Secreted kinase phosphorylates extracellular proteins that regulate biomineralization

Abstract

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of cellular life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases that localize within the Golgi apparatus and are secreted. Fam20C appears to be the Golgi casein kinase that phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and several secreted proteins implicated in biomineralization, including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs). Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Fam20C is thus a protein kinase dedicated to the phosphorylation of extracellular proteins.

Fig. 1

Fam20C is a secreted protein kinase that phosphorylates S-x-E motifs. (A) Sequence alignment of members of the Fjx1 family of proteins with Canonical (PK) and atypical (AK) protein kinases highlighting conserved residues required for protein kinase activity (black), hydrophobic residues contributing to the structure (yellow), and the SP. Conserved protein kinase secondary structural elements and sequence features are indicated above. I, ion pair residues; C, catalytic residue; M, metal-binding residues. (Single letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.) (B) Protein immunoblotting of FLAG immunoprecipitates from the cell extract and conditioned medium of U2OS cells expressing FLAG-tagged Fam20C. (C) Immunofluorescence analysis of HeLa cells overexpressing FLAG-tagged Fam20C. The Golgi resident protein GM130 is shown. (D) SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining of FLAG-tagged Fam20C immunopurified from the conditioned medium from HEK293 T cells. (E) Autoradiograph of a kinase substrate array depicting peptides (in duplicate) phosphorylated by Fam20C. (F) Sequences of peptides phosphorylated by Fam20C in (E).

Fig. 2

Fam20C is the GEF-CK. (A) Fam20C phosphorylates β28-40. Fam20C was incubated with β28-40 in the presence of divalent cations and [γ-³²P]ATP. Incorporated radioactivity was quantified by means of scintillation counting. (B) Time-dependent incorporation of ³²P from [γ-³²P]ATP into β-casein by Fam20C or Fam20C D478A (DA). Reaction products were analyzed by means of SDS-PAGE and autoradiography. (C) Protein immunoblotting of V5-immunoprecipitates from the conditioned medium of U2OS cells overexpressing V5-tagged α-casein (α-cas:V5) with either WT (20C:Flag) or catalytically inactive D478A (20C DA:Flag) FLAG-tagged Fam20C. V5-immunoprecipitates were treated with λ-phosphatase (λ-p’tase). Total extracts were analyzed for Fam20C and Fam20C D478A expression (bottom). (D and E) Fractionation of casein kinase activity from bovine milk. Whey was fractionated by means of Q-Sepharose (D) and Mono Q (E) chromatography. Fractions were assayed for casein kinase activity and Fam20C by means of protein immunoblotting.

Fig. 3

Phosphorylation of SIBLINGs by Fam20C. (A) Time-dependent incorporation of ³²P into OPN by Fam20C or the D478A mutant. Reaction products were analyzed by means of SDS-PAGE and autoradiography. (B) Phosphorylation of OPN by Fam20C. Protein immunoblotting of V5-immunoprecipitates from the medium of U2OS cells overexpressing V5-tagged OPN (OPN:V5) with either WT (20C:Flag) or catalytically inactive D478A (20C DA:Flag) FLAG-tagged Fam20C. V5-immunoprecipitates were treated with λ-phosphatase (λ-p’tase). Total extracts were analyzed for Fam20C and Fam20C D478A expression (bottom). (C) Phosphorylation of MEPE by Fam20C as in (A). (D) Protein immunoblotting of V5-immunoprecipitates from cell extracts of U2OS cells overexpressing V5-tagged MEPE (MEPE:V5) with either WT (20C:Flag) or catalytically inactive D478A (20C DA:Flag) FLAG-tagged Fam20C. Samples were analyzed as in (B).

Fig. 4

Fam20C mutations and Raine syndrome. (A) Schematic representation of the Fam20C protein depicting missense mutations associated with Raine syndrome. SP, signal peptide. Lethal and nonlethal mutants are in red and green, respectively. (B) Secretion of Fam20C mutants. Protein immunoblotting of FLAG and V5 immunoprecipitates from conditioned medium of U2OS cells cotransfected with V5-tagged OPN and FLAG-tagged mutants of Fam20C. Cell extracts were also analyzed for intracellular protein levels.