Ohmichi M, Zhu G, Saltiel A R
Department of Physiology, University of Michigan School of Medicine, Ann Arbor 48109.
Biochem J. 1993 Nov 1;295 ( Pt 3)(Pt 3):767-72. doi: 10.1042/bj2950767.
Protein kinase C (PKC) family members were examined in PC-12 rat pheochromocytoma cells to evaluate their role in the action of nerve growth factor (NGF). Immunoblot analysis of whole cell lysates using antibodies against various PKC isoforms revealed that PC-12 cells contained PKC-alpha, -delta, -epsilon and zeta. Assay of the protein kinase activity in these different anti-PKC immunoprecipitates demonstrated that NGF stimulated the kinase activity of PKC-epsilon, but not PKC-alpha, -delta and -zeta. Both histone phosphorylation and autophosphorylation of PKC-epsilon were increased by treatment of PC-12 cells with NGF. This increased phosphorylation observed in vitro is rapid, occurring maximally at 2.5 min and declining thereafter. Moreover, this effect of NGF is dose-dependent over physiological concentrations of the growth factor. Although the mechanistic basis for this specificity in PKC activation is not clear, NGF acutely stimulated the production of diacylglycerol without causing corresponding changes in intracellular Ca2+ concentrations. These results suggest that NGF may selectively stimulate the Ca(2+)-insensitive epsilon isoform of PKC by a phosphatidylinositol-independent mechanism.
在PC-12大鼠嗜铬细胞瘤细胞中检测了蛋白激酶C（PKC）家族成员，以评估它们在神经生长因子（NGF）作用中的角色。使用针对各种PKC同工型的抗体对全细胞裂解物进行免疫印迹分析显示，PC-12细胞含有PKC-α、-δ、-ε和ζ。对这些不同的抗PKC免疫沉淀物中的蛋白激酶活性进行测定表明，NGF刺激了PKC-ε的激酶活性，但未刺激PKC-α、-δ和-ζ的激酶活性。用NGF处理PC-12细胞可增加PKC-ε的组蛋白磷酸化和自身磷酸化。体外观察到的这种磷酸化增加迅速，在2.5分钟时达到最大值，此后下降。此外，在生长因子的生理浓度范围内，NGF的这种作用呈剂量依赖性。尽管PKC激活这种特异性的机制尚不清楚，但NGF可急性刺激二酰基甘油的产生，而不会引起细胞内Ca2+浓度的相应变化。这些结果表明，NGF可能通过一种不依赖磷脂酰肌醇的机制选择性地刺激PKC的Ca(2+)不敏感的ε同工型。
