Protein phosphorylation downstream of mutant EGFR kinases

突变 EGFR 激酶下游的蛋白质磷酸化

• 批准号: 8349533
• 负责人: Udayan Guha
• 金额: $ 43.07万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349533
• 关键词: Adenocarcinoma Cell; Bioinformatics; Cations; Cell Line; Cells; Collaborations; Cyclic AMP-Dependent Protein Kinases; Data Set; Digestion; EGF gene; EPHA2 gene; ERBB2 gene; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Epithelial Cells; Erlotinib; Fibroblasts; Fractionation; Goals; Human; Immunoprecipitation; KRAS2 gene; Label; Liquid Chromatography; Lung Adenocarcinoma; MAP Kinase Gene; Malignant neoplasm of lung; Mass Spectrum Analysis; Mediating; Mutation; Pathway interactions; Pharmaceutical Preparations; Phase; Phosphatidylinositols; Phosphopeptides; Phosphorylation; Phosphorylation Site; Phosphoserine; Phosphothreonine; Phosphotransferases; Phosphotyrosine; Proteins; Relative (related person); Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Solutions; Techniques; Testing; Tyrosine Kinase Inhibitor; Tyrosine Phosphorylation; Variant; Western Blotting; human FRAP1 protein; inhibitor/antagonist; interest; lung tumorigenesis; mass spectrometer; member; mutant; research study; tandem mass spectrometry; tool

2A: We asked the question: what are the changes in tyrosine phosphorylation of proteins upon EGF stimulation and tyrosine kinase inhibitor (TKI) inhibition in human lung adenocarcinoma cell lines expressing the mutant EGFRs? We used two TKIs, erlotinib, a reversible EGFR inhibitor and BIBW2992, an irreversible EGFR and ERBB2 inhibitor. Various large-scale experiments were performed using SILAC and mass spectrometry. Lung adenocarcinoma cell lines used in these experiments were H3255 and 11-18 (L858R mutation), H1975 (L858R/T790M mutation), PC9 (E746-A750 Del EGFR). In addition isogenic NR6 (a variant of 3T3 fibroblasts) and HBECs (human bronchial epithelial cells) with stable expression of WT EGFR, L858R EGFR, Del EGFR were also used for phosphorylation studies. 200-700 proteins have been identified from each of the above experiments and their relative quantitation performed. Currently we are validating the change in phosphorylation of a subset of these proteins by immunoprecipitation and western blot experiments. We also performed siRNA-mediated knockdown of proteins identified as phosphorylation targets of mutant EGFRs in lung adenocarcinoma cells harboring mutant EGFRs or mutant KRAS. It is interesting to note that several of the proteins whose tyrosine phosphorylation was inhibited by TKIs in mutant EGFR-expressing cells were also required for survival of EGFR mutant expressing cells but not KRAS mutant expressing cells. Examples of such targets that we are following more are EPHA2, SCAMP3, DAPPI1. Some of these proteins such as DAPPI1 (dual adapter for phosphotyrosine or 3-phosphoinositides) has not been implicated in EGFR signaling. However tyrosine phosphorylation at Y139 of DAPPI1 is stimulated upon EGF stimulation and inhibited 5-10 fold upon treatment with TKIs, erlotinib and BIBW2992, suggesting DAPPI1 may be an integral member of the EGFR signaling pathway. 2B: Identification of Ser/Thr phosphorylation sites on downstream targets of mutant EGFRs and quantitation of phosphorylation changes upon TKI inhibition. We initiated experiments to identify Ser/Thr phosphorylation sites by SILAC labeling of adenocarcinoma cells and mass spectrometry. We used various fractionation techniques after in-solution tryptic digestion such as strong cation exchange (SCX) or basic reverse phase. Around 30 fractions were then subjected to TiO2 enrichment for phosphopeptide isolation followed by reverse phase liquid chromatography and tandem mass spectrometry using an orbitrap velos mass spectrometer. We continued our previous collaboration with Dr. Akhilesh Pandey at Johns Hopkins to perform the mass spectrometry analysis. H3255 lung adenocarcinoma cells harboring the L858R mutation were used in a triple-SILAC experiment. A total of 3,586 phosphosites (3167 phosphoserine, 395 phosphothreonine and 24 phosphotyrosine sites) were identified from this study, which corresponds to 1,434 proteins. There were 278 phosphosites that were activated upon EGF treatment and 358 that were dephosphorylated. Majority of phosphosites (2673 sites) did not show any change on treatment with EGF. This observation is reflective of the existence of very specific signaling cascades that are disrupted on treatment with the drug. We are now analyzing this data set using several bioinformatic tools, including IPA and Ariadne pathway studio. Various canonical pathways such as p70S6, IRS, ERK/MAPK, mTOR, PKA, JAK/STAT were enriched in our dataset.

