Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan.
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan.
Biochem Biophys Res Commun. 2018 May 23;499(4):1011-1018. doi: 10.1016/j.bbrc.2018.04.039. Epub 2018 Apr 11.
Phosphoinositides mediate a large number of signaling processes in mammalian cells. Here, we report that phophatidylinositol-4-phosphate (PtdIns(4)P) downregulates the cellular glucosylceramide (GlcCer) level by inhibiting the interaction between GlcCer synthase (UGCG) and UDP-glucose in the Golgi apparatus. In this study, we used two PH domain probes to bind phosphoinositides; one derived from FAPP1 for targeting to the Golgi PtdIns(4)P and the other from PLC δ for targeting to the plasma membrane PtdIns(4,5)P. The levels of GlcCer and lactosylceramide, but not of sphingomyelin (SM), were increased following expression of the FAPP1 PH domain in cells, accompanied by an increase in UGCG activity. However, no elevated GlcCer level was observed after expression of the PLC δ PH domain. PtdIns(4)P inhibited UGCG activity, but not SMS activity, in a concentration-dependent manner, and UGCG activity was restored by the addition of UDP-glucose in the reaction mixture. These results indicate that PtdIns(4)P inhibits UGCG activity by competing with UDP-glucose. We conclude that the increase in UGCG activity due to the expression of the FAPP1 PH domain was caused by an attenuation of the inhibitory effect of PtdIns(4)P on UGCG. This study provides new insights into the regulation of GlcCer synthesis by PtdIns(4)P in the Golgi apparatus.
磷脂酰肌醇在哺乳动物细胞的许多信号转导过程中发挥作用。在这里，我们报告磷脂酰肌醇-4-磷酸（PtdIns（4）P）通过抑制内质网中葡萄糖神经酰胺（GlcCer）合成酶（UGCG）与 UDP-葡萄糖之间的相互作用，下调细胞内的葡萄糖神经酰胺（GlcCer）水平。在这项研究中，我们使用了两种 PH 结构域探针来结合磷酸肌醇；一种源自 FAPP1，用于靶向高尔基体 PtdIns（4）P，另一种源自 PLC δ，用于靶向质膜 PtdIns（4,5）P。在细胞中表达 FAPP1 PH 结构域后，GlcCer 和乳糖基神经酰胺的水平升高，但鞘磷脂（SM）水平没有升高，同时 UGCG 活性增加。然而，表达 PLC δ PH 结构域后，GlcCer 水平没有升高。PtdIns（4）P 以浓度依赖的方式抑制 UGCG 活性，但不抑制 SMS 活性，并且在反应混合物中添加 UDP-葡萄糖可恢复 UGCG 活性。这些结果表明，PtdIns（4）P 通过与 UDP-葡萄糖竞争抑制 UGCG 活性。我们得出结论，由于表达 FAPP1 PH 结构域导致 UGCG 活性增加，是由于 PtdIns（4）P 对 UGCG 的抑制作用减弱所致。本研究为 PtdIns（4）P 在高尔基体中调节 GlcCer 合成提供了新的见解。
