Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro.
Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro.
复制标题
EGF受体激酶体外鉴定人Gab-1蛋白酪氨酸磷酸化位点。
作者：
S. Lehr;J. Kotzka;A. Herkner;Elfriede Klein;C. Siethoff;B. Knebel;V. Noelle;J. Brüning;H. Klein;H. Meyer;W. Krone;D. Müller
Grb 2相关结合物-1（Gab-1）最近在胶质母细胞瘤肿瘤的cDNA文库中被鉴定，并且似乎在细胞生长反应、转化和凋亡中起核心作用。结构和功能特征表明，Gab-1是一种多底物对接蛋白下游的不同受体酪氨酸激酶，包括表皮生长因子受体（EGFR）的信号通路。因此，本研究的目的是在体外表征EGFR对重组人Gab-1（hGab-1）蛋白的磷酸化。使用pGEX系统表达整个蛋白质和hGab-1的不同结构域作为谷胱甘肽S-转移酶蛋白，这些蛋白质的磷酸化的动力学数据由麦胚凝集素纯化的EGFR和重组EGFR（rEGFR）受体激酶结构域进行了测定。我们的数据揭示了hGab-1-C对两种受体制剂的相似亲和力（rEGFR的KM = 2.7 μ M，WGA EGFR的KM = 3.2 μ M）以及对不同的重组hGab-1结构域的相似亲和力。为了鉴定特异性EGFR磷酸化位点，通过Edman降解和质谱法对hGab-1-C进行测序。整个蛋白质在八个酪氨酸残基（Y285、Y373、Y 406、Y 447、Y 472、Y 619、Y 657和Y 689）处被rEGFR磷酸化。在HPLC分析中，50%的已鉴别放射性掺入酪氨酸Y 657中作为主峰，该位点显示潜在Syp（PTP 1D）结合位点的特征。因此，用A431和HepG 2细胞裂解物的GST-下拉测定显示磷酸化的完整hGab-1能够结合Syp。这种结合似乎是特异性的，因为它通过将hGab-1的Y 657改变为F657而被消除。这些结果表明，hGab-1是EGFR的高亲和力底物，C末端的主要酪氨酸磷酸化位点Y 657是酪氨酸磷酸酶Syp的特异性结合位点。
Grb2-associated binder-1 (Gab-1) has been identified recently in a cDNA library of glioblastoma tumors and appears to play a central role in cellular growth response, transformation, and apoptosis. Structural and functional features indicate that Gab-1 is a multisubstrate docking protein downstream in the signaling pathways of different receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Therefore, the aim of the study was to characterize the phosphorylation of recombinant human Gab-1 (hGab-1) protein by EGFR in vitro. Using the pGEX system to express the entire protein and different domains of hGab-1 as glutathione S-transferase proteins, kinetic data for phosphorylation of these proteins by wheat germ agglutinine-purified EGFR and the recombinant EGFR (rEGFR) receptor kinase domain were determined. Our data revealed similar affinities of hGab-1-C for both receptor preparations (KM = 2.7 microM for rEGFR vs 3.2 microM for WGA EGFR) as well as for the different recombinant hGab-1 domains. To identify the specific EGFR phosphorylation sites, hGab-1-C was sequenced by Edman degradation and mass spectrometry. The entire protein was phosphorylated by rEGFR at eight tyrosine residues (Y285, Y373, Y406, Y447, Y472, Y619, Y657, and Y689). Fifty percent of the identified radioactivity was incorporated in tyrosine Y657 as the predominant peak in HPLC analysis, a site exhibiting features of a potential Syp (PTP1D) binding site. Accordingly, GST-pull down assays with A431 and HepG2 cell lysates showed that phosphorylated intact hGab-1 was able to bind Syp. This binding appears to be specific, because it was abolished by changing the Y657 of hGab-1 to F657. These results demonstrate that hGab-1 is a high-affinity substrate for the EGFR and the major tyrosine phosphorylation site Y657 in the C terminus is a specific binding site for the tyrosine phosphatase Syp.