IN-VIVO PHOSPHORYLATION SITES ON GASTRIC EZRIN

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。胃ezrin是一种80kDa的蛋白，高度富集于胃壁细胞的顶端微绒毛中，序列与talin和红细胞带4.1同源。Ezrin被认为在介导顶细胞分泌酸的顶表面重排中起关键作用。camp依赖性蛋白激酶A通路介导的酸分泌刺激与ezrin的磷酸化有关。这些研究表明磷酸化发生在丝氨酸/苏氨酸残基而不是酪氨酸残基。另一方面，过度表达EGF受体的A431细胞中的ezrin被认为是EGF刺激的酪氨酸激酶的底物，并且在丝氨酸和酪氨酸残基上都被磷酸化（Brestcher等，1989）。EGF抑制壁细胞的酸分泌，有人认为EGF刺激的ezrin酪氨酸磷酸化可能本身抑制刺激。在这个项目中，我们建议研究组胺和EGF分别刺激和抑制酸分泌对ezrin的位点定向磷酸化，并确定每种情况下的体内磷酸化位点。在胃腺受到不同刺激后，ezrin会被免疫沉淀纯化。携带不同磷酸盐量的Ezrin将被IEF分离并在凝胶中消化。通过MALDI质谱分析、源后衰减分析和LC MS/MS分析ezrin肽，确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.