Two PI 3-Kinases and One PI 3-Phosphatase Together Establish the Cyclic Waves of Phagosomal PtdIns(3)P Critical for the Degradation of Apoptotic Cells

Abstract

Author SummaryDuring animal development and in adulthood many cells are programmed to die by an active process called apoptosis. These dead or dying apoptotic cells are swiftly taken up by scavenger cells into membrane-bound compartments—phagosomes—where they are subsequently degraded when other intracellular organelles containing digestive enzymes fuse with phagosomes—a process called phagosome maturation. Phagocytosis of apoptotic cells is important for tissue remodeling in development and to prevent harmful inflammatory and autoimmune responses. In nematode worms—a model organism in which to study apoptosis—phagosome maturation is accompanied by two waves of the signaling molecule phosphatidylinositol 3-phosphate (PtdIns(3)P) in this compartment: one that forms soon after the formation of the phagosome and lasts for 10–15 minutes, and a second, weaker one 10 minutes later that lasts until the apoptotic cell is fully digested. In this study, we investigated the mechanism that regulates the timing and length of these two waves. We found that they are established by the sequential and combined action of three enzymes: two phosphoinositide 3-kinases, which add a phosphate group to the 3′ site of PtdIns, and one phosphoinositide 3-phosphatase, which removes it. We showed that inactivation of both kinases depleted phagosomes of PtdIns(3)P and resulted in the arrest of phagosome maturation and degradation of apoptotic cells. In addition, the timely turnover of PtdIns(3)P catalyzed by the phosphatase was critical for the step-wise progress of phagosome maturation. Our findings suggest that reversible phosphorylation of phophoinositides, catalyzed by distinct sets of kinases and phosphatases, might be a general mechanism to drive multi-step intracellular membrane trafficking events.