The role of individual SH2 domains in mediating association of phospholipase C-γ1 with the activated EGF receptor
The role of individual SH2 domains in mediating association of phospholipase C-γ1 with the activated EGF receptor
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DOI：
10.1074/jbc.274.37.26091
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发表时间：
1999-09-10
影响因子：
4.8
通讯作者：
Carpenter, G
中科院分区：
文献类型：
--
作者：
Chattopadhyay, A;Vecchi, M;Carpenter, G
测定磷脂酶C-γ 1（PLC-γ 1）中存在的两个SH 2（Src同源结构域2）结构域识别表皮生长因子受体中5个自磷酸化位点的能力。采用等离子体共振和免疫学技术来测量SH 2融合蛋白和含磷酸酪氨酸的肽之间的相互作用。N-SH 2结构域识别肽的顺序为pY 1173> pY 992> pY 1068> pY 1148，远大于pY 1086，而C-SH 2结构域识别肽的顺序为pY 992> pY 1068> pY 1148，远大于pY 1086和pY 1173。主要的自磷酸化位点pY 1173仅被N-SH 2结构域识别。在体内评估N-SH 2和C-SH 2结构域对完整PLC-γ 1分子与活化的表皮生长因子（EGF）受体的缔合的贡献。在全长表位标记的PLC-γ 1中产生每个SH 2结构域的功能丧失突变体。突变体表达后，用EGF处理细胞，并测量外源PLC-γ 1与EGF受体的结合。在这种情况下，N-SH 2是PLC-γ 1与EGF受体结合的主要贡献者。结合的结果表明，协会机制涉及N-SH 2域和pY 1173自磷酸化位点作为一个主要事件和C-SH 2域和pY 992自磷酸化位点作为一个次要事件。
The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma 1 (PLC-gamma 1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 much greater than pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 much greater than pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma 1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma 1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma 1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma 1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.