IN-VIVO PHOSPHORYLATION SITES ON GASTRIC EZRIN

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。胃ezrin是一个分子量为80 kDa的蛋白质，它高度富集于胃壁细胞的顶端微绒毛中，与talin和红细胞带4.1具有同源性。埃兹蛋白已被建议发挥关键作用，在介导的顶端表面重排参与壁细胞的酸分泌。通过cAMP依赖性蛋白激酶A途径介导的酸分泌刺激与埃兹蛋白的磷酸化相关。这些研究表明，磷酸化是在丝氨酸/苏氨酸残基，而不是在酪氨酸残基。另一方面，过表达EGF受体的A431细胞中的埃兹蛋白被表征为EGF刺激的酪氨酸激酶的底物，并且在丝氨酸和酪氨酸残基处被磷酸化（Brestcher等人，1989年）。EGF抑制壁细胞中的酸分泌，并且已经表明EGF刺激的埃兹蛋白的酪氨酸磷酸化本身可能抑制刺激。在这个项目中，我们建议检查的组胺和EGF刺激和抑制酸分泌分别在体内磷酸化位点的ezrin的定点磷酸化，并确定在每种情况下。在用不同的刺激处理胃腺后，埃兹林将通过免疫沉淀纯化。携带不同量磷酸盐的埃兹蛋白将通过IEF分离并在凝胶内消化。将通过MALDI质谱法、源后衰变分析和LC MS/MS分析ezrin肽，以鉴定不同情况下的磷酸化位点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 been suggested 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 overexpress EGF receptors were characterized as a substrate for an EGF-stimulated tyrosine kinase and were phosphorylated at both serine and tyrosine residues (Brestcher et al., 1989). EGF inhibits 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. Upon treatment of gastric glands with different stimuli, ezrin will be purified by immunoprecipitation. Ezrin carrying different amount of phosphate will be separated by IEF and in-gel digested. The ezrin peptides will be analyzed by MALDI mass spectrometry, post-source decay analysis and LC MS/MS to identify the phosphorylation site at different circumstances.