Superoxide Production at Phagosomal Cup/Phagosome through βI Protein Kinase C during FcγR-Mediated Phagocytosis in Microglia

Abstract

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcγR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. βI PKC, εPKC, and diacylglycerol kinase (DGK) β dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47phox, an essential cytosolic component of NADPH oxidase and a substrate for βI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O2−) production was profoundly inhibited by Gö6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O2− production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of βI PKC is involved in O2− production, and that O2− production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKβ plays a prominent role in regulation of O2− production during FcγR-mediated phagocytosis.

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

“…We reported that p47 phox accumulates transiently on phagocytic cups and mature phagosomes during Fc␥R-mediated phagocytosis (Ueyama et al, 2004), consistent with earlier studies showing p47 phox and p67 phox associate with both nascent and mature phagosomes in neutrophils and neutrophil-like PLB-985 cells (Allen et al, 1999;van Bruggen et al, 2004). The present study shows that GFP-p40 phox also transiently associates on phagosomes, although this targeting is initiated at later stages after phagosome sealing.…”

“…The pEGFP-C1 plasmids (Clontech) containing human p47 phox , p47 phox (W193R), p40 phox , p40 phox (PX; aa 1-167), p40 phox (PX:R105K), and p47 phox (R90K) were described previously (Ago et al, , 2003Ueyama et al, 2004). cDNA encoding p67 phox and p67 phox (⌬SH3, aa 1-457) were described previously Ueyama et al, 2004), transferred into pEGFP-C1, and designated GFP-p67 phox and GFP-p67 phox (⌬SH3), respectively.…”

“…In recent work, we described mechanisms for translocation of p47 phox (Ueyama et al, 2004) and isoform-specific translocation of Rac to the phagosome (Ueyama et al, 2005). To understand and clarify targeting of p67 phox , an essential cytoplasmic Nox2 activator that acts through Rac, we used AA as a stimulant and explored the adaptor functions of p40 phox and p47 phox in recruitment of p67 phox to membranes.…”

“…Although relatively high concentrations of AA (50 -200 M) are required for activation of Nox2 both in vitro and in vivo, functional roles for cytosolic phospholipase A 2 (cPLA 2 ), which produces AA, in Nox2 activation have been demonstrated at the cellular level (Dana et al, 1998;Zhao et al, 2002;Shmelzer et al, 2003). Furthermore, recent studies have shown that the orchestration of low concentrations of AA produced by PLA 2 together with protein kinase C (PKC)-dependent phosphorylation promotes translocation of p47 phox and enhances ROS production by Nox2 (Shiose and Sumimoto, 2000;Peng et al, 2003).In recent work, we described mechanisms for translocation of p47 phox (Ueyama et al, 2004) and isoform-specific translocation of Rac to the phagosome (Ueyama et al, 2005). To understand and clarify targeting of p67 phox , an essential cytoplasmic Nox2 activator that acts through Rac, we used AA as a stimulant and explored the adaptor functions of p40 phox and p47 phox in recruitment of p67 phox to membranes.…”

“…Therefore, we used GFP-p67 phox to monitor its interactions with other oxidase components in intact cells. Because we described the mechanism of GFP-p47 phox accumulation on phagosomal cups and phagosomes during Fc␥R-mediated phagocytosis (Ueyama et al, 2004), the present study focused on p40 phox . GFP-p40 phox is detected diffusely throughout the cytoplasm of resting RAW 264.7 cells (Figure 2A).…”