Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS
Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS
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DOI：
10.1073/pnas.0806158105
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发表时间：
2008-09-16
影响因子：
11.1
通讯作者：
Varmus, Harold E.
中科院分区：
文献类型：
--
作者：
Guha, Udayan;Chaerkady, Raghothama;Varmus, Harold E.
我们使用无偏磷酸化蛋白质组学方法，基于使用细胞培养物中氨基酸的稳定同位素标记（SILAC）的定量质谱法，来鉴定表达EGFR的两种突变等位基因中的任一种的同基因人支气管上皮细胞（HBEC）和人肺腺癌细胞系中的酪氨酸磷酸化蛋白质。（L 858 R和Del E746-A750），或突变KRAS等位基因，其在人肺腺癌中常见。在表达突变EGFR的HBEC中，信号分子的酪氨酸磷酸化程度高于表达WT EGFR或突变KRAS的细胞。受体酪氨酸激酶（如EGFR、ERBB 2、MET和IGF 1 R）和EGFR信号传导抑制剂Mig-6在表达突变型EGFR的HBEC中比表达WT EGFR或突变型RAS的细胞中磷酸化程度更高。一些蛋白的磷酸化在两种EGFR突变体表达细胞中不同;例如，一些细胞连接蛋白（β-连环蛋白、斑珠蛋白和E-钙粘蛋白）在表达L 858 R EGFR的HBEC中比在表达Del EGFR的细胞中磷酸化程度更高。蛋白质内单个酪氨酸位点的磷酸化程度也存在差异;例如，EGFR（Y 727）、ERBB 2（Y 735）或ERBB 4（Y 733）激酶结构域核苷酸结合环中先前未表征的磷酸化位点在表达缺失突变体的HBEC中的磷酸化程度显著高于表达野生型或L 858 R EGFR的细胞。以前没有参与ERBB信号传导的信号分子，如聚合酶转录物释放因子（PTRF），也在表达突变型EGFR的细胞中磷酸化。贝叶斯网络分析这些和其他数据集显示，PTRF可能是ERBB信号网络的一个潜在的重要组成部分。
We have used unbiased phosphoproteomic approaches, based on quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC), to identify tyrosine phosphorylated proteins in isogenic human bronchial epithelial cells (HBECs) and human lung adenocarcinoma cell lines, expressing either of the two mutant alleles of EGFR (L858R and Del E746-A750), or a mutant KRAS allele, which are common in human lung adenocarcinomas. Tyrosine phosphorylation of signaling molecules was greater in HBECs expressing the mutant EGFRs than in cells expressing WT EGFR or mutant KRAS. Receptor tyrosine kinases (such as EGFR, ERBB2, MET, and IGF1R), and Mig-6, an inhibitor of EGFR signaling, were more phosphorylated in HBECs expressing mutant EGFR than in cells expressing WT EGFR or mutant RAS. Phosphorylation of some proteins differed in the two EGFR mutant-expressing cells; for example, some cell junction proteins (beta-catenin, plakoglobin, and E-cadherin) were more phosphorylated in HBECs expressing L858R EGFR than in cells expressing Del EGFR. There were also differences in degree of phosphorylation at individual tyrosine sites within a protein; for example, a previously uncharacterized phosphorylation site in the nucleotide-binding loop of the kinase domains of EGFR (Y727), ERBB2 (Y735), or ERBB4 (Y733), is phosphorylated significantly more in HBECs expressing the deletion mutant than in cells expressing the wild type or L858R EGFR. Signaling molecules not previously implicated in ERBB signaling, such as polymerase transcript release factor (PTRF), were also phosphorylated in cells expressing mutant EGFR. Bayesian network analysis of these and other datasets revealed that PTRF might be a potentially important component of the ERBB signaling network.