title: TSLP signaling network revealed by SILAC-based phosphoproteomics.
abstract: 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.
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, 733 N Broadway, Baltimore, 21205 Maryland, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhong
authors.forename: Min-Sik
authors.initials: MS
authors.lastname: Kim
authors.forename: Raghothama
authors.initials: R
authors.lastname: Chaerkady
authors.forename: Xinyan
authors.initials: X
authors.lastname: Wu
authors.forename: Tai-Chung
authors.initials: TC
authors.lastname: Huang
authors.forename: Derese
authors.initials: D
authors.lastname: Getnet
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Mitchell
authors.forename: Shyam M
authors.initials: SM
authors.lastname: Palapetta
authors.forename: Jyoti
authors.initials: J
authors.lastname: Sharma
authors.forename: Robert N
authors.initials: RN
authors.lastname: O'Meally
authors.forename: Robert N
authors.initials: RN
authors.lastname: Cole
authors.forename: Akinori
authors.initials: A
authors.lastname: Yoda
authors.forename: Albrecht
authors.initials: A
authors.lastname: Moritz
authors.forename: Marc M
authors.initials: MM
authors.lastname: Loriaux
authors.forename: John
authors.initials: J
authors.lastname: Rush
authors.forename: David M
authors.initials: DM
authors.lastname: Weinstock
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Tyner
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D016207:Cytokines / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007553:Isotope Labeling; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D060066:Protein Interaction Maps; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D000094632:Thymic Stromal Lymphopoietin
chemical_list: D016207:Cytokines; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D020543:Proteome; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; C494087:Stat3 protein, mouse; C494093:Stat5a protein, mouse; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D000094632:Thymic Stromal Lymphopoietin; C000721967:TSLP protein, mouse