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

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 从对罕见疾病如X连锁低磷血症性佝偻病和肿瘤诱导的骨软化症的观察来看，FGF-23，一种由骨产生的循环激素，已成为磷和1，25二羟维生素D代谢的最重要调节剂之一。体内和体外研究表明，FGF-23通过抑制负责合成1，25二羟基维生素D的1-α羟化酶来抑制活性形式维生素D（1，25二羟基维生素D）的肾产生。在以前的实验中，我们已经表明，通过ERK 1/2激活MAPK信号通路是必要的，以诱导抑制1-α羟化酶的FGF-23在肾近端小管上皮细胞。然而，FGF-23调节1-α羟化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更好地了解肾近曲小管细胞中FGF-23激活的信号通路。我们将使用SILAC（细胞培养物中氨基酸的稳定同位素标记）比较差异标记细胞群之间的磷蛋白水平，有和没有用FGF-23处理。为了富集磷酸肽，我们将使用酪氨酸磷酸化蛋白的免疫沉淀进行亲和纯化。磷酸肽的鉴定将由加州大学旧金山分校质谱设施使用混合质谱仪（LTQ-Orbitap）进行。

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).