Characterisation of TPL-2 signalling pathways in innate immunity

作者：Olivia Mitchell · 发表于：UCL Discovery (University College London) · 年份：2015 · 研究领域：Melanoma and MAPK Pathways、Cytokine Signaling Pathways and Interactions、NF-κB Signaling Pathways

The MAP 3-kinase TPL-2 is required for activation of ERK1/2 MAP kinases in macrophages after stimulation of Toll-like receptors and the receptors for TNF and IL-1β. TPL-2 drives inflammation in a number of autoimmune and inflammatory disease models, and consequently is considered a potential anti-inflammatory drug target. Recent evidence from the Ley laboratory has indicated that TPL-2 regulates the production of the critical pro-inflammatory cytokine TNFα in an ERK1/2-independent manner. The aim of this study was to use mass spectrometry-based phosphoproteomics to investigate the signalling pathways regulated by TPL-2 in an unbiased manner to more fully understand how TPL-2 regulates innate immunity. A method for SILAC labelling of bone marrow-derived macrophages was developed and used to quantify phosphorylation changes after genetic and pharmacological perturbation of TPL-2 signalling. Across 11 pair-wise comparisons, 31,457 unique phosphosites were identified, from which a shortlist of 37 potentially TPL-2-dependent, ERK1/2-independent phosphorylations was generated. The biological consequences of the majority of these TPL-2 regulated sites remain to be determined, although bioinformatic analysis suggest that they may have roles in regulation of gene expression and the cytoskeleton. The activating residues of the MAP 2-kinases, MKK3 and MKK6 were validated as novel TPL-2 targets in LPS-stimulated macrophages and MKK6 was shown to be a direct substrate by in vitro kinase a...