Uchida C, Hagiwara M, Hidaka H
Department of Pharmacology, Nagoya University School of Medicine, Japan.
Arch Biochem Biophys. 1991 Aug 1;288(2):421-6. doi: 10.1016/0003-9861(91)90215-5.
We obtained a Ca(2+)-independent but 12-O-tetradecanoyl phorbol ester (TPA).phospholipid-activated protein kinase from rat embryo fibroblast 3Y1 cells by succeeding steps of DEAE-cellulose, H-9 affinity, and hydroxylapatite chromatography. This kinase was separated chromatography. This kinase was separated from a conventional PKC (Type III), by H-9 affinity column chromatography. The major peak from H-9 affinity column was eluted at 0.4 M of arginine and on the following step of hydroxylapatite column chromatography, at the KPO4 concentration of 0.1 M. The enzyme could be stimulated by phospholipids and by the tumor promoter TPA, but did not respond to calcium. The Ca(2+)-independent, phospholipid-activated protein kinase activity was susceptible to the protein kinase C inhibitors H-7 and K252a, but showed a phospholipid dependency and substrate specificity distinct from the conventional types of PKC. This protein kinase did not react with monoclonal antibodies against Types I, II, and III PKC. The activity of this enzyme was specifically reduced by immunoprecipitation, depending on the concentration of the polyclonal antibody, PC-delta, which was raised against a peptide synthesized according to a sequence of rat brain nPKC delta. The enzyme had a Mr of 76,000 as estimated by Western blotting. These results provide evidence for a unique type of Ca(2+)-independent, phospholipid-activated kinase, as expressed in 3Y1 cells.
我们通过二乙氨基乙基纤维素、H-9亲和及羟基磷灰石层析等后续步骤，从大鼠胚胎成纤维细胞3Y1中获得了一种不依赖钙离子但受12-O-十四烷酰佛波醇酯（TPA）和磷脂激活的蛋白激酶。该激酶通过层析进行分离。通过H-9亲和柱层析，这种激酶与传统的蛋白激酶C（III型）得以分离。H-9亲和柱的主要峰在0.4M精氨酸处洗脱，在随后的羟基磷灰石柱层析步骤中，在0.1M的磷酸钾浓度下洗脱。该酶可被磷脂和肿瘤启动子TPA刺激，但对钙离子无反应。这种不依赖钙离子、受磷脂激活的蛋白激酶活性对蛋白激酶C抑制剂H-7和K252a敏感，但表现出与传统类型蛋白激酶C不同的磷脂依赖性和底物特异性。这种蛋白激酶不与针对I型、II型和III型蛋白激酶C的单克隆抗体发生反应。根据针对按照大鼠脑nPKCδ序列合成的肽段产生的多克隆抗体PC-δ的浓度，通过免疫沉淀可特异性降低该酶的活性。通过蛋白质印迹法估计，该酶的分子量为76,000。这些结果为3Y1细胞中表达的一种独特类型的不依赖钙离子、受磷脂激活的激酶提供了证据。
