecies was markedly inhibited upon treatment with phorbol 12-myristate 13-acetate that is a potent activator of the protein kinase C family, whereas the cell lines expressing other subspecies were not significantly affected. These results suggest that each subspecies of protein kinase C family has distinct roles in growth regulation. As it was difficult to purify the zeta-subspecies form rat tissues, this subspecies was expressed with the baculovirus system and will be purified from the extract of the infected Sf9 cells. The delta- and epsilon-subspecies were also expressed in the insect cells. We are planning to prepare polyclonal antibodies against these subspecies to investigate the roles of the protein kinase C family by immunological procedures.