Endotoxin conditioning induces VCP/p97-mediated and inducible nitric-oxide synthase-dependent Tyr284 nitration in protein phosphatase 2A.
Endotoxins activate Toll-like receptors and reprogram cells to be refractory to secondary exposure. Here we found that activation of different Toll-like receptors elicited a time- and dose-dependent increase in the levels of the protein phosphatase 2A catalytic subunit (PP2Ac) but not its partner A subunit. We purified the lipopolysaccharide-induced form of PP2A by chromatography plus immunoprecipitation and used mass spectrometry to identify VCP/p97 as a novel partner for PP2Ac. Endogenous VCP/p97 and PP2Ac were co-immunoprecipitated from primary murine macrophages and human lymphocytes. GST-VCP/p97 bound purified PP2A in pulldown assays, showing direct protein-protein interaction. Endotoxin conditioning of macrophages induced formation of 3-nitrotyrosine in the PP2Ac associated with VCP/p97, a response severely reduced in macrophages from iNOS knock-out mice. The reaction of purified PP2A with peroxynitrite dissociated the A subunit, and 3-nitro-Tyr(284) was identified in PP2Ac by mass spectrometry. Myc-PP2Ac (Y284F) expressed in cells was resistant to peroxynitrite-induced nitration and reduction of A subunit binding. Transient expression of either VCP/p97 or PP2Ac was sufficient to elevate levels of the dual specificity phosphatase DUSP1, reduce p38 MAPK activation, and suppress tumor necrosis factor-alpha release. We propose that VCP/p97-mediated Tyr nitration of PP2A increases the levels of phosphatases PP2A and DUSP1 to contribute to the refractory response of conditioned cells.
Ohama
Takashi
T
Department of Microbiology, University of Virginia School of Medicine, Center for Cell Signaling, Charlottesville, Virginia 22908, USA.
Brautigan
David L
DL
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D054638:Dual Specificity Phosphatase 1 / Q000378:metabolism; D004731:Endotoxins / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D009584:Nitrogen / Q000737:chemistry; D025941:Protein Interaction Mapping; D054648:Protein Phosphatase 2 / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D004731:Endotoxins; D014443:Tyrosine; D052247:Nitric Oxide Synthase Type II; D054648:Protein Phosphatase 2; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; C517020:Dusp1 protein, mouse; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein; C000614179:Vcp protein, mouse; D009584:Nitrogen