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J Leukoc Biol. 2013 Jul;94(1):109-22. doi: 10.1189/jlb.1212634. Epub 2013 May 13.

PKC-ε pseudosubstrate and catalytic activity are necessary for membrane delivery during IgG-mediated phagocytosis.

Author information

1
Centers for Cell Biology and Cancer Researchnces, Albany Medical College, Albany, New York, USA.

Abstract

In RAW 264.7 cells, PKC-ε regulates FcγR-mediated phagocytosis. BMDM behave similarly; PKC-ε concentrates at phagosomes and internalization are reduced in PKC-ε⁻/⁻ cells. Two questions were asked: what is the role of PKC-ε? and what domains are necessary for PKC-ε concentration? Function was studied using BMDM and frustrated phagocytosis. On IgG surfaces, PKC-ε⁻/⁻ macrophages spread less than WT. Patch-clamping revealed that the spreading defect is a result of the failure of PKC-ε⁻/⁻ macrophages to add membrane. The defect is specific for FcγR ligation and can be reversed by expression of full-length (but not the isolated RD) PKC-ε in PKC-ε⁻/⁻ BMDM. Thus, PKC-ε function in phagocytosis requires translocation to phagosomes and the catalytic domain. The expression of chimeric PKC molecules in RAW cells identified the εPS as necessary for PKC-ε targeting. When placed into (nonlocalizing) PKC-δ, εPS was sufficient for concentration, albeit to a lesser degree than intact PKC-ε. In contrast, translocation of δ(εPSC1B) resembled that of WT PKC-ε. Thus, εPS and εC1B cooperate for optimal phagosome targeting. Finally, cells expressing εK437W were significantly less phagocytic than their PKC-ε-expressing counterparts, blocked at the pseudopod-extension phase. In summary, we have shown that εPS and εC1B are necessary and sufficient for targeting PKC-ε to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension.

KEYWORDS:

FcγR; macrophages; membrane fusion; patch-clamping

PMID:
23670290
PMCID:
PMC3685018
DOI:
10.1189/jlb.1212634
[Indexed for MEDLINE]
Free PMC Article
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