Protein phosphorylation downstream of mutant EGFR kinases

2A：我们提出的问题是：在表达突变的EGFR的人肺腺癌细胞株中，EGF刺激和酪氨酸激酶抑制(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，erlotinib，一种可逆的EGFR抑制剂和BIBW2992，一种不可逆的EGFR和ERBB2抑制剂。使用SILAC和质谱仪进行了各种大规模的实验。实验所用肺腺癌细胞系为H3255和11-18(L858R突变)、H1975(L858R/T790M突变)、PC9(E746-A750 DEL EGFR)。此外，还使用了稳定表达WT EGFR、L858R EGFR、Del EGFR的同基因NR6(3T3成纤维细胞的变体)和HBECs(人支气管上皮细胞)进行了磷酸化研究。从上述每个实验中鉴定了200-700个蛋白质，并进行了相对定量。目前，我们正在通过免疫沉淀和免疫印迹实验来验证这些蛋白质的一部分的磷酸化变化。我们还在携带突变的EGFR或突变的KRAS的肺腺癌细胞中进行了siRNA介导的被鉴定为突变的EGFR的磷酸化靶标的蛋白质的敲除。值得注意的是，在突变的EGFR表达细胞中，其酪氨酸磷酸化被TKI抑制的几种蛋白质也是EGFR突变表达细胞生存所必需的，但KRAS突变表达细胞不需要。我们更多关注的这些目标的例子有EphA2、SCAMP3、DAPPI1。其中一些蛋白，如DAPPI1(磷酸酪氨酸或3-磷酸肌醇的双适配子)尚未参与EGFR信号转导。然而，DAPPI1的Y139位酪氨酸磷酸化在EGF刺激下被刺激，而在TKI、erlotinib和BIBW2992处理后被抑制5-10倍，提示DAPPI1可能是EGFR信号通路中不可或缺的成员。2B：鉴定突变型EGFR下游靶标上的Ser/Thr磷酸化位点，并定量检测TKI抑制时的磷酸化变化。我们启动了通过腺癌细胞的SILAC标记和质谱学鉴定Ser/Thr磷酸化位点的实验。在溶液中胰酶消化后，我们使用了各种分级技术，如强阳离子交换(SCX)或碱性反相。然后，对大约30个组分进行二氧化钛浓缩以分离磷肽，然后使用Orbitrap Velos质谱仪进行反相液相色谱和串联质谱分析。我们继续与约翰·霍普金斯大学的阿克希莱什·潘迪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。本研究共鉴定了3,586个亚磷酶切位点(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个磷酸盐在EGF处理后被激活，358个磷酸盐被去磷酸化。大多数亚磷酸盐(2673个位点)在EGF处理后没有任何变化。这一观察结果反映了在药物治疗过程中存在非常特殊的信号级联反应。我们现在正在使用几个生物信息学工具分析这个数据集，包括IPA和Ariadne Path Studio。我们的数据集中丰富了p70S6、IRS、ERK/MAPK、mTOR、PKA、JAK/STAT等各种经典通路。

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.