Selective ceramide binding to protein kinase C-α and -δ isoenzymes in renal mesangial cells
Selective ceramide binding to protein kinase C-α and -δ isoenzymes in renal mesangial cells
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DOI：
10.1021/bi981401i
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发表时间：
1998-10-13
期刊：
影响因子：
2.9
通讯作者：
Pfeilschifter, J
中科院分区：
文献类型：
--
作者：
Huwiler, A;Fabbro, D;Pfeilschifter, J
神经酰胺是一种重要的脂质第二信使，由暴露于有限数量激动剂的细胞中的鞘脂代谢产生，进而以蛋白激酶 C (PKC) 依赖性方式触发多种细胞反应，用神经酰胺的放射性碘光亲和标记类似物刺激系膜细胞，(N-[3-[[[2-(I-125)iodo-4-[3-(三氟甲基)] [I-125]-TID-神经酰胺）将 PKC-α 和 PKC-δ 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 的结合。此外，TID-神经酰胺选择性结合重组 PKC-α 和 δ，但不结合 PKC-ε 和 -zeta 同工酶。体外激酶活性测定表明，只有神经酰胺与 PKC-α 的结合才会伴随激酶活性的增加。相比之下，测试的其他亚型（即 PKC-delta、-epsilon 和 -zeta）对于任何测试的常规底物的体外激酶活性没有变化。然而，值得注意的是，PKC-δ 在神经酰胺结合后显示出自磷酸化降低。在体内，神经酰胺对 PKC-α 的激活是通过同工型从胞质溶胶到膜部分的延迟易位来监测的，刺激 1 小时后即可检测到。相比之下，PKC-δ、-ε 和-zeta 均不被神经酰胺重新分布。系膜细胞中 PKC-α 介导的一种功能性细胞反应是配体刺激的磷酸肌醇水解的负反馈调节。当细胞用神经酰胺预处理时。 ATP 诱导的三磷酸肌醇形成呈时间依赖性减少，神经酰胺暴露 2 小时后观察到最大抑制。总之，这些结果表明神经酰胺选择性地与系膜细胞中 PKC 的 α 和 δ 亚型相互作用。 PKC-α 通过明显抑制激素刺激的磷酸肌醇信号传导而被激活，而 PKC-δ 则表现出自身磷酸化的减少，表明神经酰胺结合对 PKC-δ 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-(I-125)iodo-4-[3-(trifluoromethyl) carbonyl]propanoyl]-D-erythro-sphingosine) ([I-125]-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.