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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 从对X连锁低磷性软骨病和肿瘤引起的骨软化等罕见疾病的观察中，由骨骼产生的循环激素FGF-23已成为磷和1，25-二羟基维生素D代谢的最重要调节因子之一。体内和体外研究表明，成纤维细胞生长因子-23通过抑制合成1，25-二羟基维生素D的1-α羟基酶来抑制肾组织中活性维生素D(1，25-二羟基维生素D)的产生。在以往的实验中，我们已经证明，在肾近端小管上皮细胞中，通过ERK1/2激活MAPK信号通路是诱导抑制1-α羟基酶所必需的。然而，成纤维细胞生长因子-23调控1-α羟基酶表达的确切机制尚不清楚。 本项目的目的是利用定量磷酸蛋白质组学方法更好地了解肾小管上皮细胞中成纤维细胞生长因子-23激活的信号通路。我们将使用SILAC(细胞培养中氨基酸的稳定同位素标记)来比较使用和不使用成纤维细胞生长因子-23处理的不同标记细胞群之间的磷蛋白水平。为了丰富磷酸肽，我们将用免疫沉淀法对酪氨酸磷酸化蛋白进行亲和纯化。使用混合质谱计(LTQ-Orbitap)将由加州大学旧金山分校的质谱仪设备进行磷酸肽的鉴定。

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).