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LysoPCs induce Hck‐ and PKCδ‐mediated activation of PKCγ causing p47 phox phosphorylation and membrane translocation in neutrophils
Author(s) -
Kelher Marguerite R.,
McLaughlin Nathan J. D.,
Banerjee Anirban,
Elzi David J.,
Gamboni Fabia,
Khan Samina Y.,
Meng Xianzhong,
Mitra Sanchayita,
Silliman Christopher C.
Publication year - 2017
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1189/jlb.3a0813-420rrr
Subject(s) - protein kinase c , phosphorylation , biology , microbiology and biotechnology , immunoprecipitation , biochemistry , gene
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 Ca 2+ 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 PKCγ knockout (KO) mice were used in a 2‐event model of TRALI. LysoPCs induced Hck coprecipitation with PKCδ and PKCγ and the PKCδ:PKCγ complex also had a fluorescence resonance energy transfer (FRET) + interaction with lipid rafts and Wiskott‐Aldrich syndrome protein family verprolin‐homologous protein 2 (WAVE2). PKCγ then coprecipitated with p47 phox . Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKCγ KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCδ, which became Thr phosphorylated (Thr507). Activated PKCδ then caused activation of PKCγ, both by Tyr phosphorylation (Τyr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47 phox . In PKCγ KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET + interaction between PKCδ and PKCγ 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 PKCγ KO mice. We conclude that lysoPCs prime PMNs through Hck‐dependent activation of PKCδ, which stimulates PKCγ, resulting in translocation of phosphorylated p47 phox .