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.
Highlights
From the ‡McKusick-Nathans Institute of Genetic Medicine and Departments of Biological Chemistry, Oncology and Pathology, §Institute of Basic Biomedical Sciences, Mass Spectrometry and Proteomics Facility, ¶Graduate Program in Biochemistry, Cellular and Molecular Biology, Johns Hopkins University School of Medicine, 733 N
It has been well documented that Thymic stromal lymphopoietin (TSLP) can induce Stat3 and Stat5a phosphorylation
We first carried out time course experiments of TSLP-induced Stat3 and Stat5a phosphory
Summary
Reagents—4G10 anti-phosphotyrosine (HRP conjugated) and antiGab antibodies were purchased from Millipore (Billerica, MA). The settings used for the peptides enriched by anti-phosphotyrosine antibodies from the biological replicate 1 were: a) Precursor scans (FTMS) from 350 –1700 m/z at 30,000 resolution; b) MS2 scan (FTMS) of HCD fragmentation of the most intense nine ions (isolation width: 1.90 m/z; normalized collision energy: 35%; activation time ϭ 0.1 ms, default charge state: 2) at 7500 resolution. Growth Assays—Exponentially growing Ba/F3-IT cells were washed six times with PBS, cultured in RPMI 1640 medium without IL-3 for 6 h, and stimulated with recombinant human TSLP in the absence or presence of the Btk kinase inhibitor, PCI-32765 as the indicated doses. Kinase Inhibitor Assays—Exponentially growing Ba/F3-IT cells were washed six times with PBS, cultured in RPMI1640 without IL-3 for 6 h and resuspended in RPMI 1640 medium containing 20 ng/ml TSLP at the concentration of 80,000 cells/ml. EC50 values on TSLP-dependent cellular proliferation were calculated using second-degree polynomial regression curve fit through eight data points (average of no inhibitor wells and seven serial dilution points)
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