Zhang Xu, Belkina Natalya, Jacob Harrys Kishore Charles, Maity Tapan, Biswas Romi, Venugopalan Abhilash, Shaw Patrick G, Kim Min-Sik, Chaerkady Raghothama, Pandey Akhilesh, Guha Udayan
Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
Proteomics. 2015 Jan;15(2-3):340-55. doi: 10.1002/pmic.201400315.
Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http://proteomecentral.proteomexchange.org/dataset/PXD001101).
表皮生长因子受体（EGFR）激酶结构域突变存在于10%-30%的肺腺癌中，并与酪氨酸激酶抑制剂（TKI）敏感性相关。我们试图确定肺腺癌中突变型EGFR的直接和间接磷酸化靶点。我们采用了稳定同位素标记氨基酸在细胞培养中（SILAC）策略、磷酸肽富集和定量质谱法，以确定分别携带EGFR（L858R）和EGFR（L858R/T790M）（TKI敏感和TKI耐药突变）的肺腺癌细胞中突变型EGFR下游磷酸化的动态变化。在敏感细胞中，厄洛替尼治疗后受到抑制，但在耐药细胞中未受抑制的主要经典通路包括EGFR、胰岛素受体、肝细胞生长因子、丝裂原活化蛋白激酶、雷帕霉素作用靶点、核糖体蛋白S6激酶β1以及Janus激酶/信号转导和转录激活因子信号通路。我们在自噬网络的蛋白质中鉴定出磷酸化位点，如在这些肺腺癌细胞中组成性磷酸化的ULK1（S623）；在敏感细胞中，厄洛替尼治疗后磷酸化受到抑制，但在耐药细胞中未受抑制。最后，根据我们的数据进行的激酶-底物预测分析表明，在表皮生长因子（EGF）刺激后磷酸化增加而在TKI抑制后磷酸化降低的底物中，来自AGC、钙和钙调蛋白依赖性激酶组的嗜碱性激酶以及STE组激酶的底物显著富集，而来自CMGC和酪蛋白激酶组的脯氨酸定向激酶的底物则显著减少。这是迄今为止第一项研究厄洛替尼治疗肺腺癌细胞后整体磷酸化变化的研究，该研究结果为肺腺癌中突变型EGFR的信号转导提供了新的见解。所有质谱数据已存入蛋白质组交换库，标识符为PXD001101（http://proteomecentral.proteomexchange.org/dataset/PXD001101）。
