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Vol. 17, Issue 2, 799-813, February 2006

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Targeting of Protein Kinase C- during Fc Receptor-dependent Phagocytosis Requires the C1B Domain and Phospholipase C-1

Keylon L. Cheeseman ^*, Takehiko Ueyama , Tanya M. Michaud ^*, Kaori Kashiwagi , Demin Wang , Lindsay A. Flax ^*, Yasuhito Shirai , Daniel J. Loegering , Naoaki Saito , and Michelle R. Lennartz ^*

^* Centers for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208 Kobe University, Nada-Ku, Kobe 657-8501, Japan; Centers for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208 Kobe University, Nada-Ku, Kobe 657-8501, Japan; Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Nada-Ku, Kobe 657-8501, Japan; and The Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, WI 53226

Submitted December 21, 2004; Revised October 27, 2005; Accepted November 21, 2005
Monitoring Editor: Vivek Malhotra

Protein kinase C- (PKC-) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C- mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding C1 and C1B domains, or the C1B point mutant C259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that C259G, C1, and C1B accumulation at phagosomes was significantly less than that of intact PKC-. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC- translocation. Thus, DAG binding to C1B is necessary for PKC- translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-1, and PI-PLC-2 in PKC- accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC- localization. In contrast, the PI-PLC inhibitor U73122 [GenBank] decreased both phagocytosis and PKC- accumulation. Although expression of PI-PLC-2 is higher than that of PI-PLC-1, PI-PLC-1 but not PI-PLC-2 consistently concentrated at phagosomes. Macrophages from PI-PLC-2^-/- mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC- at the phagosome, and their sensitivity to U73122 [GenBank] . This implicates PI-PLC-1 as the enzyme that supports PKC- localization and phagocytosis. That PI-PLC-1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-1 provides DAG that binds to C1B, facilitating PKC- localization to phagosomes for efficient IgG-mediated phagocytosis.

Abbreviations used: 2,3-DPG, 2,3-diphospho-D-glyceric acid; BIgG, IgG-opsonized glass beads; BMDM, bone marrow-derived macrophage; DAG, diacylglycerol; EIgG, IgG-opsonized sheep red blood cell; GFP, green fluorescent protein; IP₃, inositol trisphosphate; PA, phosphatidic acid; PAP-1, phosphatidic acid phosphohydrolase-1; PI-PLC, phosphatidylinositol-specific phospholipase C; PKC, protein kinase C; PLD, phospholipase D.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Michelle R. Lennartz (lennarm{at}mail.amc.edu).

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