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

In RAW 264.7 cells [1], PKC-epsilon regulates FcR-mediated phagocytosis. BMDM behave similarly; PKC-epsilon concentrates at phagosomes and internalization are reduced in PKC-epsilon(-/-) cells. Two questions were asked: what is the role of PKC-epsilon? and what domains are necessary for PKC-epsilon concentration? Function was studied using BMDM and frustrated phagocytosis. On IgG surfaces, PKC-epsilon(-/-) macrophages spread less than WT. Patch-clamping revealed that the spreading defect is a result of the failure of PKC-epsilon(-/-) macrophages to add membrane. The defect is specific for FcR ligation and can be reversed by expression of full-length (but not the isolated RD) PKC-epsilon in PKC-epsilon(-/-) BMDM. Thus, PKC-epsilon function in phagocytosis requires translocation to phagosomes and the catalytic domain. The expression of chimeric PKC molecules in RAW cells identified the epsilon PS as necessary for PKC-epsilon targeting. When placed into (nonlocalizing) PKC-, epsilon PS was sufficient for concentration, albeit to a lesser degree than intact PKC-epsilon. In contrast, translocation of (epsilon PSC1B) resembled that of WT PKC-epsilon. Thus, epsilon PS and epsilon C1B cooperate for optimal phagosome targeting. Finally, cells expressing epsilon K437W were significantly less phagocytic than their PKC-epsilon-expressing counterparts, blocked at the pseudopod-extension phase. In summary, we have shown that epsilon PS and epsilon C1B are necessary and sufficient for targeting PKC-epsilon to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension.