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 assays using recombinant proteins. It was also demonstrated that TPL-2 controlled the phosphorylation of the activation loop of the MAP kinase p38γ, which is a direct target of MKK3/6. These data identified a novel signalling pathway downstream of TPL-2, increasing our understanding of how TPL-2 controls inflammatory responses.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:763051 |
| Date | January 2015 |
| Creators | Mitchell, O. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1472798/ |
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