title: TLR and TNF-R1 activation of the MKK3/MKK6-p38α axis in macrophages is mediated by TPL-2 kinase.
abstract: Previous studies suggested that Toll-like receptor (TLR) stimulation of the p38α MAP kinase (MAPK) is mediated by transforming growth factor-β-activated kinase 1 (TAK1) activation of MAPK kinases, MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor tumour progression locus 2 (TPL-2)-dependent protein phosphorylation following TLR4 stimulation with lipopolysaccharide, comparing macrophages from wild-type mice and Map3k8(D270A/D270A) mice expressing catalytically inactive TPL-2 (MAP3K8). In addition to the established TPL-2 substrates MKK1/2, TPL-2 kinase activity was required to phosphorylate the activation loops of MKK3/6, but not of MKK4. MKK3/6 activation required IκB kinase (IKK) phosphorylation of the TPL-2 binding partner nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB1) p105, similar to MKK1/2 activation. Tumour necrosis factor (TNF) stimulation of MKK3/6 phosphorylation was similarly dependent on TPL-2 catalytic activity and IKK phosphorylation of NF-κB1 p105. Owing to redundancy of MKK3/6 with MKK4, Map3k8(D270A) mutation only fractionally decreased lipopolysaccharide activation of p38α. TNF activation of p38α, which is mediated predominantly via MKK3/6, was substantially reduced. TPL-2 catalytic activity was also required for MKK3/6 and p38α activation following macrophage stimulation with Mycobacterium tuberculosis and Listeria monocytogenes Our experiments demonstrate that the IKK/NF-κB1 p105/TPL-2 signalling pathway, downstream of TAK1, regulates MKK3/6 and p38α activation in macrophages in inflammation.
authors.affiliation: The Francis Crick Institute, Immune Signalling Laboratory, Mill Hill Laboratory, London NW7 1AA, U.K.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pattison
authors.affiliation: The Francis Crick Institute, Immune Signalling Laboratory, Mill Hill Laboratory, London NW7 1AA, U.K.
authors.forename: Olivia
authors.initials: O
authors.lastname: Mitchell
authors.affiliation: The Francis Crick Institute, Protein Analysis and Proteomics Laboratory, Clare Hall Laboratory, South Mimms EN6 3LD, U.K.
authors.forename: Helen R
authors.initials: HR
authors.lastname: Flynn
authors.affiliation: The Francis Crick Institute, Immune Signalling Laboratory, Mill Hill Laboratory, London NW7 1AA, U.K.
authors.forename: Chao-Sheng
authors.initials: CS
authors.lastname: Chen
authors.affiliation: The Francis Crick Institute, Immune Signalling Laboratory, Mill Hill Laboratory, London NW7 1AA, U.K.
authors.forename: Huei-Ting
authors.initials: HT
authors.lastname: Yang
authors.affiliation: The Francis Crick Institute, Immune Signalling Laboratory, Mill Hill Laboratory, London NW7 1AA, U.K.
authors.forename: Hakem
authors.initials: H
authors.lastname: Ben-Addi
authors.affiliation: The Francis Crick Institute, Bioinformatics and Biostatistics Service, Lincoln's Inn Fields Laboratory, London WC2A 3LY, U.K.
authors.forename: Stefan
authors.initials: S
authors.lastname: Boeing
authors.affiliation: The Francis Crick Institute, Protein Analysis and Proteomics Laboratory, Clare Hall Laboratory, South Mimms EN6 3LD, U.K.
authors.forename: Ambrosius P
authors.initials: AP
authors.lastname: Snijders
authors.affiliation: The Francis Crick Institute, Immune Signalling Laboratory, Mill Hill Laboratory, London NW7 1AA, U.K.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Ley
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D008264:Macrophages / Q000201:enzymology*; D013058:Mass Spectrometry; D051379:Mice; D011494:Protein Kinases / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism*; D051193:Toll-Like Receptors / Q000378:metabolism*
chemical_list: D047888:Receptors, Tumor Necrosis Factor, Type I; D051193:Toll-Like Receptors; D011494:Protein Kinases
keywords: IKK; NF-κB1; TLR; TPL-2; p38; tumour necrosis factors