IN VIVO PHOSPHORYLATION SITES ON GASTRIC EZRIN

胃埃兹蛋白是一种80 kDa的蛋白质，其高度富集于 胃壁细胞顶端微绒毛，序列同源 talin和红细胞带4.1。 有人建议埃兹林扮演一个 在介导涉及的顶端表面重排中的关键作用 壁细胞分泌酸。 刺激的调解 通过cAMP依赖性蛋白激酶A途径的酸分泌已经被证实， 与埃兹蛋白的磷酸化有关。 这些研究表明 磷酸化是在丝氨酸/苏氨酸残基上，而不是在 酪氨酸残基。 另一方面，在A431细胞中， 表皮生长因子受体的过度表达，其特征在于作为一种底物， EGF刺激酪氨酸激酶，并在两个丝氨酸 和酪氨酸残基（Brestcher等人，1989年）。 EGF抑制剂 壁细胞中的分泌，并且已经提出， EGF刺激的ezrin酪氨酸磷酸化本身可能抑制 刺激. 在这个项目中，我们建议检查现场导向 组胺和EGF对埃兹蛋白的磷酸化， 分别抑制酸分泌，并在体内鉴定 在每种情况下的磷酸化位点。 我们的实验策略是 包括在凝胶切片中Ezrin蛋白水解，随后纯化 铁负载的磷酸肽（Fe III）次氮基乙酸（NTA） Sepaharose柱，通过HPLC分离磷酸肽， 通过以下方法鉴定磷酸化的特定Ser、Thr或Tyr MALDI质谱和源后衰变分析。

Gastric ezrin is a 80kDa protein, which is highly enriched in apical microvilli of gastric parietal cells and with sequence homology to talin and erythrocyte band 4.1. Ezrin has beensuggested to play a key role in mediating the apical surface rearrangements involved in acid secretion by parietal cells. The mediation of stimulation of acid secretion by the cAMP-dependent protein kinase A pathway has been correlated with the phosphorylation of ezrin. These studies suggest that phosphorylation is at serine/threonine residues and not at tyrosine residues. On the other hand, ezrin in A431 cells which overexpresses EGF receptors was characterized as a substrate for an EGF-stimulated tyrosine kinase and was phosphorylated at bothserine and tyrosine residues (Brestcher et al., 1989). EGF inhibited acid secretion in parietal cells and it has been suggested that EGF-stimulated tyrosine phosphorylation of ezrin might itself inhibit stimulation. In this project we propose to examine the site-directed phosphorylation of ezrin by histamine and EGF which stimulate and inhibit acid secretion respectively and identify the in vivo phosphorylation sites in each case. Our experimental strategy would involve proteolysis of ezrin in gel slices, followed by purification of phosphopeptides on iron-loaded (FeIII) nitriloacetic (NTA) sepaharose columns, the separation of the phosphopeptides by HPLC and the identification of the specific Ser, Thr or Tyr phosphorylated, by MALDI mass spectrometry and post-source decay analysis.