Quantification of Dynamic Protein Interactions and Phosphorylation in LPS Signaling Pathway by SWATH-MS

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. 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. Lipopolysaccharides (LPS) 1The abbreviations used are: LPSlipopolysaccharidesTLR4Toll-like receptor 4. 1The abbreviations used are: LPSlipopolysaccharidesTLR4Toll-like receptor 4. are major components of the outer membrane of Gram-negative bacteria. Ligation of Toll-like receptor 4 (TLR4) with LPS ultimately leads to activation of multiple signaling pathways, such as MAPK, NF-κB, and IRFs pathways (1Lu Y.C. Yeh W.C. Ohashi P.S. LPS/TLR4 signal transduction pathway.Cytokine. 2008; 42: 145-151Crossref PubMed Scopus (2021) Google Scholar). The activated signaling pathways regulate expression of a variety of cytokines, chemokines, and type I IFNs, which are essential in inflammation and host defense against microbial infection. MyD88 and TRIF are proximal signaling effectors of TLR4 that elicit MyD88-dependent and MyD88-independent (TRIF-dependent) pathways (2Lord K.A. Hoffman-Liebermann B. Liebermann D.A. Nucleotide sequence and expression of a cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6.Oncogene. 1990; 5: 1095-1097PubMed Google Scholar, 3Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.Science. 2003; 301: 640-643Crossref PubMed Scopus (2497) Google Scholar). The activation of the MyD88-dependent pathway leads to the expression of pro-inflammatory cytokines, whereas the MyD88-independent pathway mediates the induction of Type I interferons and interferon-inducible genes. lipopolysaccharides Toll-like receptor 4. lipopolysaccharides Toll-like receptor 4. On LPS stimulation, TIRAP is first recruited to TLR4 and MyD88 is then recruited to TIRAP, highlighting the importance of TIRAP in MyD88 recruitment to TLR4 (4Horng T. Barton G.M. Medzhitov R. TIRAP: an adapter molecule in the Toll signaling pathway.Nat. Immunol. 2001; 2: 835-841Crossref PubMed Scopus (826) Google Scholar, 5Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction.Nature. 2001; 413: 78-83Crossref PubMed Scopus (996) Google Scholar). The IRAK family proteins, including IRAK1–4, are then recruited to MyD88. Downstream of IRAK1 and IRAK4, TRAF6 is critical to the MyD88-dependent pathway (6Lomaga M.A. Yeh W.C. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J.M. Paige C.J. Lacey D.L. Dunstan C.R. Boyle W.J. Goeddel D.V. Mak T.W. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling.Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1077) Google Scholar). TRAF6 forms a complex with UBC13 and UEV1A, which activates TAK1 (7Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain.Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1505) Google Scholar). Subsequently TAK1 activates downstream NF-κB and MAPK pathways. The MyD88-indepent signaling pathway is mediated by TRIF, an important TIR-containing adaptor protein. TRIF recruits TRAF3 which then associates with TANK (TRAF family member-associated NF-κB activator), TBK1 and IKKi to mediate IRF3 dimerization (8Hacker H. Redecke V. Blagoev B. Kratchmarova I. Hsu L.C. Wang G.G. Kamps M.P. Raz E. Wagner H. Hacker G. Mann M. Karin M. Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6.Nature. 2006; 439: 204-207Crossref PubMed Scopus (738) Google Scholar, 9Oganesyan G. Saha S.K. Guo B. He J.Q. Shahangian A. Zarnegar B. Perry A. Cheng G. Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response.Nature. 2006; 439: 208-211Crossref PubMed Scopus (705) Google Scholar, 10Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway.Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2068) Google Scholar). IRF3, cooperating with NF-κB, activates the transcription of target genes, such as Type I interferons and interferon-inducible genes (11Moynagh P.N. TLR signalling and activation of IRFs: revisiting old friends from the NF-kappaB pathway.Trends Immunol. 2005; 26: 469-476Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Although TLR4 signaling pathway has been intensively studied in the past few years, several important questions still remain unsolved. Although LPS-induced NF-κB activation is abolished in TAK1-deficient MEF cells, it is not affected in TAK1-deficient macrophages (12Sato S. Sanjo H. Takeda K. Ninomiya-Tsuji J. Yamamoto M. Kawai T. Matsumoto K. Takeuchi O. Akira S. Essential function for the kinase TAK1 in innate and adaptive immune responses.Nat. Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (752) Google Scholar, 13Ajibade A.A. Wang Q. Cui J. Zou J. Xia X. Wang M. Tong Y. Hui W. Liu D. Su B. Wang H.Y. Wang R.F. TAK1 negatively regulates NF-kappaB and p38 MAP kinase activation in Gr-1+CD11b+ neutrophils.Immunity. 