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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。胃埃兹蛋白是一种80kDa的蛋白质，在胃壁细胞的顶端微绒毛中高度富集，并且与talin和红细胞带4.1具有序列同源性。埃兹蛋白被认为在介导壁细胞酸分泌所涉及的顶端表面重排中发挥关键作用。 cAMP 依赖性蛋白激酶 A 途径刺激酸分泌的介导与埃兹蛋白的磷酸化相关。这些研究表明磷酸化发生在丝氨酸/苏氨酸残基处，而不是酪氨酸残基处。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，1989）。 EGF 抑制壁细胞的酸分泌，并且有人提出 EGF 刺激的埃兹蛋白酪氨酸磷酸化本身可能抑制刺激。在这个项目中，我们建议检查组胺和 EGF 分别刺激和抑制酸分泌的埃兹蛋白的定点磷酸化，并确定每种情况下的体内磷酸化位点。用不同的刺激处理胃腺后，埃兹蛋白将通过免疫沉淀纯化。携带不同量磷酸盐的 Ezrin 将通过 IEF 分离并进行凝胶内消化。埃兹蛋白肽将通过 MALDI 质谱、源后衰变分析和 LC MS/MS 进行分析，以识别不同情况下的磷酸化位点。