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.