Inflammation is the body’s response to injury or infection. A hallmark of inflammation is the accumulation of leukocytes, which remove pathogens and necrotic tissue by phagocytosis and proteolytic degradation. Leukocytes/monocytes are mainly recruited by chemokine activation of chemokine receptors, resulting in leukocyte morphological changes, extravasation into the inflamed tissue and chemotaxis along the chemokine gradient to the site of injury or infection. while monocyte chemoattractant proteins (MCPs) and chemokine receptor 2 (CCR2) is the major pair, involving this process and contributing to the pathogenesis of atherosclerosis, obesity and type 2 diabetes. CCR2 is known to signal via G protein and β-arrestin-mediated pathways, the downstream signalling pathways have not been thoroughly explored.
Protein phosphorylation and dephosphorylation are crucial for cellular signal transduction. Dynamic regulation of reversible, site-specific protein phosphorylation is critical to the signalling networks. Here, we performed a data-independent acquisition (DIA) based proteome and phosphoproteome quantification workflow to investigate signal transduction and regulation in MCP-1-activated CCR2-expressing cells. This workflow showed excellent reproducibility and quantification accuracy. More importantly, in addition to some canonical signalling pathways, such as MAPK, JAK/STAT and Akt/mTOR, we have mapped and manually curated other signalling networks, including Rho guanine nucleotide exchange factors (ARHGEFs), nuclear pore complex (NPC) proteins and actin cytoskeleton. Most of the characterised and quantified phosphopeptides in these networks have never been linked to MCP/CCR2 signalling. Phosphorylation kinetic study confirmed the dynamics phosphorylation regulation of the canonical networks; furthermore, it provides the phosphorylation kinetics information of ARHGEFs, NPC and actin cytoskeleton, the consequence of activating which matches the biological function of MCP/CCR2 signalling- guiding cell migration. In light of the accurate quantification and high reproducibility provided by DIA, this study provides new insights into MCP/CCR2 signalling and may guide the identification of potential therapeutic targets.