TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics
2012; Elsevier BV; Volume: 11; Issue: 6 Linguagem: Inglês
10.1074/mcp.m112.017764
ISSN1535-9484
AutoresJun Zhong, Min‐Sik Kim, Raghothama Chaerkady, Xinyan Wu, Tai‐Chung Huang, Derese Getnet, Chris J. Mitchell, Shyam Mohan Palapetta, Jyoti Sharma, Robert N. O’Meally, Robert N. Cole, Akinori Yoda, Albrecht Moritz, Marc Loriaux, A. John Rush, David M. Weinstock, Jeffrey W. Tyner, Akhilesh Pandey,
Tópico(s)Allergic Rhinitis and Sensitization
ResumoThymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets for TSLP/TSLPR-associated diseases in humans. Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets for TSLP/TSLPR-associated diseases in humans. Thymic stromal lymphopoietin (TSLP) 1The abbreviations used are:TSLPthymic stromal lymphopoietinTSLPRthymic stromal lymphopoietin receptorIL-7RαIL-7 receptor α subunitSILACstable isotope labeling by amino acids in cell cultureBa/F3-ITBa/F3 cells coexpressing human IL-7Rα and TSLPRJakJanus kinasePtpnprotein tyrosine phosphatase non-receptor type. 1The abbreviations used are:TSLPthymic stromal lymphopoietinTSLPRthymic stromal lymphopoietin receptorIL-7RαIL-7 receptor α subunitSILACstable isotope labeling by amino acids in cell cultureBa/F3-ITBa/F3 cells coexpressing human IL-7Rα and TSLPRJakJanus kinasePtpnprotein tyrosine phosphatase non-receptor type. is an IL-7-like four-helix bundle cytokine that was originally identified as a growth factor from the conditioned medium of the Z210R.1 thymic stromal cell line to support B-cell development in the absence of IL-7 (1Friend S.L. Hosier S. Nelson A. Foxworthe D. Williams D.E. Farr A. 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Hyperactive TSLP signaling resulting from overexpression of TSLP contributes to the development of allergic inflammation observed in asthma and atopic dermatitis. Yin and colleagues have shown that the expression of TSLP is elevated in airway epithelial and mast cells of asthmatic patients (14Ying S. O'Connor B. Ratoff J. Meng Q. Mallett K. Cousins D. Robinson D. Zhang G. Zhao J. Lee T.H. Corrigan C. Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity.J. Immunol. 2005; 174: 8183-8190Crossref PubMed Scopus (625) Google Scholar) and is correlated with the severity of asthma in patients (15Ying S. O'Connor B. Ratoff J. Meng Q. Fang C. Cousins D. Zhang G. Gu S. Gao Z. Shamji B. Edwards M.J. Lee T.H. Corrigan C.J. Expression and cellular provenance of thymic stromal lymphopoietin and chemokines in patients with severe asthma and chronic obstructive pulmonary disease.J. 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Clearly, an understanding of TSLP signaling will accelerate in the development of specific therapeutics in diseases where the TSLP/TSLPR axis plays a key role in pathogenesis. It is known that TSLP can activate the JAK-STAT pathway by inducing the phosphorylation of two members of the Janus kinase family, JAK1 and JAK2, and six members of Stat transcription factor family, STAT1, 3, 4, 5a, 5b, and 6 (29Arima K. Watanabe N. Hanabuchi S. Chang M. Sun S.C. Liu Y.J. Distinct signal codes generate dendritic cell functional plasticity.Sci. Signal. 2010; 3: ra4Crossref PubMed Scopus (98) Google Scholar, 30Wohlmann A. Sebastian K. Borowski A. Krause S. Friedrich K. Signal transduction by the atopy-associated human thymic stromal lymphopoietin (TSLP) receptor depends on Janus kinase function.Biol. Chem. 2010; 391: 181-186Crossref PubMed Google Scholar). TSLP requires JAK1 and JAK2 to activate STAT5 (31Rochman Y. Kashyap M. Robinson G.W. Sakamoto K. Gomez-Rodriguez J. Wagner K.U. Leonard W.J. 