Abstract
Lipopolysaccharide (LPS)-induced macrophage activation is a prototype of innate immune response. Although key effector proteins in LPS signaling pathway have been revealed, the map of dynamic protein interactions and phosphorylation as well as the stoichiometry of protein complexes are lacking. Here we present a dynamic map of protein interactions and phosphorylation in MyD88, TRAF6 and NEMO complexes obtained by SWATH-MS. The comprehensive MS measurement leads to quantification of up to about 3,000 proteins across about 21-40 IP samples. We detected and quantified almost all known interactors of MyD88, TRAF6 and NEMO. By analyzing these quantitative data, we uncovered differential recruitment of IRAK family proteins to LPS-induced signaling complexes and determined the stoichiometry of the Myddosome complex. In addition, quantitative phosphoproteomics analysis identified a number of unreported high-confidence phosphosites on the key proteins in LPS signaling pathway. Collectively, data of dynamic protein interactions and phosphorylation presented by this study could be a resource for further study of the LPS signaling pathway.
Highlights
Lipopolysaccharide (LPS)-induced macrophage activation is a prototype of innate immune response
We present a dynamic map of protein interactions and phosphorylation in MyD88, TRAF6 and NEMO complexes obtained by SWATH-MS
Ligation of Tolllike receptor 4 (TLR4) with LPS leads to activation of multiple signaling pathways, such as MAPK, NF-B, and IRFs pathways [1]
Summary
Lipopolysaccharide (LPS)-induced macrophage activation is a prototype of innate immune response. Key effector proteins in LPS signaling pathway have been revealed, the map of dynamic protein interactions and phosphorylation as well as the stoichiometry of protein complexes are lacking. We present a dynamic map of protein interactions and phosphorylation in MyD88, TRAF6 and NEMO complexes obtained by SWATH-MS. We detected and quantified almost all known interactors of MyD88, TRAF6 and NEMO By analyzing these quantitative data, we uncovered differential recruitment of IRAK family proteins to LPS-induced signaling complexes and determined the stoichiometry of the Myddosome complex. These reports indicate that other unknown players downstream of TRAF6, but not TAK1, are essential for LPS-induced NF-B activation in macrophages Another intriguing question is how TIRAPMyD88-IRAK complex (named Myddosome) is assembled and what the stoichiometry of this complex is
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