PAK2 in Superoxide Generation by LAP Neutrophils

LAP PMN exhibit increased secretion of superoxide (O2-) and pro-inflammatory mediators. p47phox, a subunit of the NADPH oxidase and central in O2- generation is phosphorylated in resting LAP PMN. P21-activated kinases (PAKs) are ubiquitous serine/threonine protein kinases downstream of activated Rac or Cdc42 involved in regulation of cytoskeletal architecture, cell cycle progression, motility, and stress response in a variety of cell types. In vitro PAK2 directly phosphorylates p47phox; however, PAK2 pathways in neutrophils are largely unknown. Objectives: to determine the role of PAK2 in O2- generation by LAP PMN. Methods: Peripheral blood neutrophils were isolated by Ficoll-Hypaque gradient centrifugation; protein phosphorylation was assessed by Western blot; O2- generation was assessed by superoxide dismutase-inhibitable cytochrome c reduction. Co-immunoprecipitation and confocal microscopy were employed to assess protein-protein interactions. Since, human PMN are short lived cells in vitro and transfection experiments are impractical, Tat, an HIV protein functional domain that transduces proteins through plasma membrane, and PAK2 fused constructs were produced by classical cloning techniques to introduce active PAK2 into human PMN. Results: PAK2 is activated (hyperphosphorylated) in LAP neutrophils. PAK2 co-localizes with p47phox in situ, by both co-immunoprecipitation and confocal microscopy. PAK2 is downstream of PI3-kinase in human neutrophils as demonstrated by wortmannin inhibition. Transduction of Tat-PAK2 constructs (PAK2 auto-inhibitory domain, constitutively active or kinase dead) revealed intracellular protein after only 5 minutes of incubation. Transduced PAK2 phosphorylates p47phox in human neutrophils, with subsequent O2- generation. Conclusion: TAT-fusion protein transduction technology is a valuable method for investigating signaling pathways in short-lived cells. LAP neutrophils are primed to produce excessive O2-. PAK2 is upstream of p47phox leading to O2- production. PAK2 is pre-activated in LAP PMN and is in the direct pathway leading to priming of LAP PMN and pre-assembly of the NADPH-oxidase through early phosphorylation of p47phox. Grants DE016191, DE016893, M01 RR00533