Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis

Abstract

The family with sequence similarity 20, member C (Fam20C) has recently been identified as the Golgi casein kinase. Fam20C phosphorylates secreted proteins on Ser-x-Glu/pSer motifs and loss-of-function mutations in the kinase cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia. Fam20C is potentially an upstream regulator of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), because humans with FAM20C mutations and Fam20C KO mice develop hypophosphatemia due to an increase in full-length, biologically active FGF23. However, the mechanism by which Fam20C regulates FGF23 is unknown. Here we show that Fam20C directly phosphorylates FGF23 on Ser 180 , within the FGF23 R 176 XXR 179 /S 180 AE subtilisin-like proprotein convertase motif. This phosphorylation event inhibits O-glycosylation of FGF23 by polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3), and promotes FGF23 cleavage and inactivation by the subtilisin-like proprotein convertase furin. Collectively, our results provide a molecular mechanism by which FGF23 is dynamically regulated by phosphorylation, glycosylation, and proteolysis. Furthermore, our findings suggest that cross-talk between phosphorylation and O-glycosylation of proteins in the secretory pathway may be an important mechanism by which secreted proteins are regulated.phosphate homeostasis | rickets | Fam20 | familial tumoral calcinosis | chronic kidney disease P rotein kinases are evolutionarily conserved enzymes that regulate numerous cellular processes by transferring a molecule of phosphate from ATP to target substrates (1, 2). The vast majority of theses enzymes function within the nucleus and cytosol. In contrast, there are several examples of phosphorylated proteins that are secreted from the cell, which raises the question: What are the kinases that phosphorylate these secreted phosphoproteins? We recently identified a small family of secretory pathway kinases that phosphorylate secreted proteins and proteoglycans (3). These enzymes have N-terminal signal sequences that direct them to the lumen of the endoplasmic reticulum, where they encounter the proteins or proteoglycans that they phosphorylate. One member of this atypical kinase family is the family with sequence similarity 20, member C (Fam20C), which phosphorylates secreted proteins on Ser(S)-xGlu(E)/pSer(pS) (S-x-E/pS) motifs (3, 4). Because Fam20C localizes within the secretory pathway and the vast majority of secreted phosphoproteins are phosphorylated on S-x-E/pS motifs, Fam20C has been proposed to play a major role in the generation of the secreted phosphoproteome (5-7). For example, ∼75% of human serum and cerebrospinal fluid phosphoproteins are phosphorylated on S-x-E/pS motifs (8, 9). This includes proteins important for tooth and bone formation, as well as numerous hormones. In most cases, the functional importance of these phosphorylation events is unknown.Loss-of-function mutations in the human FAM20C gene cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia (10, 11)....

Development of Isoform-specific Sensors of Polypeptide GalNAc-transferase Activity

“…Generally, peptide preferences of the GalNAc-transferases are sensitive to residues from Ϫ3 to ϩ3 relative to the glycosylation site with a preference for proline at positions ϩ1 and ϩ3 (27). Furthermore, as mentioned above, the residue at ϩ2 (Ser 180 ) impedes glycosylation when phosphorylated (31). Consistent with these considerations, substitution of glycine at ϩ2 and proline at ϩ3 caused a significant drop in background activation of the T3 sensor (Fig.…”

“…To improve the signal to noise ratio we decided to modify the FGF23 insert sequence with the goal of making it a better substrate of T3 glycosylation. Fortunately, extensive in vitro assays were previously used to assess glycosylation of various peptide sequences (27) and recent work identified a phosphorylation site (Ser 180 ) that negatively regulates glycosylation (31). Based on these results, we tested the single mutation H177V and the triple mutation H177V/S180G/A181P.…”

Dysregulation of phosphate metabolism and conditions associated with phosphate toxicity

“…This observation suggests that the interplay between phosphorylation and O-glycosylation of FGF23 may be a critical posttranslational modification by which the activity of secreted FGF23 protein is determined. 21 Once secreted, FGF23 protein binds its receptors to exert diverse functions.…”

O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking

“…Recent studies have demonstrated a role for O-glycans in controlling prohormone convertase processing of FGF 23 (8,10), Tango-1 (9), pro-brain natriuretic peptide (56), and angiopoietin-like protein-3 (ANGPTL3) (57). Lacking O-glycans in exon 16, a 68-kDa product that contained part of exon 16, PAL, the transmembrane and cytosolic domains of PAM, was generated from PAM-1/OSX.…”