TSLP signaling network revealed by SILAC-based phosphoproteomics.

Jun Zhong, Min Sik Kim, Raghothama Chaerkady, Xinyan Wu, Tai Chung Huang, Derese Getnet, Christopher J. Mitchell, Shyam M. Palapetta, Jyoti Sharma, Robert N. O'Meally, Robert N. Cole, Akinori Yoda, Albrecht Moritz, Marc Loriaux, John Rush, David M. Weinstock, Jeffrey Tyner, Akhilesh Pandey

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
JournalMolecular & cellular proteomics : MCP
Volume11
Issue number6
DOIs
StatePublished - Jun 2012

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Isotope Labeling
thymic stromal lymphopoietin
Phosphorylation
src-Family Kinases
Cell culture
Isotopes
Labeling
Phosphotransferases
Cell Culture Techniques
Interleukin-7 Receptors
Activation analysis
Activation Analysis
Amino Acids
Phosphopeptides
Proteins
Phosphoprotein Phosphatases
Phosphoproteins
Phosphatidylinositol 3-Kinases
Immunoprecipitation
Assays

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Zhong, J., Kim, M. S., Chaerkady, R., Wu, X., Huang, T. C., Getnet, D., ... Pandey, A. (2012). TSLP signaling network revealed by SILAC-based phosphoproteomics. Molecular & cellular proteomics : MCP, 11(6). https://doi.org/10.1074/mcp.M112.017764

TSLP signaling network revealed by SILAC-based phosphoproteomics. / Zhong, Jun; Kim, Min Sik; Chaerkady, Raghothama; Wu, Xinyan; Huang, Tai Chung; Getnet, Derese; Mitchell, Christopher J.; Palapetta, Shyam M.; Sharma, Jyoti; O'Meally, Robert N.; Cole, Robert N.; Yoda, Akinori; Moritz, Albrecht; Loriaux, Marc; Rush, John; Weinstock, David M.; Tyner, Jeffrey; Pandey, Akhilesh.

In: Molecular & cellular proteomics : MCP, Vol. 11, No. 6, 06.2012.

Research output: Contribution to journalArticle

Zhong, J, Kim, MS, Chaerkady, R, Wu, X, Huang, TC, Getnet, D, Mitchell, CJ, Palapetta, SM, Sharma, J, O'Meally, RN, Cole, RN, Yoda, A, Moritz, A, Loriaux, M, Rush, J, Weinstock, DM, Tyner, J & Pandey, A 2012, 'TSLP signaling network revealed by SILAC-based phosphoproteomics.', Molecular & cellular proteomics : MCP, vol. 11, no. 6. https://doi.org/10.1074/mcp.M112.017764
Zhong, Jun ; Kim, Min Sik ; Chaerkady, Raghothama ; Wu, Xinyan ; Huang, Tai Chung ; Getnet, Derese ; Mitchell, Christopher J. ; Palapetta, Shyam M. ; Sharma, Jyoti ; O'Meally, Robert N. ; Cole, Robert N. ; Yoda, Akinori ; Moritz, Albrecht ; Loriaux, Marc ; Rush, John ; Weinstock, David M. ; Tyner, Jeffrey ; Pandey, Akhilesh. / TSLP signaling network revealed by SILAC-based phosphoproteomics. In: Molecular & cellular proteomics : MCP. 2012 ; Vol. 11, No. 6.
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