A role for PKC-epsilon in Fc gammaR-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- epsilon in Fc gamma receptor (Fc gammaR)-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- epsilon, but not PKC-delta, concentrated around the beads. PKC- epsilon accumulation was transient; apparent as a "flash" on target ingestion. Similarly, endogenous PKC- epsilon was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC- epsilon, but not PKC-alpha, PKC-delta, or PKC-gamma enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC- epsilon expressors was twice that of cells expressing GFP PKC-delta. Expression of the regulatory domain ( epsilon RD) and the first variable region ( epsilon V1) of PKC- epsilon inhibited uptake, whereas the corresponding PKC-delta region had no effect. Actin polymerization was enhanced on expression of GFP PKC- epsilon and epsilon RD, but decreased in cells expressing epsilon V1, suggesting that the epsilon RD and epsilon V1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC- epsilon in Fc gammaR-mediated phagocytosis that is independent of its effects on actin assembly.
Larsen
Elaine C
EC
Centers for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA.
Ueyama
Takehiko
T
Brannock
Pamela M
PM
Shirai
Yasuhito
Y
Saito
Naoaki
N
Larsson
Christer
C
Loegering
Daniel
D
Weber
Peter B
PB
Lennartz
Michelle R
MR
D000818:Animals; D002460:Cell Line; D003911:Dextrans / Q000494:pharmacology; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D007074:Immunoglobulin G / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism / Q000502:physiology*; D008164:Luminescent Proteins / Q000378:metabolism; D008264:Macrophages; D051379:Mice; D010587:Phagocytosis; D010590:Phalloidine / Q000494:pharmacology; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000378:metabolism*; D012235:Rhodamines / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D003911:Dextrans; D007074:Immunoglobulin G; D007527:Isoenzymes; D008164:Luminescent Proteins; D020033:Protein Isoforms; D017452:Receptors, IgG; D012235:Rhodamines; D049452:Green Fluorescent Proteins; D010590:Phalloidine; C497259:Prkcd protein, mouse; C497256:Prkce protein, mouse; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon