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 Golgiassociated 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.