Mukai H, Kitagawa M, Shibata H, Takanaga H, Mori K, Shimakawa M, Miyahara M, Hirao K, Ono Y
Department of Biology, Faculty of Science, Kobe University, Japan.
Biochem Biophys Res Commun. 1994 Oct 14;204(1):348-56. doi: 10.1006/bbrc.1994.2466.
PKN, a novel protein kinase with catalytic domain homologous to PKC family and unique amino terminal leucine zipper-like sequences, was purified partially from COS7 cells transfected with the cDNA construct encoding human PKN for enzymatic characterization of the enzyme. Using serine containing synthetic peptides based on PKC pseudosubstrate sites as the phosphate acceptors, kinase activities estimated from partially purified PKN were not stimulated by Ca2+/phosphatidylserine/diolein but were activated several-fold to several tens-fold by 40 microM unsaturated fatty acids, such as arachidonic acid, linoleic acid, and oleic acid. Autophosphorylation of the immunoprecipitates using anti-PKN antiserum was also stimulated by various unsaturated fatty acids. Limited proteolysis of PKN with trypsin induced an enhancement of the peptide kinase activity that was almost independent of arachidonic acid.
PKN是一种新型蛋白激酶，其催化结构域与蛋白激酶C（PKC）家族同源，且具有独特的氨基末端亮氨酸拉链样序列。从转染了编码人PKN的cDNA构建体的COS7细胞中部分纯化出PKN，用于该酶的酶学特性研究。以基于PKC假底物位点的含丝氨酸合成肽作为磷酸受体，部分纯化的PKN的激酶活性不受Ca2+/磷脂酰丝氨酸/二油精的刺激，但被40微摩尔的不饱和脂肪酸（如花生四烯酸、亚油酸和油酸）激活了几倍到几十倍。使用抗PKN抗血清对免疫沉淀物进行的自磷酸化也受到各种不饱和脂肪酸的刺激。用胰蛋白酶对PKN进行有限的蛋白水解可诱导肽激酶活性增强，这几乎与花生四烯酸无关。
