Lehr S, Kotzka J, Herkner A, Klein E, Siethoff C, Knebel B, Noelle V, Brüning J C, Klein H W, Meyer H E, Krone W, Müller-Wieland D
Klinik II und Poliklinik für Innere Medizin am Zentrum für Molekulare Medizin Köln, Institut für Biochemie der Universit at zu Köln, Germany.
Biochemistry. 1999 Jan 5;38(1):151-9. doi: 10.1021/bi9818265.
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.
Grb2相关结合蛋白1（Gab-1）最近在胶质母细胞瘤肿瘤的cDNA文库中被鉴定出来，并且似乎在细胞生长反应、转化和凋亡中发挥核心作用。结构和功能特征表明，Gab-1是不同受体酪氨酸激酶信号通路下游的多底物对接蛋白，包括表皮生长因子受体（EGFR）。因此，本研究的目的是在体外表征EGFR对重组人Gab-1（hGab-1）蛋白的磷酸化作用。使用pGEX系统表达hGab-1的全长蛋白和不同结构域作为谷胱甘肽S-转移酶蛋白，测定了麦胚凝集素纯化的EGFR和重组EGFR（rEGFR）受体激酶结构域对这些蛋白磷酸化的动力学数据。我们的数据显示hGab-1-C对两种受体制剂具有相似的亲和力（rEGFR的KM = 2.7 microM，WGA EGFR的KM = 3.2 microM），对不同的重组hGab-1结构域也是如此。为了鉴定特定的EGFR磷酸化位点，通过埃德曼降解和质谱对hGab-1-C进行测序。全长蛋白被rEGFR在八个酪氨酸残基（Y285、Y373、Y406、Y447、Y472、Y619、Y657和Y689）处磷酸化。在HPLC分析中，50%的已鉴定放射性以酪氨酸Y657作为主要峰被掺入，该位点表现出潜在的Syp（PTP1D）结合位点的特征。相应地，用A431和HepG2细胞裂解物进行的GST下拉试验表明，磷酸化的完整hGab-1能够结合Syp。这种结合似乎是特异性的，因为将hGab-1的Y657突变为F657后结合被消除。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Y657是酪氨酸磷酸酶Syp的特异性结合位点。
