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

Huang, Cheng; Foster, Simon R.; Shah, Anup D.; Kleifeld, Oded; Canals, Meritxell; Schittenhelm, Ralf B.; Stone, Martin J.

Abstract

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein-coupled receptors (GPCRs). 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 (DIA) 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 (GEFs) and GTPase activating proteins (GAPs). 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.

Citation

Huang, C., Foster, S. R., Shah, A. D., Kleifeld, O., Canals, M., Schittenhelm, R. B., & Stone, M. J. (2020). Phosphoproteomic characterization of the signaling network resulting from activation of chemokine receptor CCR2. Journal of Biological Chemistry, 295, 6518-6531. https://doi.org/10.1074/jbc.ra119.012026

• Journal Article Type: Article
• Acceptance Date: Apr 2, 2020
• Online Publication Date: Apr 2, 2020
• Publication Date: May 8, 2020
• Deposit Date: Apr 3, 2020
• Publicly Available Date: Apr 3, 2020
• Journal: Journal of Biological Chemistry
• Print ISSN: 0021-9258
• Electronic ISSN: 1083-351X
• Publisher: American Society for Biochemistry and Molecular Biology
• Peer Reviewed: Peer Reviewed
• Volume: 295
• Pages: 6518-6531
• DOI: https://doi.org/10.1074/jbc.ra119.012026
• Keywords: Cell Biology; Biochemistry; Molecular Biology
• Public URL: https://nottingham-repository.worktribe.com/output/4247624
• Publisher URL: https://www.jbc.org/content/295/19/6518
• Additional Information: This research was originally published in the Journal of Biological Chemistry. Cheng Huang Simon R. Foster Anup D. Shah, Oded Kleifeld, Meritxell Canals, Ralf B. Schittenhelm, Martin J. Stone. Phosphoproteomic characterization of the signaling network resulting from activation of the chemokine receptor CCR2. J Biol Chem. 2020; 295 : p. 6518-6531. © the American Society for Biochemistry and Molecular Biology or © the Author(s).