Endotoxin Conditioning Induces VCP/p97-mediated and Inducible Nitric-oxide Synthase-dependent Tyr284 Nitration in Protein Phosphatase 2A
Endotoxin Conditioning Induces VCP/p97-mediated and Inducible Nitric-oxide Synthase-dependent Tyr284 Nitration in Protein Phosphatase 2A
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DOI：
10.1074/jbc.m109.099788
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发表时间：
2010-03-19
影响因子：
4.8
通讯作者：
Brautigan, David L.
中科院分区：
文献类型：
--
作者：
Ohama, Takashi;Brautigan, David L.
内毒素激活Toll样受体并重新编程细胞使其对二次暴露难以抵抗。在这里，我们发现，激活不同的Toll样受体引起的蛋白磷酸酶2A催化亚基（PP 2Ac）的水平的时间和剂量依赖性的增加，但不是它的伴侣A亚基。我们纯化的脂多糖诱导形式的PP 2A的层析加免疫沉淀和质谱鉴定VCP/p97作为一个新的合作伙伴PP 2Ac。从原代鼠巨噬细胞和人淋巴细胞中共免疫沉淀内源性VCP/p97和PP 2Ac。GST-VCP/p97结合纯化的PP 2A在下拉测定，显示直接的蛋白质-蛋白质相互作用。巨噬细胞的内毒素调节诱导与VCP/p97相关的PP 2Ac中3-硝基酪氨酸的形成，这一反应在iNOS敲除小鼠的巨噬细胞中严重降低。纯化的PP 2A与过氧亚硝酸根反应解离A亚基，并通过质谱鉴定PP 2Ac中的3-硝基-Tyr（284）。Myc-PP 2Ac（Y284 F）在细胞中表达的耐过氧亚硝酸盐诱导的硝化和减少的A亚基结合。VCP/p97或PP 2Ac的瞬时表达足以提高双特异性磷酸酶DUSP 1的水平，减少p38 MAPK的激活，并抑制肿瘤坏死因子-α的释放。我们建议，VCP/p97介导的酪氨酸硝化的PP 2A增加磷酸酶PP 2A和DUSP 1的水平，以促进条件细胞的难治性反应。
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.