Department of Thoracic Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.
Nat Chem Biol. 2010 Apr;6(4):291-9. doi: 10.1038/nchembio.332. Epub 2010 Feb 28.
We describe a strategy for comprehending signaling pathways that are active in lung cancer cells and that are targeted by dasatinib using chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine-phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SRC-family kinase (SFK) members (LYN, SRC, FYN, LCK and YES), nonreceptor tyrosine kinases (FRK, BRK and ACK) and receptor tyrosine kinases (Ephrin receptors, DDR1 and EGFR). Using quantitative phosphoproteomics, we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug-resistant gatekeeper mutants, we show that SFKs (particularly SRC and FYN), as well as EGFR, are relevant targets for dasatinib action. The combined mass spectrometry-based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and a rationale for combinatorial therapeutic strategies.
我们描述了一种策略，用于使用化学蛋白质组学来识别直接相互作用的蛋白质，并结合免疫亲和纯化对应于激活的酪氨酸激酶的酪氨酸磷酸化肽，从而理解在肺癌细胞中活跃且被达沙替尼靶向的信号通路。我们鉴定出达沙替尼的近 40 种不同激酶靶标。这些靶标包括 SRC 家族激酶（SFK）成员（LYN、SRC、FYN、LCK 和 YES）、非受体酪氨酸激酶（FRK、BRK 和 ACK）和受体酪氨酸激酶（Ephrin 受体、DDR1 和 EGFR）。使用定量磷酸蛋白质组学，我们鉴定出与这些酪氨酸激酶的自身磷酸化位点相对应的肽，这些肽被达沙替尼以浓度依赖性方式抑制。使用耐药性“守门员”突变体，我们表明 SFK（尤其是 SRC 和 FYN）以及 EGFR 是达沙替尼作用的相关靶标。这里描述的基于质谱的组合方法提供了达沙替尼在癌细胞中作用的系统水平视图，并为组合治疗策略提供了功能靶标和理论依据。
