Phosphoproteomic characterization of the signaling network resulting from activation of the chemokine receptor CCR2

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Abstract

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein-coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.

Original languageEnglish
Pages (from-to)6518-6531
Number of pages14
JournalJournal of Biological Chemistry
Volume295
Issue number19
DOIs
Publication statusPublished - 8 May 2020

Keywords

  • actin
  • C-C chemokine ligand type 2 (CCL2)
  • C-C chemokine receptor type 2 (CCR2)
  • CCL2
  • CCR2
  • cell signaling
  • chemokine
  • cytoskeleton
  • data-independent acquisition
  • data-independent acquisition mass spectrometry (DIA-MS)
  • G protein-coupled receptor (GPCR)
  • GTPase-activating protein (GAP)
  • guanine nucleotide exchange factor (GEF)
  • mass spectrometry (MS)
  • nuclear pore
  • phosphoproteomics
  • signaling networks

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