Differential requirement for classic and novel PKC isoforms in respiratoryburst and phagocytosis in RAW 264.7 cells

The binding of Ab (IgG)-opsonized particles by Fc gamma Rs on macrophages r esults in phagocytosis of the particles and generation of a respiratory bur st. Both IgG-stimuiated phagocytosis and respiratory burst involve activati on of protein kinase C (PKC), However, the specific PKC isoforms required f or these responses have yet to be identified. We have studied the involveme nt of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in th e mouse macrophage-like cell line, RAW 264.7, Like primary monocyte/macroph ages, their IgG-mediated phagocytosis was calcium independent and diacylgly cerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphost in C as well as by the classic PKC-selective inhibitors Go 6976 and CGP 412 51, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs . RAW 264.7 cells expressed PKCs alpha, betaI, delta, epsilon, and zeta, Su bcellular fractionation demonstrated that PKCs alpha, delta ,and epsilon tr anslocate to membranes during phagocytosis, In Ca2+-depleted cells, only no vel PKCs delta and epsilon increased in membranes, and the time course of t heir translocation was consistent with phagosome formation. Confocal micros copy of cells transfected with green fluorescent protein-conjugated PKC alp ha or epsilon confirmed that these isoforms translocated to the forming pha gosome in Ca-replete cells, but only PKC epsilon colocalized with phagosome s in Ca2+-depleted cells. Taken together, these results suggest that the cl assic PKC alpha mediates IgG-stimulated respiratory burst in macrophages, w hereas the novel PKCs delta and/or epsilon are necessary for phagocytosis.