Protein phosphorylation downstream of mutant EGFR kinases

2A：我们提出了这样一个问题：在表达突变EGFR的人肺腺癌细胞系中，EGF刺激和酪氨酸激酶抑制剂（TKI）抑制后蛋白质酪氨酸磷酸化的变化是什么？我们使用了两种TKI，厄洛替尼（一种可逆的EGFR抑制剂）和BIBW 2992（一种不可逆的EGFR和ERBB 2抑制剂）。使用SILAC和质谱法进行各种大规模实验。在这些实验中使用的肺腺癌细胞系是H3255和11-18（L 858 R突变）、H1975（L 858 R/T790 M突变）、PC 9（E746-A750 Del EGFR）。此外，具有WT EGFR、L 858 R EGFR、Del EGFR稳定表达的同基因NR 6（3 T3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。从上述每个实验中鉴定了200-700种蛋白质，并进行了它们的相对定量。目前，我们正在验证这些蛋白质的一个子集的免疫沉淀和蛋白质印迹实验磷酸化的变化。 我们还进行了siRNA介导的蛋白质敲低，这些蛋白质被鉴定为携带突变型EGFR或突变型KRAS的肺腺癌细胞中突变型EGFR的磷酸化靶标。值得注意的是，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突变型表达细胞而不是KRAS突变型表达细胞存活所需的。我们正在关注的这些目标的例子是EPHA 2，SCAMP 3，DAPPI 1。这些蛋白质中的一些，如DAPPI 1（磷酸酪氨酸或3-磷酸肌醇的双重衔接子），与EGFR信号传导无关。然而，DAPPI 1的Y139处的酪氨酸磷酸化在EGF刺激时被刺激，并且在用TKI、厄洛替尼和BIBW 2992处理时被抑制5-10倍，这表明DAPPI 1可能是EGFR信号传导途径的组成成员。2B：突变EGFR下游靶点上Ser/Thr磷酸化位点的鉴定和TKI抑制后磷酸化变化的定量。 我们启动了实验，以确定丝氨酸/苏氨酸磷酸化位点的SILAC标记的腺癌细胞和质谱。我们在溶液中胰蛋白酶消化后使用各种分级技术，如强阳离子交换（SCX）或碱性反相。然后对约30个馏分进行TiO 2富集，用于磷酸肽分离，然后使用轨道阱velos质谱仪进行反相液相色谱和串联质谱。我们继续与约翰霍普金斯大学的Akhilesh Pandey博士合作进行质谱分析。将携带L 858 R突变的H3255肺腺癌细胞用于三重SILAC实验。本研究共鉴定了3，586个磷酸化位点（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个磷酸化位点在EGF处理后被激活，358个被去磷酸化。大多数磷酸化位点（2673个位点）在EGF治疗后没有显示出任何变化。这一观察结果反映了在药物治疗中被破坏的非常特异性的信号级联的存在。我们现在正在使用几种生物信息学工具分析这个数据集，包括IPA和Ariadne pathway studio。在我们的数据集中富集了各种典型途径，如p70 S6，IRS，ERK/MAPK，mTOR，PKA，JAK/STAT。

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.