Larsson C, Saermark T, Mau S, Simonsson P
Department of Psychiatry and Neurochemistry, Lund University, Sweden.
J Neurochem. 1992 Aug;59(2):644-51. doi: 10.1111/j.1471-4159.1992.tb09418.x.
The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.
通过测量特异性蛋白激酶C底物髓鞘碱性蛋白4 - 14的磷酸化，研究了洋地黄皂苷通透处理的人神经母细胞瘤SH - SY5Y细胞中蛋白激酶C的激活情况。磷酸化受到蛋白激酶C抑制肽PKC19 - 36的抑制，并且与该酶向SH - SY5Y细胞膜组分的转位有关。1,2 - 二辛酰 - sn - 甘油对蛋白激酶C活性没有影响，除非钙浓度升高到刺激细胞中发现的浓度（高于100 nM）。在没有其他激活剂的情况下，钙不会刺激蛋白激酶C。佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯对蛋白激酶C的激活或转位不依赖于钙。诱导的激活持续10分钟，此后只能检测到底物的少量净磷酸化。单独应用钙或二辛酰甘油时，仅引起轻微的转位，而联合应用时则观察到明显的转位。在亚最大浓度的钙和二辛酰甘油存在下，花生四烯酸（10 microM）增强了蛋白激酶C的活性。在被认为足以抑制磷脂酶A2的浓度下，奎纳克林和对溴苯甲酰溴不会抑制钙和二辛酰甘油诱导的蛋白激酶C活性。
