LysoPCs induce Hck- and PKCδ-mediated activation of PKCγ causing p47phox phosphorylation and membrane translocation in neutrophils

Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion-related acute lung injury (TRALI). LysoPCs cause ligation of the G2A receptor, cytosolic Ca2+ flux, and activation of Hck. We hypothesize that lysoPCs induce Hck-dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47(phox)). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole-cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild-type (WT) and PKC gamma knockout (KO) mice were used in a 2-event model of TRALI. LysoPCs induced Hck coprecipitation with PKC delta and PKC gamma and the PKC delta: PKC gamma complex also had a fluorescence resonance energy transfer (FRET)(+) interaction with lipid rafts and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2). PKC gamma then coprecipitated with p47(phox). Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKC gamma KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKC delta, which became Thr phosphorylated (Thr507). Activated PKC delta then caused activation of PKC gamma, both by Tyr phosphorylation (.yr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47(phox). In PKC gamma KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET+ interaction between PKC delta and PKC gamma nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2-event in vivo model of TRALI but did not induce TRALI in PKC gamma KO mice. We conclude that lysoPCs prime PMNs through Hck-dependent activation of PKC delta, which stimulates PKC gamma, resulting in translocation of phosphorylated p47(phox).