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Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (908) Google Scholar) have been developed to pinpoint the phosphorylation sites in the phosphoproteome. These enrichment methods have also been combined with the SILAC strategy to quantitate phosphorylation changes in various biological systems. For example, Gruhler et al. combined SCX/IMAC phosphopeptide enrichment with SILAC to study the pheromone-regulated phosphorylation in yeast (51Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar) and Nguyen et al. combined IMAC with SILAC and label-free quantitation methods to study temporal dynamics of the phosphoproteome in T-cell receptor signaling (52Nguyen V. Cao L. Lin J.T. Hung N. Ritz A. Yu K. Jianu R. Ulin S.P. Raphael B.J. Laidlaw D.H. Brossay L. Salomon A.R. A new approach for quantitative phosphoproteomic dissection of signaling pathways applied to T cell receptor activation.Mol. Cell. Proteomics. 2009; 8: 2418-2431Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Olsen and colleagues combined SILAC with TiO2-based enrichment to characterize the EGFR-mediated temporal changes of the phosphoproteome in HeLa cells (53Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2583) Google Scholar). Rigbolt and colleagues also combined SCX/TiO2 with SILAC to characterize the temporal changes of the phosphoproteome during human embryonic stem cell differentiation (54Rigbolt K.T. Prokhorova T.A. Akimov V. Henningsen J. Johansen P.T. Kratchmarova I. Kassem M. Mann M. Olsen J.V. Blagoev B. System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation.Sci. Signal. 2011; 4: rs3Crossref PubMed Scopus (322) Google Scholar). Guha et al. used antiphosphotyrosine antibodies to enrich tyrosine-phosphorylated peptides and quantitated the changes of the tyrosine phosphoproteome in cells expressing lung cancer-specific alleles of EGFR and KRAS by SILAC (55Guha U. Chaerkady R. Marimuthu A. Patterson A.S. Kashyap M.K. Harsha H.C. Sato M. Bader J.S. Lash A.E. Minna J.D. Pandey A. Varmus H.E. Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 14112-14117Crossref PubMed Scopus (93) Google Scholar). Rubbi et al. combined antiphosphotyrosine antibodies with SILAC to reveal crosstalk between Bcr-Abl and negative feedback mechanisms controlling Src signaling (56Rubbi L. Titz B. Brown L. Galvan E. Komisopoulou E. Chen S.S. Low T. Tahmasian M. Skaggs B. Müschen M. Pellegrini M. Graeber T.G. Global phosphoproteomics reveals crosstalk between bcr-abl and negative feedback mechanisms controlling SRC signaling.Sci. Signal. 2011; 4: ra18Crossref PubMed Scopus (54) Google Scholar). Thus, SILAC-based quantitative phosphoproteomic approaches are a powerful tool to dissect phosphorylation-mediated signaling pathways. Previously, we undertook a systematic mutagenesis approach to evaluate the role of tyrosine residues in the TSLP receptor complex in mediating TSLP-induced cellular proliferation (57Zhong J. Pandey A. Site-directed mutagenesis reveals a unique requirement for tyrosine residues in IL-7R alpha and TSLPR cytoplasmic domains in TSLP-dependent cell proliferation.BMC Immunol. 2010; 11: 5Crossref PubMed Scopus (0) Google Scholar). In contrast to IL-4, 7, or 9 signaling, our data revealed that any single cytoplasmic tyrosine residue of either human IL-7Rα chain or TSLPR could mediate TSLP-induced cellular proliferation. Mutation of all four cytoplasmic tyrosine residues of human TSLP receptor complex to phenylalanine was required to completely abolish TSLP-dependent cell proliferation and Stat5a phosphorylation delineating the importance of tyrosine phosphorylation in TSLP signaling. Here, we applied a SILAC-based strategy to study the phosphoproteomic changes induced by TSLP in cells. By using TiO2- and antiphosphotyrosine antibody-based phosphopeptide enrichment, we identified 4,164 phosphopeptides on 1,670 p
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