Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

Delineation of the monocyte chemoattractant protein-dependent CCR2 signalling network using phosphoproteomics (#89)

Simon Foster 1 , C Huang 1 , A Shah 1 , O Kleifeld 1 , R_B Schittenhelm 1 , M.J Stone 1
  1. Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia

A universal feature of inflammatory diseases is the excessive migration of leukocytes to the affected tissues.  Leukocyte recruitment is regulated by chemokines, which are secreted at the site of injury or infection and then activate chemokine receptors, G protein-coupled receptors expressed on the target leukocytes. CCR2 is the major chemokine receptor on monocytes and macrophages, for which the primary cognate ligands are the monocyte chemoattractant proteins (CCL2/MCP-1, CCL7/MCP-3 and CCL8/MCP-2). Genetic deletion of CCR2 or silencing of CCL2 protects mice from developing atherosclerosis, yet numerous clinical trials of CCR2 antagonists have failed, in many cases due to lack of efficacy in phase II or III. One contributing factor for this failure is the complexity and potential compensatory mechanisms inherent in chemokine signalling networks. Thus, to identify appropriate targets for more effective therapeutic approaches, we need a deeper understanding of the chemokine-mediated signalling networks. Here, we describe a large-scale proteomic study using data-independent acquisition (DIA) mass spectrometry to quantify the differences in protein and phosphopeptide levels between untreated and CCL2-treated human endothelial kidney cells stably expressing CCR2. We also conduct a time course study of CCL2 activation to investigate and characterise the kinetics of signalling events. To the best of our knowledge, this is the first time that DIA has been applied to globally quantify the phosphoproteome between multiple conditions. We successfully confirmed the involvement of known canonical pathways (MAPK, JAK/STAT and Akt/mTOR) as downstream effectors of CCL2/CCR2 signalling and we also identified, mapped and manually curated additional signalling networks that have not been associated with the CCL2/CCR2 signalling cascade. These include Rho guanine nucleotide exchange factors (ARHGEFs), nuclear pore complex (NPC) proteins and many actin cytoskeletal proteins.