A role for PKC-ε in FcγR-mediated phagocytosis by RAW 264.7 cells

Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC-ε in Fcγ receptor (FcγR)–dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG–opsonized beads. PKC-ε, but not PKC-δ, concentrated around the beads. PKC-ε accumulation was transient; apparent as a “flash” on target ingestion. Similarly, endogenous PKC-ε was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-ε, but not PKC-α, PKC-δ, or PKC-γ enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC-ε expressors was twice that of cells expressing GFP PKC-δ. Expression of the regulatory domain (εRD) and the first variable region (εV1) of PKC-ε inhibited uptake, whereas the corresponding PKC-δ region had no effect. Actin polymerization was enhanced on expression of GFP PKC-ε and εRD, but decreased in cells expressing εV1, suggesting that the εRD and εV1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC-ε in FcγR-mediated phagocytosis that is independent of its effects on actin assembly.