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The ABCs of the atypical Fam20 secretory pathway kinases

Open AccessPublished:January 07, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100267
      The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome. Fam20B is a glycan kinase that phosphorylates xylose residues and triggers peptidoglycan biosynthesis, a role conserved from sponges to human. The protein kinase, Fam20C, conserved from nematodes to humans, phosphorylates well over 100 substrates in the secretory pathway with overall functions postulated to encompass endoplasmic reticulum homeostasis, nutrition, cardiac function, coagulation, and biomineralization. The preferred phosphorylation motif of Fam20C is SxE/pS, and structural studies revealed that related member Fam20A allosterically activates Fam20C by forming a heterodimeric/tetrameric complex. Fam20A, a pseudokinase, is observed only in vertebrates. Loss-of-function genetic alterations in the Fam20 family lead to human diseases such as amelogenesis imperfecta, nephrocalcinosis, lethal and nonlethal forms of Raine syndrome with major skeletal defects, and altered phosphate homeostasis. Together, these three members of the Fam20 family modulate a diverse network of secretory pathway components playing crucial roles in health and disease. The overarching theme of this review is to highlight the progress that has been made in the emerging field of extracellular phosphorylation and the key roles secretory pathway kinases play in an ever-expanding number of cellular processes.

      Keywords

      Abbreviations:

