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.

Keywords: Fam20; chronic kidney disease; familial tumoral calcinosis; phosphate homeostasis; rickets.

Fig. 1.

Fam20C phosphorylates FGF23 on Ser¹⁸⁰. (A) Schematic representation of human FGF23 indicating the signal peptide (SP), intact FGF23 (iFGF23), N-terminal fragment (nFGF23), and C-terminal fragment (cFGF23). The residues surrounding the SPC cleavage site (downward arrow) are shown. Mutations that replace the Arg in patients with autosomal dominant hypophosphatemic rickets (ADHR) are also shown. A potential Fam20C phosphorylation site is in red. (B and C) Representative MS/MS fragmentation spectra of a tryptic peptide (FGF23 180–196) depicting Ser¹⁸⁰ phosphorylation of FGF23 R176Q purified from conditioned medium of HEK293T cells. (D) Time-dependent incorporation of ³²P from [γ-³²P]ATP into FGF23 R176Q by Fam20C or Fam20C D478A (DA). Reaction products were analyzed by SDS/PAGE and autoradiography.

Fig. 2.

Fam20C phosphorylates FGF23 in mammalian cells (A) Protein immunoblotting of trichloroacetic acid precipitates from conditioned medium of control and Fam20C KO cells (C5, clone 5; C9, clone 9) using an affinity-purified rabbit anti-Fam20C polyclonal antibody. GAPDH from cell extracts is shown as loading control (Lower). (B) Control and Fam20C KO cells were metabolically labeled with ³²PO₄³⁻ and transfected with V5-tagged OPN. Protein immunoblotting of V5-immunoprecipitates from conditioned medium (Top) and autoradiography depicting ³²P incorporation into OPN (Middle). Fam20C protein levels in conditioned medium are also shown (Bottom). (C) Protein immunoblotting of V5-immunoprecipitates from the conditioned medium of U2OS cells expressing V5-tagged FGF23. V5-immunoprecipitates were treated with O-glycosidase and α-(2→3,6,8,9)-neuraminidase to remove O-linked glycosylation or PNGase F to remove N-linked glycosylation. (D) Control and Fam20C KO cells were metabolically labeled with ³²PO₄³⁻ and transfected with V5-tagged FGF23. Protein immunoblotting of V5-immunoprecipitates from conditioned medium (Top) and autoradiography depicting ³²P incorporation into FGF23 (Middle). Intact FGF23 and C-terminal fragments are shown. Fam20C protein levels in conditioned medium are also shown (Bottom). IB, immunoblotting; IP, immunoprecipitation.

Fig. 3.

Phosphorylation of FGF23 at Ser¹⁸⁰ inhibits O-glycosylation by GalNAc-T3. (A) Protein immunoblotting of V5-immunoprecipitates from the conditioned medium of U2OS cells expressing V5-tagged FGF23 or mutants (S180A, T178A, S180D) with either WT (20C) or catalytically inactive D478A (20C D478A) FLAG-tagged Fam20C. V5-immunoprecipitates were treated with λ-phosphatase (λp’tase) (Upper). Extracts were analyzed for Fam20C, Fam20C D478A, FGF23, and GAPDH (Lower). (B and C) sGalNAc-T3 was incubated overnight (o/n) with FGF23(172–190) (B) or Ser¹⁸⁰-phosphorylated FGF23(172–190) (C) and UDP-GalNAc. The products were analyzed by MALDI-TOF MS.

Fig. 4.

Furin cleaves phosphorylated FGF23. (A) Protein immunoblotting of V5-immunoprecipitates from the conditioned medium of U2OS cells expressing V5-tagged FGF23 or mutants (S180D and T178A) with FLAG-tagged PCSK1, PCSK2, or PCSK3 (furin) (Upper). Extracts were analyzed for PCSK1–3, FGF23, and GAPDH expression (Lower). (B) Protein immunoblotting of V5-immunoprecipitates from the conditioned medium of control or FURIN KO U2OS cells (C17, clone 17; C82, clone 82) (Upper). Extracts were analyzed for FGF23 and GAPDH expression (Lower). (C and D) Time-dependent cleavage of FGF23(172–190) (C) or Ser¹⁸⁰-phosphorylated FGF23(172–190) (D). The products were analyzed by MALDI-TOF MS.

Fig. 5.

Deficient inhibition of FGF23 O-glycosylation by Fam20C T268M. (A) Structural representation of the active site of Fam20 from C. elegans highlighting the position of Thr¹⁷⁵ (human Thr²⁶⁸). The glycine-rich loop and nucleotide (ADP) are shown. (B) Sequence alignment of Fam20C from human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), fly (Drosophila melanogaster), and worm (Caenorhabditis elegans) depicting the conservation of the Thr. (C and D) Protein immunoblotting of V5 and Flag-immunoprecipitates from conditioned medium of U2OS cells expressing V5-tagged OPN (C) or FGF23 (D) with Flag-tagged D478A (20C D478A), WT (20C WT), or mutant Fam20C found in patients with FGF23-related hypophosphatemia (20C T268M) (Upper). Extracts were analyzed for Fam20C and GAPDH expression (Lower).