Golgi-Associated Protein Kinase C-ε Is Delivered to Phagocytic Cups: Role of Phosphatidylinositol 4-Phosphate.
Golgi-Associated Protein Kinase C-ε Is Delivered to Phagocytic Cups: Role of Phosphatidylinositol 4-Phosphate.
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DOI：
10.4049/jimmunol.1700243
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发表时间：
2017-07-01
期刊：
影响因子：
--
通讯作者：
Lennartz MR
中科院分区：
文献类型：
--
作者：
Hanes CM;D'Amico AE;Ueyama T;Wong AC;Zhang X;Hynes WF;Barroso MM;Cady NC;Trebak M;Saito N;Lennartz MR
吞噬杯处的蛋白激酶C - ε（PKC - ε）浓度介导了有效免疫球蛋白G（IgG）介导的吞噬作用所必需的膜融合。C1B和假底物（εPS）结构域对于这种浓度是必需且足够的。C1B结合二酰基甘油；εPS的对接伙伴未知。脂质体分析显示，εPS结合磷脂酰肌醇 - 4 - 磷酸（PI4P）和磷脂酰肌醇 - 4,5 - 二磷酸（PIP2）。渥曼青霉素（wortmannin），而非LY294002，抑制吞噬杯处的PKC - ε浓度，并显著降低吞噬速率。由于渥曼青霉素抑制PI4激酶，我们假设PI4P介导吞噬杯处的PKC - ε浓度和吞噬速率。PKC - ε与反式高尔基体网络PI4P报告分子P4M共定位，表明其在高尔基体反面网状结构（TGN）处的系留。表达绿色荧光蛋白 - PKC - ε的巨噬细胞的实时成像显示，在吞噬过程中高尔基体相关的PKC - ε缺失，这与高尔基体到吞噬体的易位一致。用PI4K抑制剂PIK93处理，会减少高尔基体反面网状结构和吞噬杯处的PKC - ε，降低吞噬作用，并阻止伴随膜融合的电容增加。最后，高尔基体导向的PI4P磷酸酶hSac1 - K2A的表达重现了PIK93的表型，证实高尔基体相关的PI4P对于有效吞噬至关重要。总之，这些数据与一个模型相符，在该模型中，PKC - ε通过εPS - PI4P相互作用系留在高尔基体反面网状结构。高尔基体反面网状结构相关的PKC - ε池在吞噬杯处聚集，在那里它介导吞噬作用所必需的膜融合。这些数据的新颖之处在于证明了εPS结合PI4P和PIP2，以及PI4P对于PKC - ε在高尔基体反面网状结构的定位、其向吞噬杯的易位以及有效FcγR介导的吞噬作用所需的膜融合是必需的。
PKC-ε concentration at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (εPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for εPS is unknown. Liposome assays revealed that the εPS binds phosphatidylinositol 4-phosphate (PI4P) and PIP2. Wortmannin, but not LY294002, inhibits PKC-ε concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ε concentration at cups and the rate of phagocytosis. PKC-ε co-localizes with the Trans Golgi network PI4P reporter the P4M, suggesting its' tethering at the TGN. Real time imaging of GFP-PKC-ε expressing macrophages revealed a loss of Golgi-associated PKC-ε during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4K inhibitor, reduces PKC-ε at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgi-associated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ε is tethered to the TGN via an εPS-PI4P interaction. The TGN-associated pool of PKC-ε concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that εPS binds PI4P and PIP2 and that PI4P is necessary for PKC-ε localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient FcγR-mediated phagocytosis.
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