ALBERT

Description: We showed that ceramide-1-phosphate (C1P) is formed by ceramide kinase (CERK). In this study the mechanism by which C1P enhanced phagocytosis and phagolysosomal formation in COS-1 cells expressing hCERK was evaluated. We hypothesized that CERK has a unique role in inducing Ca2+ signaling during Fc-mediated phagocytosis via its generation of C1P. Furthermore, we also hypothesize that CERK mediated changes in Ca2+ levels were not direct but required the participation of a store operated channels (SOC). In some systems, evidence suggests that SOC might be related to transient receptor potential channel (TRP) homologues. This hypothesis was tested using cell lines with different levels of expression of CERK and by using series of pharmacological inhibitors. To monitor subcellular localization of hCERK, red fluorescent protein (RFP)-tagged hCERK was created and confocal microscopy was performed. To study dynamic changes in Ca2+ during phagocytosis, imaging microscopy was used. Phagolysosome formation was evaluated by labeling the cells with lysotracker red DND-99 at 37oC and followed by imaging. When challenged with EIgG, hCERK transfected cells increased phagocytosis and simultaneously increased C1P. During subcellular fractionation of hCERK transfected cells, CERK translocates during activation from the cytosol to a lipid raft fraction. We also observed that TRP-1 accumulated at the site of CERK translocation in lipid rafts. Microfluorimetry of indo-1 revealed that Ca2+ oscillations in COS-1 cells, stably transfected with Fcγ RIIA/hCERK, were much higher compared to Fcγ RIIA transfected cells. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. hCERK transfected cells had a mean rate of fusion of 90%, compare to 76% ( catalytic inactive mutant of CERK-G198DhCERK), 65% (Fcγ RIIA), and 70% (Vector) transfected cells. The indo-1 intensity spikes demonstrated significantly higher Ca2+ intensities for phagosomes associated with the hCERK transfectants. The SOC inhibitor SKF96365 blocked enhanced Ca2+ signaling in the hCERK transfected cells. Similarly SOC inhibitors reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. Our data showed that there is a co-localization of EIgG and CERK as well as caveolin-1 and CERK on the surface of the phagocytic cells. Site directed mutagenesis demonstrated that the kinase activity of hCERK was required for higher Ca2+ signals. Pharmacological inhibitors revealed that this Ca2+ signal operates through SOC. Our results show that introduction of the hCERK gene in cells alters the functional behavior of intracellular signals and supports the role of C1P in promoting membrane fusion by modulating Ca2+ signaling through activation of SOC