title: PKC-ε pseudosubstrate and catalytic activity are necessary for membrane delivery during IgG-mediated phagocytosis.
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.
authors.affiliation: Centers for Cell Biology and Cancer Researchnces, Albany Medical College, Albany, New York, USA.
authors.forename: Tiffany R
authors.initials: TR
authors.lastname: Wood
authors.forename: Rachel Y
authors.initials: RY
authors.lastname: Chow
authors.forename: Cheryl M
authors.initials: CM
authors.lastname: Hanes
authors.forename: Xuexin
authors.initials: X
authors.lastname: Zhang
authors.forename: Kaori
authors.initials: K
authors.lastname: Kashiwagi
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Shirai
authors.forename: Mohamed
authors.initials: M
authors.lastname: Trebak
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Loegering
authors.forename: Naoaki
authors.initials: N
authors.lastname: Saito
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Lennartz
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry; D007074:Immunoglobulin G / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000378:metabolism*; D010957:Plasmids; D051744:Protein Kinase C-epsilon / Q000502:physiology*; D021381:Protein Transport; D011554:Pseudopodia / Q000502:physiology*; D017452:Receptors, IgG / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D050505:Mutant Proteins; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D051744:Protein Kinase C-epsilon
keywords: FcγR; macrophages; membrane fusion; patch-clamping