Endotoxin Conditioning Induces VCP/p97-mediated and Inducible Nitric-oxide Synthase-dependent Tyr284 Nitration in Protein Phosphatase 2A

Abstract

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 dosedependent 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-␣ 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.Endotoxins are microbial components that engage Toll-like receptors (TLRs) 2 to activate MAPK signaling and stimulate cytokine release from cells of the innate immune system. Cells adapt to endotoxin and become resistant to subsequent stimulation, a response called preconditioning or tolerance. This resistant state is not well understood, but it involves the induction and suppression of many genes and has been studied in both cellular and in vivo models (1). Reduced cytokine release by preconditioned cells is attributed to diminished activation of MAPK, in particular p38 MAPK (1). The MAPKs are under the control of different classes of protein phosphatases, including protein phosphatase 2A (PP2A) and dual specificity phosphatases (DUSPs). PP2A is an essential serine/threonine phosphatase conserved from yeast to human that regulates signal transduction pathways, cell cycle, cell growth, cytoskeleton dynamics, and cell mobility (2). PP2A exists in cells as a heterotrimer comprised of a 36-kDa catalytic subunit (PP2Ac), a 65-kDa scaffolding subunit (A subunit), plus one regulatory B subunit from four different families of genes. There is good evidence that mammalian cells contain a pool of the AC dimer, as well as heterotrimer forms of PP2A (3). Biochemical and genetic analyses have shown that the regulatory B subunits control PP2A specificity by targeting the AC core dimer to substrates. Crystal structures of heterotrimeric PP2A holoenzymes revealed that the regulatory B subunits interact with PP2Ac while bound side by side on the same face...

Ser70 phosphorylation of Bcl-2 by selective tyrosine nitration of PP2A-B56δ stabilizes its antiapoptotic activity

“…In fact, more is not always better: others have found that high levels of exogenous ONOO 2 (.75 mM) can nitrate and activate the catalytic subunit of PP2A. 34,35 This could account for the biphasic effect of ONOO 2 observed in Figure 4C and supplemental Figure 4D, where initial increase in S70pBcl-2 and cell viability at low doses of ONOO 2 was followed by falling Bcl-2 phosphorylation and cell death at high-dose ONOO 2 .…”

Mammalian Ste20-like Kinase (Mst2) Indirectly Supports Raf-1/ERK Pathway Activity via Maintenance of Protein Phosphatase-2A Catalytic Subunit Levels and Consequent Suppression of Inhibitory Raf-1 Phosphorylation

“…Lipopolysaccharide treatment of macrophages renders cells refractory to repeated exposure in part through an agonist-induced stabilization of PP2A-C. This is mediated in part by valosin-containing protein/p97, which is required to maintain elevated PP2A-C levels, consequent to lipopolysaccharide treatment, through a process involving Tyr nitration of the PP2A-C polypeptide (54). Whether or not Mst2 operates in a similar manner to maintain PP2A-C remains to be determined.…”

Evaluation of Vitamin C for Adjuvant Sepsis Therapy

“…Peroxynitrite has a short half-life of appearance and thus the site of its production dictates which tyrosine residues it can access. iNOS is known to interact with PP2Ac directly (42). If eNOS does not interact with PP2Ac similarly, then the discrepant interactions of the NOS isoforms with PP2Ac may account for the increase in PP2A nitration that occurs in sepsis, when iNOS activity is elevated and eNOS activity is depressed.…”