Selective ceramide binding to protein kinase C-alpha and -delta isoenzymes in renal mesangial cells.
Selective ceramide binding to protein kinase C-alpha and -delta isoenzymes in renal mesangial cells.
复制标题
选择性神经酰胺与肾系膜细胞中的蛋白激酶 C-α 和 δ 同工酶结合。
作者：
A. Huwiler;D. Fabbro;J. Pfeilschifter
神经酰胺是一种重要的脂类第二信使，在有限数量的激动剂作用下，神经酰胺通过鞘磷脂代谢产生，进而以蛋白激酶C(PKC)依赖的方式触发多种细胞反应。用一种神经酰胺的放射性光亲和标记类似物(N-[3-[[[2-(125I)iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benz yl]oxy]carbonyl]propanoyl]-D-erythro-sphingosine)([125I]-TID-神经酰胺)刺激系膜细胞，将PKC-α和PKC-Delta定义为神经酰胺的直接靶标。未检测到神经酰胺与PKC-epsilon和PKC-Zeta结合。此外，TID-神经酰胺选择性地与重组PKC-α和-Delta结合，而不与PKC-epsilon和-Zeta同工酶结合。体外的激酶活性测定表明，只有神经酰胺与PKC-α结合时，才会伴随着激酶活性的增加。相反，相对于任何常规底物，其他被测试的异构体，即PKC-Delta、-epsilon和-Zeta的体外激酶活性没有变化。然而，值得注意的是，PKC-Delta在神经酰胺结合时显示出自磷酸化减少。在体内，神经酰胺对PKC-α的激活是通过一种延迟的异构体从胞浆到膜部分的移位来监测的，在刺激1小时后就可以检测到。相反，无论是PKC-Delta、Nor-epsilon还是Nor-Zeta都不会被神经酰胺重新分布。肾小球系膜细胞中由PKC-α介导的一种功能反应是配体刺激的肌醇磷脂水解物的负反馈调节。当细胞被神经酰胺预处理时，ATP诱导的三磷酸肌醇的形成随时间而减少。神经酰胺作用2小时后抑制作用最强。综上所述，这些结果表明神经酰胺选择性地与系膜细胞中PKC的α-和β-亚型相互作用。虽然PKC-α被激活，但激素刺激的肌醇磷脂信号明显受到抑制，而PKC-Delta的自磷酸化程度降低，这表明神经酰胺结合对PKC-Delta的活性具有负面作用。
Ceramide is an important lipid second messenger produced by sphingolipid metabolism in cells exposed to a limited number of agonists and in turn triggers several cell responses in a protein kinase C (PKC)-dependent manner. Stimulation of mesangial cells with a radioiodinated photoaffinity labeling analogue of ceramide, (N-[3-[[[2-(125I)iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benz yl] oxy]carbonyl]propanoyl]-D-erythro-sphingosine) ([125I]-TID-ceramide), defines PKC-alpha and PKC-delta as direct targets of ceramide. No binding of ceramide to PKC-epsilon and PKC-zeta could be detected. Moreover, TID-ceramide selectively binds to recombinant PKC-alpha and -delta but not to PKC-epsilon and -zeta isoenzymes. In vitro kinase activity assays reveal that only the binding of ceramide to PKC-alpha is accompanied by an increase in kinase activity. In contrast, there is no change in in vitro kinase activity of the other isoforms tested, i.e., PKC-delta, -epsilon, and -zeta, toward any of the conventional substrates tested. However, it is noteworthy that PKC-delta shows a decreased autophosphorylation upon ceramide binding. In vivo, activation of PKC-alpha by ceramide is monitored by a delayed translocation of the isoform from the cytosol to the membrane fraction, detectable after 1 h of stimulation. In contrast, neither PKC-delta, nor -epsilon nor -zeta is redistributed by ceramide. One functional cell response mediated by PKC-alpha in mesangial cells is a negative feedback regulation of ligand-stimulated phosphoinositide hydrolysis. When cells are pretreated with ceramide, ATP-induced inositol trisphosphate formation is time-dependently reduced. A maximal inhibition is observed after 2 h of ceramide exposure. In summary, these results suggest that ceramide selectively interacts with the alpha- and delta-isoforms of PKC in mesangial cells. Whereas PKC-alpha is activated with pronounced inhibition of hormone-stimulated phosphoinositide signaling, PKC-delta displays a decrease in its autophosphorylation, suggesting a negative role of ceramide binding on PKC-delta activity.