The Raine syndrome protein FAM20C is a Golgi kinase that phosphorylates bio-mineralization proteins

Abstract

Raine syndrome is caused by mutations in FAM20C, which had been reported to encode a secreted component of bone and teeth. We found that FAM20C encodes a Golgi-localized protein kinase, distantly related to the Golgi-localized kinase Four-jointed. Drosophila also encode a Golgi-localized protein kinase closely related to FAM20C. We show that FAM20C can phosphorylate secreted phosphoproteins, including both Casein and members of the SIBLING protein family, which modulate biomineralization, and we find that FAM20C phosphorylates a biologically active peptide at amino acids essential for inhibition of biomineralization. We also identify autophosphorylation of FAM20C, and characterize parameters of FAM20C's kinase activity, including its Km, pH and cation dependence, and substrate specificity. The biochemical properties of FAM20C match those of an enzymatic activity known as Golgi casein kinase. Introduction of point mutations identified in Raine syndrome patients into recombinant FAM20C impairs its normal localization and kinase activity. Our results identify FAM20C as a kinase for secreted phosphoproteins and establish a biochemical basis for Raine syndrome.

Figure 1. Localization of FAM20C proteins.

a) Relative amino acid sequence similarity amongst Golgi kinases in flies (red) and humans (blue). Sequences compared were: Drosophila CG3631 (UniProt Q95T10), CG31145 (Q9VCK5), Four-jointed (Fj, P54360), human FAM20A (Q96MK3), FAM20B (O75063), FAM20C (Q8IXL6), and Four-jointed box protein 1 (Fjx1, Q86VR8). b) Western blot showing that FAM20C:V5 expressed in cultured cells is detected in both lysate (L) and medium (M), GFP:V5 serves as a non-secreted control. c) CG31145:V5 expressed in S2 cells is detected in both lysate (L) and medium (M), CG3631:V5 is only detected in lysate. d,e) in situ hybridization showing d) saggital view and e) ventral view of expression of CG31145 in stage 16 Drosophila embryo; expression is detected in salivary gland (sg), dorsal vessel (dv), intestine (in), and midline glia (mg). Reproduced with permission from http://insitu.fruitfly.org/ . f) Localization of FAM20C:V5 (green) in HEK293T cells overlaps a Golgi marker (Giantin, magenta). g) FAM20C:V5 localization is distinct from an ER marker (anti-KDEL, magenta). h) Localization of CG31145:V5 (green) in S2 cells overlaps a Golgi marker (p120-Golgi, magenta). i) Localization of CG31145:V5 (green) in S2 cells is distinct from an ER marker (anti-KDEL, magenta). j) Localization of FAM20C (green) in MC3T3 cells overlaps a Golgi marker (Giantin, magenta). For f–j, panels marked by prime symbols show single channels of the merged images to the left.

Figure 2. Kinase activity of FAM20C.

a) Autoradiogram on protein gel showing the results of in vitro kinase assays in the presence (+) or absence (−) of FAM20C:V5 and the proteins indicated at bottom as substrates; the mobilities of FAM20C and Casein are indicated. Panels b−d,g,h show the results of kinase assays using dephosphorylated alpha casein as a substrate, and affinity purified FAM20C:V5 as the enzyme. b) Time course of FAM20C kinase reaction. c) Relationship between kinase activity and amount of wild-type or D453G mutant FAM20C:V5. d) Relationship between kinase activity and amount of Casein, curve fitting (using Graphpad Prism software) was used to determine Km. e) Autophosphorylation of FAM20C, as shown by autoradiogram on protein gel showing the results of in vitro kinase assays with the indicated proteins. A GST:FAM20C fusion protein expressed in bacteria is phosphorylated in a bacterial lysate, whereas an isoform with the D453G mutation is not. FAM20C:V5 can autophosphorylate, and FAM20C:V5 can phosphorylate GST:FAM20C with the D453G mutation. f) Autophosphorylation of FAM20C is concentration dependent, as the fraction of FAM20C phosphorylated increases at higher enzyme concentrations, consistent with a bimolecular reaction. Reactions were conducted in 10 µL volume, using 5, 10, 25, or 50 ng FAM20C. g) Relationship between kinase activity and amount of ATP, curve fitting (using Graphpad Prism software) was used to determine Km. h) Dependency of kinase activity on divalent cation concentration.

Figure 3. FAM20C phosphorylates SIBLING proteins.

a) Activity of FAM20C on peptides reported to be uniquely recognized by G-CK, CK1, or CK2 (23). Phosphocellulose assays were performed using 100 µm of the indicated peptides, and 5 ng FAM20C, 5 units CK1, or 1 unit CK2, as indicated. Phosphorylation sites are underlined, with the Ser in blue. b) Western blot (anti-FLAG) showing mobility shift of Opn:FLAG induced by co-transfection of FAM20C:V5 (as indicated by +) in HEK293T cells, and its reversal by Antarctic phosphatase (An. phosphatase). c) Western blots (anti-FLAG) showing mobility shifts of Bsp:FLAG and MEPE:FLAG induced by co-transfection of FAM20C:V5, but not FAM20A:V5 (as indicated by +), in HEK293T cells. d) Autoradiogram on protein gel showing the results of in vitro kinase assays in the presence (+) or absence (−) of FAM20C:V5 and the proteins indicated at bottom as substrates; the mobility of FAM20Cis indicated. The substrate proteins were affinity purified on FLAG beads, mock purification of conditioned medium from untransfected cells was used as a negative control. The mobilities of labeled protein bands are indicated at right. e) Results of kinase assays using the indicated OPN-ASARM peptides (− indicates no peptide), and using the indicated kinases. The names and sequence of peptides used are indicated at top right. First three amino acids (gray) were added to enable assays using phosphocellulose paper. Ser residues are highlighted in blue and G-CK consensus sites are underlined.

Figure 4. Localization and activity of FAM20C mutant isoforms.

A–d Examples of localization of the indicated V5-tagged FAM20C mutant proteins (green) transfected into 293 cells, as compared to a Golgi markers (Giantin, a,c, red) or an ER marker (anti-KDEL, b,d red). Nuceli are labeled by DAPI (blue). Panels marked by prime symbols show single channels of the merged image to the left. e) Western blot showing localization of FAM20C mutant proteins in the lysate (L) and/or medium (M) of 293 cells. f) Results of kinase assays using the indicated FAM20C mutant proteins and the OPN-ASARM peptide as a substrate.