Previous studies suggested that TLR stimulation of the p38α MAP kinases is mediated by TAK1 activation of MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor 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 MKK4. MKK3/6 activation required IKK phosphorylation of the TPL-2 binding partner NF-κB1 p105, similar to MKK1/2 activation. TNF stimulation of MKK3/6 phosphorylation was similarly dependent on TPL-2 catalytic activity and IKK phosphorylation of NF-κB1 p105. Due to redundancy of MKK3/6 with MKK4, Map3k8 D270A mutation only fractionally decreased LPS activation of p38α. However, TNF activation of p38α, which is mediated only by 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 signaling pathway, downstream of TAK1, regulates MKK3/6 and p38α activation in macrophages in inflammation.
- tumour necrosis factors
- ©2016 The Author(s)
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