Phosphoproteomic analysis sheds light on intracellular signaling cascades triggered by Formyl-Peptide Receptor 2

Abstract

Formyl peptide receptors (FPRs) belong to the family of seven transmembrane Gi-protein coupled receptors (GPCR). FPR2 is considered the most promiscuous member of this family since it recognizes a wide variety of ligands. It plays a crucial role in several physio-pathological processes and different studies highlighted the correlation between its expression and the higher propensity to invasion and metastasis of some cancers. FPR2 stimulation by its synthetic agonist WKYMVm triggers multiple phosphorylations of intracellular signaling molecules, such as ERKs, PKC, PKB, p38MAPK, PI3K, PLC, and of non-signaling proteins, such as p47phox and p67phox which are involved in NADPH oxidase-dependent ROS generation. Biological effects of FPR2 stimulation include intracellular Ca2+ mobilization, cellular proliferation and migration, and wound healing. A systematic analysis of the phosphoproteome in FPR2-stimulated cells has not been yet reported. Herein, we describe a large-scale phosphoproteomic study in WKYMVm-stimulated CaLu-6 cells. By using high resolution MS/MS we identified 290 differentially phosphorylated proteins and 53 unique phosphopeptides mapping on 40 proteins. Phosphorylations on five selected phospho-proteins were further validated by western blotting, confirming their dependence on FPR2 stimulation. Interconnection between some of the signalling readout identified was also evaluated. Furthermore, we show that FPR2 stimulation with two anti-inflammatory agonists induces the phosphorylation of selected differentially phosphorylated proteins, suggesting their role in the resolution of inflammation. These data provide a promising resource for further studies on new signaling networks triggered by FPR2 and on novel molecular drug targets for human diseases.

Pro-Resolving FPR2 Agonists Regulate NADPH Oxidase-Dependent Phosphorylation of HSP27, OSR1, and MARCKS and Activation of the Respective Upstream Kinases

“…Previously, we applied a phospho-proteomics approach to analyse phosphorylated proteins in WKYMVm-stimulated CaLu-6 cells and identified 290 phosphoproteins and 53 unique phospho-peptides mapping on 40 proteins [ 56 ]. We also demonstrated that the selective phosphorylation on Ser82 of HSP-27, Ser339 of OSR1, and Ser170 of MARCKS uniquely depends on FPR2 stimulation by WKYMVm.…”

“…Previously, we demonstrated a potential FPR2 homodimerization upon WKYMVm stimulation, which indicates the triggering of anti-inflammatory signals; moreover, we showed that stimulation of CaLu-6 cells with ANXA1 induces HSP-27(S82), OSR1(S339), and MARCKS(S170) phosphorylation [ 56 ].…”

“…Whole lysates were obtained from 24-h serum-starved CaLu-6 cells stimulated or not for 5 min with 10 μM WKYMVm or 10 nM ANXA1 in the presence or absence of the appropriate amounts of NADPH oxidase specific inhibitors, as described elsewhere [ 56 ]. Whole lysates were also obtained from p22phox Crispr/Cas9 and CTR cells.…”

“…Therefore, FPR1 binds efficiently N-fMLP and other formylated peptides, whereas FPR2 is a promiscuous receptor activated by the synthetic peptide WKYMVm [ 55 ], LXA4, ANXA1 [ 56 ], SAA, and by peptides or lipids of different origin. FPR3 shares some non-formylated peptide ligands with FPR2 [ 57 ].…”

“…By using a phospho-proteomic analysis we demonstrated that FPR2 stimulation by WKYMVm or ANXA1 induces the phosphorylation of several proteins involved in metabolic processes and biological regulation [ 56 , 83 ], including heat shock protein 27 (HSP27), oxidative stress responsive kinase 1 (OSR1), and myristolated alanine-rich C-kinase substrate (MARCK). Herein, we analyse the mechanism linking pro-resolving agonist stimulation of FPR2 with specific phosphorylation of HSP27(Ser82), OSR1(Ser339), MARCK(Ser170), and their respective upstream kinases.…”