McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, 733 N Broadway, Baltimore, 21205 Maryland, USA.
Mol Cell Proteomics. 2012 Jun;11(6):M112.017764. doi: 10.1074/mcp.M112.017764. Epub 2012 Feb 16.
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.
胸腺基质淋巴细胞生成素（TSLP）是一种细胞因子，在调节免疫反应中发挥多种作用。TSLP 需要由白细胞介素 7 受体 α 亚基和其独特的 TSLP 受体（基因符号 CRLF2）组成的异二聚体受体复合物在细胞中传递信号。异常的 TSLP 信号（例如 TSLP 或其独特受体 TSLPR 的过表达）导致许多疾病的发展，包括哮喘和白血病。然而，对 TSLP 激活的信号通路的详细了解仍然难以捉摸。在这项研究中，我们使用稳定同位素标记的氨基酸在细胞培养中进行了 TSLP 信号网络的全局定量磷酸蛋白质组学分析。通过采用二氧化钛以及抗磷酸酪氨酸抗体作为富集方法，我们在 1670 个磷酸化蛋白上鉴定到了 4164 个磷酸肽。使用稳定同位素标记的氨基酸在细胞培养中的定量方法，我们确定了 226 种蛋白质的磷酸化状态被 TSLP 刺激所调节。我们的分析首次发现 TSLP 激活了 Src 和 Tec 家族激酶的几个成员，包括 Btk、Lyn 和 Tec。此外，我们报告了 TSLP 诱导的蛋白磷酸酶的磷酸化，如 Ptpn6（SHP-1）和 Ptpn11（Shp2），这在以前也没有报道过。免疫共沉淀实验表明，Shp2 以 TSLP 依赖的方式与接头蛋白 Gab2 结合。这是 TSLP 信号中诱导的蛋白质复合物的首次证明。激酶抑制剂筛选显示，PI-3 激酶、Jak 家族激酶、Src 家族激酶或 Btk 的药理学抑制抑制了 TSLP 依赖性细胞增殖，使它们成为由于异常 TSLP 信号导致的疾病的候选治疗靶点。我们的研究是 TSLP 信号通路的首次磷酸蛋白质组学分析，极大地扩展了我们对 TSLP 信号的理解，并为人类 TSLP/TSLPR 相关疾病提供了新的治疗靶点。
