IN-VIVO PHOSPHORYLATION SITES ON GASTRIC EZRIN

胃 Ezrin 的体内磷酸化位点

• 批准号: 7369095
• 负责人: JOHN G FORTE
• 金额: $ 0.76万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369095
• 关键词: VIVO; PHOSPHORYLATION; SITES; GASTRIC; EZRIN

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.

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。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。胃EZRIN是一种80kDa蛋白，它高度富含胃顶细胞的顶端微绒毛，并且与塔林和红细胞带4.1具有序列同源性。已建议埃兹林在介导顶叶细胞酸分泌的顶部表面重排方面发挥关键作用。依赖CAMP依赖性蛋白激酶A途径刺激酸分泌的介导与Ezrin的磷酸化有关。这些研究表明，磷酸化是丝氨酸/苏氨酸残基，而不是酪氨酸残基。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磷酸化（Brestcher等，1989）。 EGF抑制顶叶细胞中的酸分泌，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，我们建议通过组胺和EGF检查Ezrin的位置定向的磷酸化，这些磷酸分别刺激和抑制酸分泌，并在每种情况下鉴定体内磷酸化位点。用不同刺激治疗胃腺后，Ezrin将通过免疫沉淀纯化。携带不同磷酸盐的埃兹林将被IEF分离，并消化凝胶。 Ezrin肽将通过MALDI质谱法，后源衰减分析和LC MS/MS分析，以在不同情况下识别磷酸化位点。