Tanimura Akihiko, Nezu Akihiro, Morita Takao, Hashimoto Noboru, Tojyo Yosuke
Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
J Biol Chem. 2002 Aug 9;277(32):29054-62. doi: 10.1074/jbc.M201130200. Epub 2002 May 7.
The function of protein kinase C (PKC) is closely regulated by its subcellular localization. We expressed PKCalpha fused to green fluorescent protein (PKCalpha-GFP) and examined its translocation in living and permeabilized cells of the human parotid cell line, HSY-EB. ATP induced an oscillatory translocation of PKCalpha-GFP to and from the plasma membrane that paralleled the appearance of repetitive Ca2+ spikes. Staurosporine attenuated the relocation of PKCalpha-GFP to the cytosol and caused a stepwise accumulation of PKCalpha-GFP at the plasma membrane during ATP stimulation. Diacylglycerol enhanced the amplitude and duration of the ATP-induced oscillatory translocation of PKCalpha-GFP. Ionomycin induced a transient translocation of PKCalpha-GFP to the plasma membrane despite the continuous elevation of cytosolic Ca2+. The ionomycin-induced transient translocation of PKCalpha-GFP was prolonged by staurosporine, diacylglycerol, and phorbol myristate acetate. Experiments using permeabilized cells showed that staurosporine or the elimination of ATP and Mg2+ decreases the rate of dissociation of PKCalpha-GFP from the membrane. Diacylglycerol slowed the dissociation of PKCalpha-GFP from the membrane regardless of the Ca2+ concentration. The effect of diacylglycerol was attenuated by ATP plus Mg2+ at low concentrations of Ca2+ (<500 nm) but not at high concentrations of Ca2+ (>1000 nm). These data suggest a complex interplay between Ca2+, diacylglycerol, and phosphorylation in the regulation of the membrane binding of PKCalpha.
蛋白激酶C（PKC）的功能受其亚细胞定位的密切调控。我们表达了与绿色荧光蛋白融合的PKCα（PKCα-GFP），并检测了其在人腮腺细胞系HSY-EB的活细胞和透化细胞中的转位情况。ATP诱导PKCα-GFP在质膜内外进行振荡性转位，这与重复性Ca2+尖峰的出现平行。星形孢菌素减弱了PKCα-GFP向细胞质的重新定位，并在ATP刺激期间导致PKCα-GFP在质膜上逐步积累。二酰基甘油增强了ATP诱导的PKCα-GFP振荡性转位的幅度和持续时间。离子霉素尽管使细胞质Ca2+持续升高，但仍诱导PKCα-GFP向质膜的短暂转位。星形孢菌素、二酰基甘油和佛波醇肉豆蔻酸酯延长了离子霉素诱导的PKCα-GFP短暂转位。使用透化细胞的实验表明，星形孢菌素或去除ATP和Mg2+会降低PKCα-GFP从膜上解离的速率。无论Ca2+浓度如何，二酰基甘油都会减缓PKCα-GFP从膜上的解离。在低Ca2+浓度（<500 nm）时，ATP加Mg2+会减弱二酰基甘油的作用，但在高Ca2+浓度（>1000 nm）时则不会。这些数据表明，在PKCα膜结合的调控中，Ca2+、二酰基甘油和磷酸化之间存在复杂的相互作用。
