title: LysoPCs induce Hck- and PKCδ-mediated activation of PKCγ causing p47phox phosphorylation and membrane translocation in neutrophils.
abstract: 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 (p47phox). 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 p47phoxImmunoblotting, 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 p47phoxIn 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 p47phox.
authors.affiliation: Research Laboratory, Bonfils Blood Center, Denver, Colorado, USA.|Department of Surgery, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; and.
authors.forename: Marguerite R
authors.initials: MR
authors.lastname: Kelher
authors.affiliation: Department of Surgery, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; and.
authors.forename: Nathan J D
authors.initials: NJ
authors.lastname: McLaughlin
authors.affiliation: Department of Surgery, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; and.
authors.forename: Anirban
authors.initials: A
authors.lastname: Banerjee
authors.affiliation: Research Laboratory, Bonfils Blood Center, Denver, Colorado, USA.|Department of Surgery, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; and.
authors.forename: David J
authors.initials: DJ
authors.lastname: Elzi
authors.affiliation: Department of Surgery, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; and.
authors.forename: Fabia
authors.initials: F
authors.lastname: Gamboni
authors.affiliation: Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA.
authors.forename: Samina Y
authors.initials: SY
authors.lastname: Khan
authors.affiliation: Department of Surgery, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; and.
authors.forename: Xianzhong
authors.initials: X
authors.lastname: Meng
authors.affiliation: Department of Surgery, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; and.
authors.forename: Sanchayita
authors.initials: S
authors.lastname: Mitra
authors.affiliation: Research Laboratory, Bonfils Blood Center, Denver, Colorado, USA; christopher.silliman@ucdenver.edu.|Department of Surgery, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; and.|Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Silliman
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D055370:Lung Injury / Q000473:pathology; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D051379:Mice; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D051572:Proto-Oncogene Proteins c-hck / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D008244:Lysophosphatidylcholines; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C087857:protein kinase C gamma; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D002118:Calcium
keywords: WAVE; acute lung injury; lipid rafts