IN-VIVO PHOSPHORYLATION SITES ON GASTRIC EZRIN

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。胃Ezrin是一种80 kDa的蛋白质，在胃壁细胞顶端微绒毛中高度丰富，与talin和红细胞带4.1的序列具有同源性。Ezrin被认为在介导顶面重排中发挥关键作用，该重排涉及壁细胞的酸分泌。CAMP依赖的蛋白激酶A途径介导的促酸分泌作用与Ezrin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，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Ezrin被表征为EGF刺激的酪氨酸激酶的底物，并在丝氨酸和酪氨酸残基上被磷酸化(Brestcher等人，1989)。表皮生长因子抑制壁细胞的酸分泌，有研究表明，表皮生长因子刺激的Ezrin酪氨酸磷酸化本身可能抑制刺激。在这个项目中，我们建议检测组胺和EGF分别刺激和抑制酸分泌的Ezrin的定点磷酸化，并确定每种情况下的体内磷酸化位点。用不同的刺激物处理胃腺后，Ezrin将通过免疫沉淀得到纯化。携带不同磷酸盐的Ezrin将被离子交换膜分离并凝胶内消化。利用MALDI质谱仪、源后衰变分析和LC-MS/MS对Ezrin多肽进行分析，以确定不同环境下的磷酸化位点。

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.