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

Tiffany R. Wood, Rachel Y. Chow, Cheryl M. Hanes, Xuexin Zhang, Kaori Kashiwagi, Yasuhito Shirai, Mohamed Trebak, Daniel J. Loegering, Naoaki Saito, Michelle R. Lennartz

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7 Scopus citations

Abstract

In RAW 264.7 cells [1], 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. Patchclamping 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.

Original languageEnglish (US)
Pages (from-to)109-122
Number of pages14
JournalJournal of Leukocyte Biology
Volume94
Issue number1
DOIs
StatePublished - Jul 2013

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology
  • Cell Biology

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    Wood, T. R., Chow, R. Y., Hanes, C. M., Zhang, X., Kashiwagi, K., Shirai, Y., Trebak, M., Loegering, D. J., Saito, N., & Lennartz, M. R. (2013). PKC-ε pseudosubstrate and catalytic activity are necessary for membrane delivery during IgG-mediated phagocytosis. Journal of Leukocyte Biology, 94(1), 109-122. https://doi.org/10.1189/jlb.1212634