title: FGF23 is endogenously phosphorylated in bone cells.
abstract: Levels of serum phosphate are controlled by the peptide hormone FGF23, secreted from bone osteocytes. Elevated levels of circulating FGF23 are a key factor in several hypophosphatemic disorders and play a role in chronic kidney disease. Posttranslational processing of FGF23 includes multi-site O-glycosylation, which reduces intracellular cleavage by proprotein convertases. The FGF23 protein also contains four serine phosphorylation consensus sequences (S-X-D/E); in this work, we asked whether FGF23 is a substrate for secretory phosphorylation. Both HEK cells as well as IDG-SW3 cells, an osteocyte model, incorporated radiolabeled orthophosphate into intact FGF23, as well as into the 14-kDa carboxy-terminal-but not the 17-kDa N-terminal-fragment. Sequential serine-to-alanine site-directed mutagenesis of four kinase consensus sites showed that labeling occurred on three serines within the carboxy-terminal fragment, Ser180 (adjacent to the cleavage site), Ser207, and Ser212. Liquid chromatography-coupled mass spectroscopy indicated the presence of phosphate at Ser212 in recombinant R&D mouse FGF23(R179Q) , confirming labeling results. A phosphopeptide-specific antibody was raised against phospho-Ser212 and exhibited immunoreactivity in osteocytes present in mouse long bone, providing further evidence that FGF23 is naturally phosphorylated in bone. Bone SIBLING proteins are serine-phosphorylated by the ubiquitous Golgi secretory kinase FAM20C. Cotransfection of HEK and MC3T3 cells with FGF23 and active, but not inactive, FAM20C kinase increased the storage and release of FGF23 in radiolabeling experiments, indicating potential effects of phosphorylation on FGF23 stability. Collectively, these data point to an important role for phosphorylation of FGF23 in bone.
authors.affiliation: Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Iris
authors.initials: I
authors.lastname: Lindberg
authors.forename: Hong Weng
authors.initials: HW
authors.lastname: Pang
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Stains
authors.forename: David
authors.initials: D
authors.lastname: Clark
authors.forename: Austin J
authors.initials: AJ
authors.lastname: Yang
authors.forename: Lynda
authors.initials: L
authors.lastname: Bonewald
authors.forename: Kevin Z
authors.initials: KZ
authors.lastname: Li
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism*; D002460:Cell Line; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation
chemical_list: C000717427:FGF23 protein, human; C000717428:Fgf23 protein, mouse; D005346:Fibroblast Growth Factors; D000089703:Fibroblast Growth Factor-23
keywords: BONE; FAM20C; FGF23; KINASE; OSTEOCYTE; PHOSPHATE HOMEOSTASIS