Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors
Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors
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DOI：
10.1073/pnas.97.1.179
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发表时间：
2000-01-04
影响因子：
11.1
通讯作者：
Lodish, HF
中科院分区：
文献类型：
--
作者：
Pandey, A;Podtelejnikov, AV;Lodish, HF
受体蛋白酪氨酸激酶如表皮生长因子受体（EGFR）被其同源配体寡聚化导致受体的激活，受体亚基的转磷酸化随后是含有src同源2 （SH2）或磷酸酪氨酸相互作用域（PID）的信号分子的募集。此外，一些细胞质蛋白可能与受体相关，也可能不相关，它们经历酪氨酸磷酸化。为了在一个步骤中确定EGFR信号通路的几个组成部分，我们利用免疫沉淀的酪氨酸磷酸化的分子响应于EGF，并通过一维凝胶电泳和质谱分析对它们进行了分析，结合基质辅助激光解吸/电离（MALDI）和纳米电喷雾串联质谱（MS/MS）鉴定了9个信号分子，其中7个先前与EGFR信号通路有关。这些分子中有几个是从装载到凝胶上的低飞摩尔水平的蛋白质中鉴定出来的。我们确定了Vav-2，一种最近发现的鸟苷核苷酸交换因子，普遍表达，作为EGFR的底物，我们证明了Vav-2在酪氨酸残基上磷酸化，以响应EGF并与EGFR在体内相关，Vav-2与EGFR的结合是由Vav-2的SH2结构域介导的。与其普遍表达一致，Vav-2似乎是一种一般的信号分子，因为它也与血小板衍生生长因子（PDGF）受体相关，并在PDGF刺激下在成纤维细胞中经历酪氨酸磷酸化。本文提出的策略可用于哺乳动物细胞中细胞表面受体下游组分的常规鉴定。
Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor, Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry, Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling, Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR, We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo, Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.