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

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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).