PKC-ε regulates vesicle delivery and focal exocytosis for efficient IgG-mediated phagocytosis.

Protein kinase C (PKC)-ε is required for membrane addition during IgG-mediated phagocytosis, but its role in this process is ill defined. Here, we performed high-resolution imaging, which reveals that PKC-ε exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-ε colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-ε and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles were not delivered in macrophages from PKC-ε null mice, or upon dissociation of the Golgi-associated pool of PKC-ε, implies that Golgi-tethered PKC-ε is a driver of Golgi-to-phagosome trafficking. Finally, we established that the regulatory domain of PKC-ε is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-ε in focal exocytosis - its regulatory domain drives Golgi-derived vesicles to the phagosome, whereas catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain. This article has an associated First Person interview with the first author of the paper.

Golgi-Associated Protein Kinase C-ε Is Delivered to Phagocytic Cups: Role of Phosphatidylinositol 4-Phosphate.

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)P2 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 trans-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)P2 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.