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。项目的这一部分已经完成。目前，我们正在验证这些蛋白质的一个子集的磷酸化的变化，通过免疫沉淀，蛋白质印迹实验，并试图了解这些蛋白质的磷酸化的意义。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。我们目前正在跟踪两个这样的目标：DAPP 1和SCAMP 3。在过去的几个月里，我们已经证明DAPP 1和SCAMP 3都与突变的EGFR相互作用。我们已经做了几个磷酸化位点特异性突变体的这些蛋白质，目前进行生物测定，研究这些蛋白质的磷酸化改变的意义和这些蛋白质在突变EGFR驱动的肺肿瘤发生中发挥的整体作用。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。我们已经通过生物化学实验表明，DAPP 1与EGFR相互作用。我们还发现Y139是DAPP 1的主要磷酸化位点。我们已经计划了实验来分析EGFR是否是磷酸化DAPP 1的激酶，因为有一些证据表明SRC可能是参与的激酶。SCAMP 3（分泌型生涯膜蛋白）已被证明与EGFR相互作用。我们已经证明突变EGFR比WT EGFR更多地磷酸化SCAMP 3中的特异性位点。SCAMP 3参与受体再循环。我们目前正在研究SCAMP 3在突变型EGFR转运中的作用。2B：突变型EGFR下游靶点的Ser/Thr磷酸化位点的鉴定和TKI抑制后对EGFR靶向TKI耐药的磷酸化变化的定量该项目的这一部分已经完成。正在编写一份手稿，以传达这一项目的成果。我们已经完成了一系列的实验，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。在三重SILAC实验中使用携带L 858 R突变的H3255肺腺癌细胞和携带L 858 R/T790 M突变的H1975细胞。从H3255细胞中鉴定了总共约8000个磷酸化位点，在H1975细胞中鉴定了约6000个位点。我们证明了这些细胞在EGF刺激和TKI抑制后动态磷酸化变化的不同模式。我们使用几种生物信息学工具分析了这个数据集，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。我们目前正在与哥伦比亚大学的Andrea Califano博士合作，利用他们独特的生物信息学工具来询问数据，以产生新的假设，然后通过实验验证。

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. This part of the project is complete. Currently we are validating the changes in phosphorylation of a subset of these proteins by immunoprecipitation, western blot experiments and trying to understand the significance of phosphorylation of these proteins. 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. We are currently following two such targets: DAPP1 and SCAMP3. We have showed in the last several months that both DAPP1 and SCAMP3 interact with mutant EGFRs. We have made several phosphorylation site specific mutants of these proteins and currently conducting biological assays to study the significance of altered phosphorylation of these proteins and the overall role played by these proteins in mutant EGFR-driven lung tumorigenesis. 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. We have shown by biochemical experiments that DAPP1 interacts with EGFR. We have also shown that Y139 is a major phosphorylation site of DAPP1. We have planned experiments to assay whether EGFR is the kinase phosphorylating DAPP1, since there is some evidence that SRC may be the kinase involved. SCAMP3 (secretory career membrane protein) has been shown to interact with EGFR. We have demonstrated that mutant EGFRs phosphorylate specific sites in SCAMP3 more than WT EGFR. SCAMP3 is involved in receptor recycling. We are currently studying the role of SCAMP3 in mutant EGFR trafficking. 2B: Identification of Ser/Thr phosphorylation sites on downstream targets of mutant EGFRs and quantitation of phosphorylation changes upon TKI inhibition resistant to EGFR-directed TKIs This part of the project is complete. A manuscript is in preparation to convey the results of this project. We have completed a series of 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. H3255 lung adenocarcinoma cells harboring the L858R mutation and H1975 cells harboring L858R/T790M mutations were used in a triple-SILAC experiment. A total of around 8000 phosphosites were identified from H3255 celss and around 6000 sites identified in H1975 cells. We demonstrated different patterns of dynamic phosphorylation changes upon EGF stimulation and TKI inhibition in these cells. TWe have analyzed 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. We are currently collaborating with Dr. Andrea Califano in Columbia University to use their unique bioinformatics tools to interrogate the data to generate new hypothesis that can then be validated experimentally.