Compare substrate specificities of wild type and mutant EGFR kinases.

1A：在蛋白质阵列上使用体外激酶测定来鉴定突变的EGFR底物（预算的5%）我们试图在蛋白质阵列上使用体外激酶测定来鉴定突变的EGFR激酶的靶标。我们使用了由Invitrogen公司开发的人类蛋白质阵列，最近与约翰霍普金斯医学研究所的朱恒博士的实验室合作，使用了他们小组开发的定制蛋白质阵列。我们使用WTEGFR， L858R EGFR， L858R/T790M EGFR在酪氨酸激酶抑制剂埃洛替尼存在或不存在的情况下对这些蛋白阵列进行体外激酶检测。我们发现了几种在体外被突变激酶磷酸化的蛋白，但不被WTEGFR磷酸化。例如，我们发现STK3， PKCtheta, PAK3, MST4 (MASK), TBK1， LynA可被L585R EGFR磷酸化，而不是WT EGFR。朱博士实验室的蛋白质阵列在玻片上发现了大约2万个全长的人类蛋白质。我们已经完成了筛选，并分析了过去一年的激酶测定数据。我们目前正在验证通过该试验确定的突变EGFR的一些靶点。我们采用了生物化学和遗传方法来验证目标。其中一个目标是蛋白DLG1（大圆盘）。我们还通过基于质谱的磷酸化蛋白质组学实验（见下文）确定了该蛋白是突变EGFR的磷酸化靶点。我们已经证明DLG1确实与EGFR相互作用。进一步表征这种相互作用和磷酸化的意义正在进行中。1B：比较WT EGFR、L858R EGFR和Del E746-A750 EGFR相关蛋白的相互作用程度（占预算的5%）。两种最常见的tki致敏肺癌特异性EGFR突变是组成型活性的。然而，两项接受厄洛替尼治疗的患者的小型临床试验表明，携带Del EGFR的患者的预后优于携带L858R EGFR的患者。与携带L858R EGFR的患者相比，携带Del EGFR的患者反应更好，无进展生存期更长。我们采用了一种通过质谱法鉴定egfr相互作用蛋白的方法。我们在NR6细胞中表达WT EGFR、L858R EGFR和Del E746-A750 EGFR， NR6细胞是小鼠3T3成纤维细胞的一种变体，不表达内源性EGFR。我们使用细胞培养中氨基酸的稳定同位素标记（SILAC）来区分标记表达每种EGFR变体的细胞。用EGFR细胞外结构域特异性单克隆抗体对EGFR进行免疫沉淀，分离EGFR相互作用蛋白，并通过质谱法进一步鉴定。细胞的SILAC标记能够相对定量与WT型EGFR和突变型EGFR相互作用的蛋白的相互作用程度。我们确定了几种与突变egfr特异性相互作用的蛋白质。特别令人感兴趣的是，与WT型EGFR相比，受体酪氨酸激酶与L858R和Del EGFR的相互作用更多（例如EPHA2、DDR2、AXL），这表明在表达突变型EGFR的细胞中，受体酪氨酸激酶存在串扰。在重复这些相互作用的研究时，我们遇到了一些特殊的问题。在NR6细胞中表达WT型EGFR和突变型EGFR的初始稳定细胞系随着生长的持续，相应EGFR的表达发生了变化。因此，我们分离了这些稳定系的单细胞克隆，目前表征了egfr的表达。我们将重复与突变型egfr特异性抗体的质谱相互作用研究，该研究是我们与德国Nanotools合作完成的。我们计划将相互作用研究数据与体外激酶测定数据相结合，以相互补充，并进一步缩小突变型egfr的特异性靶点列表。

1A: Identify mutant EGFR substrates using in vitro kinase assays on protein arrays (5% of budget)We sought to identify targets of mutant EGFR kinases using in vitro kinase assays on protein arrays. We have used human protein arrays developed by Invitrogen and more recently collaborated with the laboratory of Dr. Heng Zhu at Johns Hopkins Medical Institute to use the custom protein arrays developed by their group. We used WTEGFR, L858R EGFR, L858R/T790M EGFR in presence or absence of tyrosine kinase inhibitor, erlotinib to perform in vitro kinase assays on these protein arrays. We identified several proteins that are phosphorylated in vitro by mutant kinases, but not WTEGFR. For example, we found STK3, PKCtheta, PAK3, MST4 (MASK), TBK1, LynA to be phosphorylated by L585R EGFR, but not WT EGFR. The protein arrays from Dr. Zhu laboratory have around 20,000 full-length human proteins spotted on glass slides. We have completed the screening and have analyzed the kinase assay data this past year. We are currently validating some of the targets of mutant EGFR identified with this assay. We have undertaken both biochemical and genetic approaches to validate the targets. One such target is the protein DLG1 (disc large). We also identified this protein as a phosphorylation target of mutant EGFR from our mass spectrometry-based phosphoproteomics experiments (see below). We have shown that DLG1 indeed interacts with EGFR. Further characterization of this interaction and the significance of the phosphorylation is underway. 1B: Compare the degree of interaction of proteins that associate with WT EGFR, L858R EGFR and Del E746-A750 EGFR (5% of budget)The two most common TKI-sensitizing lung cancer-specific EGFR mutations are constitutively active. However two small clinical trials of patients treated with erlotinib have shown that the prognosis of patients harboring the Del EGFR is better than those with L858R EGFR. Patients with Del EGFR respond better and have more prolonged progression free survival than those harboring the L858R EGFR. We undertook an approach of identifying EGFR-interacting proteins by mass spectrometry. We expressed WT EGFR, L858R EGFR and Del E746-A750 EGFR in NR6 cells, a variant of mouse 3T3 fibroblasts that do not express endogenous EGFR. We used stable isotope labeling with amino acids in cell culture (SILAC) to differentially label cells expressing each of the EGFR variants. Immunoprecipitation of EGFR with a monoclonal antibody specific to the extracellular domain of EGFR was performed to isolate EGFR-interacting proteins that were further identified by mass spectrometry. SILAC labeling of cells enabled relative quantitation of degree of interaction of proteins that interacted with both WT EGFR and mutant EGFRs. We identified several proteins that interacted specifically with mutant EGFRs. Of particular interest is receptor tyrosine kinases that interacted more with L858R and Del EGFR compared to WT EGFR (e.g. EPHA2, DDR2, AXL), suggesting cross talk of receptor tyrosine kinases in cells expressing mutant EGFRs. We encountered specific problems while repeating these interaction studies. The initial stable cell lines expressing WT EGFR and the mutant EGFRs in NR6 cells altered their expression of corresponding EGFRs with continued growth. Hence we have isolated single cell clones of these stable lines and currently characterizing the expression of EGFRs. We will repeat the mass spectrometry-based interaction studies with mutant EGFR-specific antibodies that we have made in collaboration with Nanotools in Germany. We plan to integrate the interaction study data with in vitro kinase assay data to complement each other and further narrow down the list of specific targets of mutant EGFRs.