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Dynamic phosphotyrosine-dependent signaling profiling in living cells by two-dimensional proximity proteomics

ABSTRACT: Tyrosine phosphorylation (pTyr)-dependent signaling pathways play a vital role in various biological processes, which are spatiotemporally assembled and dynamically regulated on minute-scale by pTyr in living cells. Studying these pTyr-mediated signaling complexes is therefore challenging due to the highly dynamic nature of the protein complexes and the low abundance of pTyr. In this study, we adopted minute-resolution APEX2-based proximity labeling (PL) in living cells and Src superbinder-based pTyr peptide enrichment for simultaneously profiling these protein complexes and associated pTyr sites from the same affinity-purified sample. Upon different time-course of EGF stimulation of the living cells stably expressed with APEX2-fused adaptor protein GRB2, we constructed two-dimensional time-course curves for both GRB2 interactome and tyrosine phosphoproteome. Most of the known GRB2 interacting proteins and more than 40 pTyr sites were identified from each time point with acceptable quantification precision. Well-annotated pTyr signaling complexes in EGFR signaling and located at endosome were quantified with tightly correlated time-course curves for both interacting proteins and pTyr sites. Importantly, the correlated time-course curves for EGFR and endosomal HGS were well validated by PRM-based targeted MS analysis. Taking advantage of the high sensitivity of the PRM assay, the low abundant pTyr peptide EGFR pY1110 that cannot be identified in the DDA analysis could be well detected and quantified. Collectively, this two-dimensional proximity proteomic strategy is promising for comprehensively characterizing pTyr-mediated protein complexes with high sensitivity in living cells.

INSTRUMENT(S): Q-Extractive HF-X

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ruijun Tian

PROVIDER: MSV000089826 | MassIVE | Wed Jul 06 18:16:00 BST 2022

REPOSITORIES: MassIVE

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Project description:Macrophage polarization between the M2 (repair, pro-tumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of CSF-1R that contribute to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain-of-function and loss-of-function with bioinformatic analysis of the macrophage CSF-1R pTyr-721-regulated transcriptome, we uncovered miR-21 as a downstream molecular switch controlling macrophage activation and identified ERK1/2 and NF-M-NM-:B as CSF-1R pTyr-721-regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the proinflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21-regulated mRNAs revealed that 80% of the CSF-1-regulated canonical miR-21 targets are pro-inflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R-mediated activation of ERK1/2 and NF-M-NM-:B. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide (LPS). M-bM-^@M-^C These results identify the macrophage miR-21 network as a novel target for controlling macrophage polarization. We performed microarray-based analysis on four mouse macrophage cell lines, two CSF-1R pTyr-721-expressing cell lines (M-/-.WT and M-/-.3ABY721) and two CSF-1R Tyr-721-deficient lines (M-/-.Y721F and M-/-.3AB). Total RNA (two biological replicates) was extracted from CSF-1-starved cells (UR) or from cells constitutively grown in CSF-1 (CONST). Additionally, CSF-1-starved M-/-.WT and M-/-.Y721F cell lines were stimulated with CSF-1 for 0min, 20 min, 60 min and 180 min and used for total RNA extraction. Total RNA preparations with Ribosomal Integrity Numbers (RIN) > 9.5 were used for microarray analysis. A total of 100 ug/cell line/replicate was used for gene expresion analysis on the Affymetrix Mouse Gene ST1.0 chips at the Genomics Core at Einstein, according to manufacturerM-bM-^@M-^Ys instructions. Differential expression analysis was performed using the M-bM-^@M-^XlimmaM-bM-^@M-^Y package of R/Bioconductor to identify significantly differentially expressed mRNAs over time, in response to CSF-1 treatment and to the genotype. CSF1-regulated genes were identified according to the cutoff with both expression folder change > 1.5 and p-value < 0.05.

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Dynamic phosphotyrosine-dependent signaling profiling in living cells by two-dimensional proximity proteomics

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