Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor
Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor
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DOI：
10.1016/s1044-0305(03)00206-x
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发表时间：
2003-09-01
影响因子：
3.2
通讯作者：
Johnson, RS
中科院分区：
文献类型：
--
作者：
Guo, L;Kozlosky, CJ;Johnson, RS
表皮生长因子受体（EGFR）是一种参与许多动物细胞（包括癌细胞）生长调节的受体酪氨酸激酶。EGFR胞质结构域内特定酪氨酸残基的磷酸化是配体结合后发生的初始活化过程的一部分，这些磷酸酪氨酸残基随后用作细胞内信号分子的对接位点。为了研究每个单独位点上的磷酸化，通过质谱分析从人表皮样癌细胞系（A431）产生的EGFR。采用液相色谱-串联质谱法（LC/MS/MS）鉴定胰蛋白酶磷酸肽及其磷酸化位点（Y 992、Y1045、Y1068、Y1086、S1142、Y1148和Y1173）。测量含有磷酸化位点的磷酸化和未磷酸化胰蛋白酶肽的离子强度，并使用强度比来评估每个位点的磷酸化程度。表皮生长因子（EGF）和转化生长因子α（TGF α）作为刺激物的配体浓度不同，因此发现所有EGFR酪氨酸位点均表现出磷酸化水平升高，尽管速率和程度不同。Y 992的磷酸化似乎在较低浓度的配体下达到平台，而其他位点在广泛的浓度范围内继续具有增加的磷酸化。在EGF和TGF α诱导的EGFR磷酸化之间只能检测到很小的差异。用小分子EGFR抑制剂预处理A431细胞几乎消除了除Y 992和Y1068外的所有位点上的配体诱导的磷酸化。
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Phosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR is part of the initial activation process that occurs upon ligand binding, and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules. To study the phosphorylation on each individual site, EGFR generated from a human epidermoid carcinoma cell line (A431) was analyzed by mass spectrometry. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) was used to identify the tryptic phosphopeptides and their sites of phosphorylation (Y992, Y1045, Y1068, Y1086, S1142, Y1148, and Y1173). Ion intensities for the phosphorylated and unphosphorylated tryptic peptides containing the sites of phosphorylation were measured, and the intensity ratios were used to assess the degree of phosphorylation at each site. Ligand concentrations were varied for epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) as stimuli, and all of the EGFR tyrosine sites were consequently found to exhibit increased levels of phosphorylation, although at different rates and to different extents. Phosphorylation of Y992 appeared to plateau at lower concentrations of ligand, whereas the other sites continued to have increased phosphorylation throughout a wide range of concentrations. Only small differences could be detected between the EGF and the TGFalpha-induced EGFR phosphorylation. Pretreatment of A431 cells with a small molecule EGFR inhibitor nearly eliminated the ligand-induced phosphorylation on all of the sites except for Y992 and Y1068.