Lisboa Felipe A, Peng Ze, Combs Christian A, Beaven Michael A
Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1760, USA.
J Immunol. 2009 Oct 15;183(8):5104-12. doi: 10.4049/jimmunol.0802728. Epub 2009 Sep 30.
Initial IgE-dependent signaling events are associated with detergent-resistant membrane microdomains. Following Ag stimulation, the IgE-receptor (Fc(epsilon)RI ) accumulates within these domains. This facilitates the phosphorylation of Fc(epsilon)RI subunits by the Src kinase, Lyn, and the interaction with adaptor proteins, such as the linker for activation of T cells. Among the phospholipases (PL) subsequently activated, PLD is of interest because of its presence in lipid microdomains and the possibility that its product, phosphatidic acid, may regulate signal transduction and membrane trafficking. We find that in Ag-stimulated RBL-2H3 mast cells, the association of Fc(epsilon)RI with detergent-resistant membrane fractions is inhibited by 1-butanol, which subverts production of phosphatidic acid to the biologically inert phosphatidylbutanol. Furthermore, the knockdown of PLD2, and to a lesser extent PLD1 with small inhibitory RNAs, also suppressed the accumulation of Fc(epsilon)RI and Lyn in these fractions as well as the phosphorylation of Src kinases, Fc(epsilon)RI , linker for activation of T cells, and degranulation. These effects were accompanied by changes in distribution of the lipid microdomain component, ganglioside 1, in the plasma membrane as determined by binding of fluorescent-tagged cholera toxin B subunit and confocal microscopy in live cells. Collectively, these findings suggest that PLD activity plays an important role in promoting IgE-dependent signaling events within lipid microdomains in mast cells.
初始的IgE依赖性信号转导事件与抗去污剂膜微区相关。抗原刺激后，IgE受体（Fc(ε)RI）在这些微区内聚集。这促进了Src激酶Lyn对Fc(ε)RI亚基的磷酸化，以及与衔接蛋白的相互作用，如T细胞活化连接蛋白。在随后被激活的磷脂酶（PL）中，PLD备受关注，因为它存在于脂质微区，且其产物磷脂酸可能调节信号转导和膜运输。我们发现，在抗原刺激的RBL-2H3肥大细胞中，1-丁醇抑制了Fc(ε)RI与抗去污剂膜组分的结合，1-丁醇将磷脂酸的产生转变为生物惰性的磷脂丁醇。此外，用小干扰RNA敲低PLD2以及在较小程度上敲低PLD1，也抑制了这些组分中Fc(ε)RI和Lyn的积累，以及Src激酶、Fc(ε)RI、T细胞活化连接蛋白的磷酸化和脱颗粒。这些效应伴随着质膜中脂质微区成分神经节苷脂1分布的变化，这是通过荧光标记的霍乱毒素B亚基结合和活细胞共聚焦显微镜确定的。总体而言，这些发现表明PLD活性在促进肥大细胞脂质微区内IgE依赖性信号转导事件中起重要作用。
