Hanes Cheryl M, D'Amico Anna E, Ueyama Takehiko, Wong Alexander C, Zhang Xuexin, Hynes W Frederick, Barroso Margarida M, Cady Nathaniel C, Trebak Mohamed, Saito Naoaki, Lennartz Michelle R
Department of Regenerative and Cancer Cell Biology, Albany Medical College, Albany, NY 12208.
Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
J Immunol. 2017 Jul 1;199(1):271-277. doi: 10.4049/jimmunol.1700243. Epub 2017 May 24.
Protein kinase C-ε (PKC-ε) 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 PI(3,5)P 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 colocalizes with the -Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered 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 PI4 kinase 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 PI(3,5)P 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 Fc [γ] receptor-mediated phagocytosis.
吞噬杯处的蛋白激酶C-ε（PKC-ε）介导高效IgG介导的吞噬作用所需的膜融合。C1B和假底物（εPS）结构域对于这种聚集是必需且足够的。C1B结合二酰基甘油；εPS的对接伴侣未知。脂质体分析显示，εPS结合磷脂酰肌醇4-磷酸（PI4P）和PI(3,5)P，渥曼青霉素可抑制，但LY294002不能抑制PKC-ε在吞噬杯处的聚集，并显著降低吞噬速率。由于渥曼青霉素抑制PI4激酶，我们推测PI4P介导PKC-ε在吞噬杯处的聚集和吞噬速率。PKC-ε与高尔基体网络（TGN）PI4P报告基因P4M共定位，表明它与TGN相连。对表达绿色荧光蛋白（GFP）-PKC-ε的巨噬细胞进行实时成像显示，吞噬过程中高尔基体相关的PKC-ε减少，这与从高尔基体向吞噬体的转运一致。用PI4激酶抑制剂PIK93处理可降低TGN和吞噬杯处的PKC-ε，减少吞噬作用，并阻止膜融合时伴随的电容增加。最后，高尔基体靶向的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对高效吞噬作用至关重要。这些数据共同支持了一个模型，即PKC-ε通过εPS-PI4P相互作用与TGN相连。TGN相关的PKC-ε池聚集在吞噬杯处，在那里它介导吞噬作用所需的膜融合。这些数据的新颖之处在于证明了εPS结合PI4P和PI(3,5)P，并且PI4P对于PKC-ε在TGN处的定位、其向吞噬杯的转运以及高效Fc[γ]受体介导的吞噬作用所需的膜融合是必需的。
