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 Map3k8D270A/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, Map3k8D270A 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.

Innate immune receptors in development of Mycobacterium tuberculosis infection

“…TLRs экспрессируются на различных иммунных клетках, включая альвеолярные макрофаги, моноциты, дендритные клетки, В-клетки, специфические типы Т-клеток и даже на фибробластах и эпителиальных клетках [51]. M. tuberculosis и компоненты ее клеточной стенки распознаются TLR1, TLR2, TLR4, TLR7 и TLR9 [45,49,79,103], что приводит к MyD88-зависимой активации антибактериальных эффекторных путей и продукции NO-оксидазы, а также провоспалительных цитокинов, таких как TNFα, IL-12, хемокинов [73]. Такие гликолипиды M. tuberculosis, как липо арабиноманнан, липоманнан и фосфатидилинозитол маннозид, а также белок пролин-пролин-глутаминовой кислоты (PPE)-17 подают сигнал через гетеродимеры TLR2-TLR1 [41,105].…”

“…TLR2 участвуют в рекрутировании в легкие Т-регуляторных клеток (Treg), которые ограничивают приток нейтрофилов и макрофагов, тем самым способствуя ограничению воспаления [67]. Накопление Treg в легких способствует поддержанию целостности туберкулезной гранулемы при хронической инфекции [ [73].…”

Mapping out p38MAPK

“…For example, molecules such as tumor progression locus 2 (TPL-2) TPL2 participate in a circuit that is dependent on p38 activation and itself activates the MKK3/6 which in turn activates p38 55 . For another example, stressors that generate p38 activation can also result in expression of High Mobility Group B1 protein (HMGB1), but this in turn can act via internal pathways to activate p38.…”

Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

“…Pattison et al, 2016; Touati et al, 2018) with minor modifications. 1 mg each of "light" and "heavy" labelled lysates were mixed and dried.…”

“…1 mg each of "light" and "heavy" labelled lysates were mixed and dried. Protease digestion and phosphopeptide enrichment was done as described with minor modifications(Pattison et al, 2016; Touati et al, 2018). Peptides were dissolved in 35 μl of 1% TFA, with sonication, and fractionated on a 50 cm, 75 μm I.D.…”