2A：我们提出了这样的问题：在表达突变型 EGFR 的人肺腺癌细胞系中，EGF 刺激和酪氨酸激酶抑制剂 (TKI) 抑制后蛋白质的酪氨酸磷酸化有何变化？我们使用了两种 TKI：厄洛替尼（一种可逆 EGFR 抑制剂）和 BIBW2992（一种不可逆 EGFR 和 ERBB2 抑制剂）。使用 SILAC 和质谱法进行了各种大规模实验。这些实验中使用的肺腺癌细胞系是H3255和11-18（L858R突变）、H1975（L858R/T790M突变）、PC9（E746-A750 Del EGFR）。此外，稳定表达WT EGFR、L858R EGFR、Del EGFR的同基因NR6（3T3成纤维细胞的变体）和HBEC（人支气管上皮细胞）也用于磷酸化研究。从上述每个实验中已鉴定出 200-700 种蛋白质，并进行了相对定量。目前，我们正在通过免疫沉淀和蛋白质印迹实验验证这些蛋白质子集磷酸化的变化。 我们还在携带突变 EGFR 或突变 KRAS 的肺腺癌细胞中对被确定为突变 EGFR 磷酸化靶标的蛋白质进行了 siRNA 介导的敲低。有趣的是，在突变型 EGFR 表达细胞中，酪氨酸磷酸化被 TKI 抑制的几种蛋白质也是 EGFR 突变型表达细胞生存所必需的，但 KRAS 突变型表达细胞则不需要。我们更多关注的此类目标的例子有 EPHA2、SCAMP3、DAPPI1。其中一些蛋白质，例如 DAPPI1（磷酸酪氨酸或 3-磷酸肌醇的双接头）尚未涉及 EGFR 信号转导。然而，DAPPI1 Y139 的酪氨酸磷酸化在 EGF 刺激下受到刺激，而在 TKI、厄洛替尼和 BIBW2992 治疗后被抑制 5-10 倍，表明 DAPPI1 可能是 EGFR 信号传导通路的重要成员。图2B：突变EGFR下游靶标上Ser/Thr磷酸化位点的鉴定以及TKI抑制时磷酸化变化的定量。 我们启动了通过腺癌细胞的 SILAC 标记和质谱法来鉴定 Ser/Thr 磷酸化位点的实验。我们在溶液内胰蛋白酶消化后使用了各种分级技术，例如强阳离子交换 (SCX) 或碱性反相。然后对大约 30 个级分进行 TiO2 富集以分离磷酸肽，然后使用 Orbitrap velos 质谱仪进行反相液相色谱和串联质谱分析。我们继续之前与约翰霍普金斯大学的 Akhilesh Pandey 博士合作进行质谱分析。携带 L858R 突变的 H3255 肺腺癌细胞用于三重 SILAC 实验。本研究共鉴定出 3,586 个磷酸位点（3167 个磷酸丝氨酸、395 个磷酸苏氨酸和 24 个磷酸酪氨酸位点），对应于 1,434 个蛋白质。有 278 个磷酸位点在 EGF 处理后被激活，358 个被去磷酸化。大多数磷酸位点（2673 个位点）在 EGF 处理后没有表现出任何变化。这一观察结果反映了非常特殊的信号级联的存在，这些级联在药物治疗时被破坏。我们现在正在使用多种生物信息学工具分析该数据集，包括 IPA 和 Ariadne Pathway Studio。我们的数据集中丰富了各种经典途径，例如 p70S6、IRS、ERK/MAPK、mTOR、PKA、JAK/STAT。