A role for PKC-ɛ 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-ɛ 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-ɛ, but not PKC-δ, concentrated around the beads. PKC-ɛ accumulation was transient; apparent as a “flash” on target ingestion. Similarly, endogenous PKC-ɛ was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-ɛ, but not PKC-α, PKC-δ, or PKC-γ enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC-ɛ expressors was twice that of cells expressing GFP PKC-δ. Expression of the regulatory domain (ɛRD) and the first variable region (ɛV1) of PKC-ɛ inhibited uptake, whereas the corresponding PKC-δ region had no effect. Actin polymerization was enhanced on expression of GFP PKC-ɛ and ɛRD, but decreased in cells expressing ɛV1, suggesting that the ɛRD and ɛV1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC-ɛ in FcγR-mediated phagocytosis that is independent of its effects on actin assembly.