PHOSPHOPROTEOMICS OF FGF-23 SIGNALING PATHWAY IN 1 ALPHA-HYDROXYLASE REGULATION

1 α-羟化酶调节中 FGF-23 信号通路的磷酸蛋白质组学

• 批准号: 8169811
• 负责人: ANTHONY A PORTALE
• 金额: $ 0.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-12 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169811
• 关键词: Affinity Chromatography; Amino Acids; Cell Culture Techniques; Cells; Computer Retrieval of Information on Scientific Projects Database; Dihydroxycholecalciferols; Enzymes; Epithelial Cells; Familial hypophosphatemic bone disease; Funding; Grant; Hormones; Hybrids; Immunoprecipitation; In Vitro; Institution; Kidney; Label; MAPK Signaling Pathway Pathway; Mass Spectrum Analysis; Metabolism; Mixed Function Oxygenases; Osteomalacia; Phosphopeptides; Phosphoproteins; Phosphorus; Population; Production; Proteins; Proximal Kidney Tubules; Rare Diseases; Regulation; Research; Research Personnel; Resources; Signal Pathway; Source; Stable Isotope Labeling; Tyrosine; United States National Institutes of Health; Vitamin D; bone; in vivo; mass spectrometer; research study; tumor

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. From the observation of rare diseases like X-linked hypophosphatemic rickets and tumor-induced osteomalacia, FGF-23, a circulating hormone produced by bone has emerged as one of the most important regulators of phosphorus and 1,25 dihydroxyvitamin D metabolism. In vivo and in vitro studies have shown that FGF-23 inhibits the renal production of active form of vitamin D (1,25 dihydroxyvitamin D), by suppression of the 1-alpha hydroxylase enzyme which is responsible for the synthesis of 1,25 dihydroxyvitamin D. In previous experiments, we have shown that activation of MAPK signaling pathway via ERK1/2 is necessary to induce suppression of the 1-alpha hydroxylase enzyme by FGF-23 in renal proximal tubule epithelial cells. However, the exact mechanism of regulation of 1-alpha hydroxylase enzyme expression by FGF-23 is unknown. The aim of this project is to better understand the signaling pathway activated by FGF-23 in renal proximal tubule cells using quantitative phosphoproteomics. We will use SILAC (stable isotope labeling with amino acids in cell culture) to compare phosphoprotein levels between differentially labelled cell populations, with and without treatment with FGF-23. In order to enrich for phosphopeptides, we will perform affinity purification with immunoprecipitation of tyrosine phosphorylated proteins. Identification of phosphopeptides will be performed by the UCSF Mass Spectrometry Facility using a hybrid mass spectrometer (LTQ-Orbitap).

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。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。列出的机构是 对于中心，这不一定是调查员的机构。 从观察到罕见的疾病，例如X连锁的次磷酸易患性r和肿瘤诱导的骨质乳酸，FGF-23（骨产生的循环激素）已成为磷的循环激素，是磷的最重要的调节剂之一和1,25二羟基羟耐抗毒素D-D-DHYVITAMIN D-D-D-D-N-N-N-Cranabolism。体内和体外研究表明，FGF-23通过抑制1-α羟基酶酶的肾脏产生活性形式（1,25二羟基维生素D），该酶通过1,25二氢二氢二氧化胺的合成来抑制1-α羟基酶酶，该酶的合成通过了先前的实验。 ERK1/2对于通过FGF-23在肾近端小管上皮细胞中诱导1-α羟基酶抑制1-α羟化酶。然而，FGF-23的1-α羟化酶表达调节的确切机制尚不清楚。 该项目的目的是更好地了解使用定量磷酸蛋白质组学在肾近端小管细胞中FGF-23激活的信号传导途径。我们将使用SILAC（在细胞培养中使用氨基酸稳定的同位素标记）来比较差异标记的细胞群体之间的磷酸蛋白水平，并与FGF-23进行处理。为了富集磷酸肽，我们将通过免疫沉淀酪氨酸磷酸化蛋白进行亲和力纯化。磷酸肽的鉴定将使用杂种质谱仪（LTQ-Orbitap）通过UCSF质谱设施进行。