2012; 36: Full Text Full Text PDF PubMed Scopus Google Scholar). that downstream of not are essential for LPS-induced NF-κB activation in is complex is and the stoichiometry of this complex of all is a MS which the of MS and L.C. P. S. H. L. R. R. data of the by a for and 2012; Full Text Full Text PDF PubMed Scopus Google Scholar, C. L. G. S. R. for quantitative a PubMed Scopus Google Scholar). and protein quantification across multiple samples. protein quantification for of protein complex stoichiometry M. I. G. R. M. A. van A. M. R. by PubMed Scopus Google Scholar). 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Wang Q. Cui J. Zou J. Xia X. Wang M. Tong Y. Hui W. Liu D. Su B. Wang H.Y. Wang R.F. TAK1 negatively regulates NF-kappaB and p38 MAP kinase activation in Gr-1+CD11b+ neutrophils.Immunity. 2012; 36: Full Text Full Text PDF PubMed Scopus Google for of protein detected with whereas identified with the identified detected by and detected and the that not in the TIRAP and which are known to a role in LPS signaling identified by the not by TRAF6 data, we these and detected about the of and proteins with detected by the protein and the unique to proteins we an of TRAF6 data, we and of the identified proteins to be in LPS-induced activation (1Lu Y.C. Yeh W.C. Ohashi P.S. 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In to of key components in MyD88, TRAF6 and NEMO we identified and quantified the phosphorylation in these in IP by MS and analysis The identified by these and of and distinct identified in MyD88, TRAF6 and NEMO these and be quantified across the of LPS in MyD88, TRAF6 and NEMO We then on quantified of high-confidence we and and We identified phosphosites in these high-confidence interactors and of have not been We first the with the stoichiometry of the phosphorylation phosphorylation of with protein and on and on and on quantified across LPS in MyD88 data on TAK1 and on a with proteins, the phosphorylation to protein on TAK1 distinct with the protein in an quantified phosphosites on phosphosites a with protein. and phosphorylation in from that of the phosphorylation on these recruitment to MyD88 In TRAF6 phosphorylation of on TRAF6 whereas phosphorylation and phosphorylation on IRAK1 in TRAF6 complex and whereas IRAK1 protein in complex from and the from IRAK1 protein and and phosphorylation of IRAK1 the on IRAK1 in TRAF6 complex not that IRAK1 first recruited to MyD88 and then to and phosphorylation on IRAK1 in MyD88 complex by to TRAF6 In NEMO phosphorylation of not in an and on on and and on as in an on to be by which the function of to pro-inflammatory J.E. J.M. A. L.C. IkappaB kinase the to of the NF-kappaB PubMed Scopus Google Scholar). and TAK1 in NF-κB we to the identified phosphosites on affected the to NF-κB this we and phosphorylation and determined to NF-κB of phosphorylation not these protein function to NF-κB we that of not to NEMO induced NF-κB gene expression that phosphorylation on NEMO a role in NF-κB have an of LPS-induced signaling we the data of of signaling and the phosphoproteomics data a LPS signaling pathway and in all known key in LPS signaling pathway are in the identified proteins We identified several high-confidence LPS-induced phosphorylation in which about not in in study as a resource for of LPS signaling pathway. In this we protein interactions and phosphorylation in LPS signaling pathway SWATH-MS. is on the that is used to and The are by data In this we used a comprehensive proteins to the IP with this not all in data from IP analysis an from to the of key proteins that are in LPS signaling pathway. that of IP a of the and the this is required for the of data from complex such as further We quantified about proteins in MyD88, and NEMO IP data across about 21–40 IP samples. these proteins, the of as not LPS we in a of of LPS We detected proteins in IP proteins and which from interactors of the the number of identified proteins in identified in MyD88, TRAF6 and NEMO data that in of the proteins in the IP the protein. We the proteins in the on LPS on this has high-confidence interactors of the proteins, and of the interactors have been in are for LPS-induced recruitment that to of in protein in the is that the of several proteins LPS stimulation, which could then the of these is that proteins to LPS Although the of protein not in cells, we to the The the interactors that are to and that are of NEMO are not in this the of these proteins of all interactors is In addition, we that analysis by requires be the of be of which are the for to the results by we the proteins quantitative in LPS on the we differential of IRAK family proteins in MyD88, TRAF6 and NEMO and a of and of in the signaling complexes of LPS and data IRAK1 recruitment and phosphorylation not in is that with NEMO LPS it is well known that and a complex in the in NEMO complex of a complex from the the stoichiometry in could be the of protein in and to the of MyD88, TIRAP, and in We identified high-confidence phosphosites in the interactors in MyD88 and NEMO of not phosphosites on of which is in the We that of the identified phosphosites on NEMO affected the of NEMO to NF-κB we the data of analysis an important resource for further LPS pathway MS data, all and have been through the We and for in with