Targeting of protein kinase C-ε during Fcγ receptor-dependent phagocytosis requires the εC1B domain and phospholipase C-γ1

Protein kinase C-epsilon (PKC-epsilon) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-epsilon mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding epsilon C1 and epsilon C1B domains, or the epsilon C1B point mutant epsilon C259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that epsilon C259G, epsilon C1, and epsilon C1B accumulation at phagosomes was significantly less than that of intact PKC-epsilon. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-epsilon translocation. Thus, DAG binding to epsilon C1B is necessary for PKC-epsilon translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-gamma 1, and PI-PLC-gamma 2 in PKC-epsilon accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-epsilon localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-epsilon accumulation. Although expression of PI-PLC-gamma 2 is higher than that of PI-PLC-gamma 1, PI-PLC-gamma 1 but not PI-PLC-gamma 2 consistently concentrated at phagosomes. Macrophages from PI-PLC-gamma 2(-/-) mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-epsilon at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-gamma 1 as the enzyme that supports PKC-epsilon localization and phagocytosis. That PI-PLC-gamma 1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-gamma 1 provides DAG that binds to epsilon C1B, facilitating PKC-epsilon localization to phagosomes for efficient IgG-mediated phagocytosis.