TLR and TNF-R1 activation of the MKK3/MKK6-p38α axis in macrophages is mediated by TPL-2 kinase.

Michael J Pattison,Chao-Sheng Chen,Olivia Mitchell,Stefan Boeing,Steven C Ley,Ambrosius P Snijders,Hakem Ben-Addi,Huei-Ting Yang,Helen R Flynn

doi:10.1042/bcj20160502

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.

Highlights

Toll-like receptors (TLRs) play a central role in the initiation of innate immune responses to pathogen infection, recognizing invariant pathogen molecules from bacteria, fungi and viruses [1]
Two pairwise comparisons were made between LPS-stimulated wild-type (WT) Bone marrow-derived macrophages (BMDM) and Map3k8D270A/D270A BMDM, which express catalytically inactive tumour progression locus 2 (TPL-2) [21]: a forward comparison, in which WT cells were labelled with R0K0 medium and Map3k8D270A/D270A BMDM with R10K8 amino acids, and a reverse comparison, in which each cell genotype was labelled with the alternate medium
The present study has demonstrated that, in addition to its established substrates MKK1 and MKK2, a novel major output of TPL-2 signalling following TLR and tumour necrosis factor (TNF)-R1 stimulation of macrophages was the phosphorylation and activation of MKK3 and MKK6

Summary

Introduction

Toll-like receptors (TLRs) play a central role in the initiation of innate immune responses to pathogen infection, recognizing invariant pathogen molecules from bacteria, fungi and viruses [1]. Lipopolysaccharide (LPS) from Gram-negative bacteria stimulates TLR4 receptors on the plasma membranes of macrophages and dendritic cells, rapidly triggering the expression of multiple genes [2]. The products of these genes can directly target the invading pathogen (e.g. antimicrobial peptides) or induce recruitment of additional immune cells (e.g. cytokines and chemokines). This inflammatory response is essential for direct killing of pathogens by phagocytic cells and the subsequent induction of the adaptive immune response [3]. TPL-2 mediates ERK1/2 activation by tumour necrosis factor (TNF) receptor 1 (TNF-R1) and interleukin-1 receptor, emphasizing the important role of TPL-2 in innate immune responses [6]

Methods

Results

Conclusion