title: Phosphoproteome profiling of the macrophage response to different toll-like receptor ligands identifies differences in global phosphorylation dynamics.
abstract: Toll-like receptors (TLRs) are among the first sensors that detect infection and drive immune response. Macrophages encountering a pathogen are usually stimulated not by one TLR, but by a combination of TLRs engaged by distinct microbe ligands. To understand the integrated signaling under complex conditions, we investigated the differences in the phosphoprotein signaling cascades triggered by TLR2, TLR4, and TLR7 ligands using a single responding cell population. We performed a global, quantitative, early poststimulation kinetic analysis of the mouse macrophage phosphoproteome using stable isotope labeling with amino acids coupled to phosphopeptide enrichment and high-resolution mass spectrometry. For each TLR ligand, we found marked elevation of phosphorylation of cytoskeleton components, GTPases of the Rho family, and phospholipase C signaling pathway proteins. Phosphorylation of proteins involved in phagocytosis was only seen in response to TLR2 and TLR4 but not to TLR7 activation. Changes in the phosphorylation of proteins involved in endocytosis were delayed in response to TLR2 as compared to TLR4 ligands. These findings reveal that the phosphoproteomic response to stimulation of distinct TLRs varies both in the major modification targets and the phosphorylation dynamics. These results advance the understanding of how macrophages sense and respond to a diverse set of TLR stimuli.
authors.affiliation: Laboratory of Systems Biology, and §Research Technology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Virginie
authors.initials: V
authors.lastname: Sjoelund
authors.forename: Margery
authors.initials: M
authors.lastname: Smelkinson
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Nita-Lazar
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D007093:Imidazoles / Q000494:pharmacology; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D010587:Phagocytosis; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051193:Toll-Like Receptors / Q000378:metabolism*
chemical_list: D007093:Imidazoles; D008024:Ligands; D008070:Lipopolysaccharides; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D010750:Phosphoproteins; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; C402365:resiquimod
keywords: SILAC; TLR2; TLR4; TLR7; innate immunity; macrophage; phosphoproteomics; toll-like receptors