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

¹ Centers for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208
² Centers for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208
³ Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
⁴ Department of Laboratory Medicine, Molecular Medicine, Malmo University Hospital, S-205 02 Malmo, Sweden
⁵ Waste Reduction by Waste Reduction, Inc., 1 University Place, Rensselaer, NY 12144

Address correspondence to Michelle R. Lennartz, Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208. Tel.: (518) 262-5217. Fax: (518) 262-5669. E-mail: lennarm{at}mail.amc.edu

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- in Fc receptor (FcR)–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-, but not PKC-, concentrated around the beads. PKC- accumulation was transient; apparent as a "flash" on target ingestion. Similarly, endogenous PKC- was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-, but not PKC-, PKC-, or PKC- enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC- expressors was twice that of cells expressing GFP PKC-. Expression of the regulatory domain (RD) and the first variable region (V1) of PKC- inhibited uptake, whereas the corresponding PKC- region had no effect. Actin polymerization was enhanced on expression of GFP PKC- and RD, but decreased in cells expressing V1, suggesting that the RD and V1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC- in FcR-mediated phagocytosis that is independent of its effects on actin assembly.

Key Words: protein kinase C-epsilon; macrophage; confocal; signal transduction; immunoglobulin

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