      AI (amelogenesis imperfecta), DCM (dilated cardiomyopathy), DMP1 (dentin matrix protein-1), EC (ectopic calcification), ER (endoplasmic reticulum), FGF-23 (fibroblast growth factor-23), Fj (four-jointed), LDLR (low-density lipoprotein receptor), NC (nephrocalcinosis), PCSK9 (proprotein convertase subtilisin-kexin), SIBLING (small integrin binding ligand-N-linked glycoprotein), T2D (type 2 diabetic), VLK (vertebrate lonesome kinase)
      The study of protein phosphorylation began as early as 1883 to 1900, when phosphorous was detected in milk casein (
      • Hammarsten O.
      Zur Frage ob das Caseïn ein einheitlicher Stoff sei.
      ) and egg-yolk phosvitin (
      • Leven P.A.
      • Alsberg C.
      Zur chemie der paranucleinsaure.
      ) respectively, thus making them the two earliest known phosphoproteins. Intriguingly, both these phosphoproteins are secreted from cells. Casein is secreted in milk (
      • Meggio F.
      • Boulton A.P.
      • Marchiori F.
      • Borin G.
      • Lennon D.P.
      • Calderan A.
      • Pinna L.A.
      Substrate-specificity determinants for a membrane-bound casein kinase of lactating mammary gland. A study with synthetic peptides.
      ) while phosvitin, a cleaved form of vitellogenin, is synthesized in the liver and secreted into the oviduct (
      • Byrne B.M.
      • van het Schip A.D.
      • van de Klundert J.A.
      • Arnberg A.C.
      • Gruber M.
      • Ab G.
      Amino acid sequence of phosvitin derived from the nucleotide sequence of part of the chicken vitellogenin gene.
      ,
      • Cozza G.
      • Moro E.
      • Black M.
      • Marin O.
      • Salvi M.
      • Venerando A.
      • Tagliabracci V.S.
      • Pinna L.A.
      The Golgi 'casein kinase' Fam20C is a genuine 'phosvitin kinase' and phosphorylates polyserine stretches devoid of the canonical consensus.
      ). Since these initial discoveries, casein and phosvitin have been used as common artificial substrates in the study of numerous kinases (
      • Burnett G.
      • Kennedy E.P.
      The enzymatic phosphorylation of proteins.
      ,
      • Rabinowitz M.
      • Lipmann F.
      Reversible phosphate transfer between yolk phosphoprotein and adenosine triphosphate.
      ,
      • Rodnight R.
      • Lavin B.E.
      Phosvitin kinase from brain: Activation by ions and subcellular distribution.
      ). In fact, the first evidence for the existence of protein kinases was provided by the pioneering study of George Burnett and Eugene Kennedy where they used rat mitochondrial extract to provide ATP and casein as the substrate to demonstrate the covalent addition of phosphate to casein in vitro (
      • Burnett G.
      • Kennedy E.P.
      The enzymatic phosphorylation of proteins.
      ). Since that time, many investigators have added to the number and complexity of kinases leading to the compilation of the kinome in 2002 (
      • Manning G.
      • Whyte D.B.
      • Martinez R.
      • Hunter T.
      • Sudarsanam S.
      The protein kinase complement of the human genome.
      ). This list of the human kinome included 540 individual members and represented kinases that could phosphorylate proteins as well as other biological molecules such as lipids and carbohydrates primarily within the cytosol and nucleus of the cell. But what about the kinases that phosphorylate resident proteins in the secretory pathway or proteins destined for secretion? This question was partially answered when the physiological secretory pathway kinase phosphorylating casein, family of sequence similarity 20C (Fam20C), was discovered in 2012 (
      • Ishikawa H.O.
      • Xu A.
      • Ogura E.
      • Manning G.
      • Irvine K.D.
      The Raine syndrome protein FAM20C is a Golgi kinase that phosphorylates bio-mineralization proteins.
      ,
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ). This same kinase was found to phosphorylate phosvitin in 2018 and thereby is accountable for the phosphorylation of the first identified secreted phosphoproteins (
      • Cozza G.
      • Moro E.
      • Black M.
      • Marin O.
      • Salvi M.
      • Venerando A.
      • Tagliabracci V.S.
      • Pinna L.A.
      The Golgi 'casein kinase' Fam20C is a genuine 'phosvitin kinase' and phosphorylates polyserine stretches devoid of the canonical consensus.
      ).
      The first clue for recognizing the secretory pathway kinases came from the identification of the Drosophila protein, four-jointed (Fj), as a secretory pathway kinase that phosphorylated the extracellular domains of atypical cadherins (
      • Ishikawa H.O.
      • Takeuchi H.
      • Haltiwanger R.S.
      • Irvine K.D.
      Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains.
      ). Using Fj as a BLAST query revealed a small family of related proteins that included Fam20A, B, and C (
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ). Since little was known about these proteins, they were designated “Fams” based on shared but limited sequence similarity. They all harbor a signal peptide that would direct them into the secretory pathway, but due to a lack of sequence similarity with canonical kinases, none of these atypical kinases were represented in the human kinome. The other domain these proteins share, which is also the sequence of highest homology, is the C-terminal Fam20 domain. Unexpectedly, the conserved Fam20 domain in each of these proteins has a very different function. Fam20C is the Golgi casein kinase responsible for phosphorylating secreted proteins on SxE/pS motifs (
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ). Fam20A is a pseudokinase that interacts with Fam20C and increases its activity (
      • Cui J.
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Rahdar M.
      • Dixon J.E.
      A secretory kinase complex regulates extracellular protein phosphorylation.
      ), and Fam20B is a xylose kinase involved in proteoglycan biosynthesis (
      • Wen J.
      • Xiao J.
      • Rahdar M.
      • Choudhury B.P.
      • Cui J.
      • Taylor G.S.
      • Esko J.D.
      • Dixon J.E.
      Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.
      ,
      • Koike T.
      • Izumikawa T.
      • Tamura J.
      • Kitagawa H.
      FAM20B is a kinase that phosphorylates xylose in the glycosaminoglycan-protein linkage region.
      ).
      Over the past few decades, multiple proteins in the extracellular and secretory space have been found to be phosphorylated. Many of these phospho-proteins are secreted into milk, serum, plasma, and cerebrospinal fluid (reviewed in (
      • Tagliabracci V.S.
      • Xiao J.
      • Dixon J.E.
      Phosphorylation of substrates destined for secretion by the Fam20 kinases.
      )) and have defined roles in diverse cellular processes from signaling, coagulation, migration, extracellular matrix formation, proteolysis, and biomineralization. The majority of these secreted proteins exhibit a phospho-motif of SxE/pS but to date, we have limited knowledge of the function of the majority of these extracellular phosphorylation events (reviewed in (
      • Tagliabracci V.S.
      • Xiao J.
      • Dixon J.E.
      Phosphorylation of substrates destined for secretion by the Fam20 kinases.
      )). Interestingly, out of the 540 kinases in the human kinome, only two kinases have been found localized in the secretory pathway: protein O-mannosyl kinase (POMK/SGK196) (
      • Zhu Q.
      • Venzke D.
      • Walimbe A.S.
      • Anderson M.E.
      • Fu Q.
      • Kinch L.N.
      • Wang W.
      • Chen X.
      • Grishin N.V.
      • Huang N.
      • Yu L.
      • Dixon J.E.
      • Campbell K.P.
      • Xiao J.
      Structure of protein O-mannose kinase reveals a unique active site architecture.
      ,
      • Yoshida-Moriguchi T.
      • Willer T.
      • Anderson M.E.
      • Venzke D.
      • Whyte T.
      • Muntoni F.
      • Lee H.
      • Nelson S.F.
      • Yu L.
      • Campbell K.P.
      SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function.
      ) and the tyrosine kinase, vertebrate lonesome kinase (VLK/SGK493) (
      • Bordoli M.R.
      • Yum J.
      • Breitkopf S.B.
      • Thon J.N.
      • Italiano Jr., J.E.
      • Xiao J.
      • Worby C.
      • Wong S.K.
      • Lin G.
      • Edenius M.
      • Keller T.L.
      • Asara J.M.
      • Dixon J.E.
      • Yeo C.Y.
      • Whitman M.
      A secreted tyrosine kinase acts in the extracellular environment.
      ), both of which do not phosphorylate SxE/pS motifs. Because the identity of the kinase(s) responsible for the majority of extracellular phosphorylation events remained elusive, the study of extracellular phosphorylation has lagged behind that of intracellular phosphorylation. It is increasingly clear that extracellular phosphorylation events play just as important roles in cellular regulation as their intracellular counterparts.
      To date, there are 13 known secretory pathway kinases (or kinase-like proteins), and we know very little about some of them. In a handful of cases, we do not know their substrate specificity or even if they are active kinases. This review focuses on Fam20A, B, C, the small subfamily of secretory pathway kinases for which we have made significant progress. In particular, we will address their cellular functions, reported substrates, structure/function relationships, and importance in human disease.

      Secreted kinases

      VLK family and POMK

      VLK and POMK are two secreted kinases that can be found at the root of the kinome tree. Therefore, their amino acid sequences were well enough conserved with the canonical kinases for them to be classified as kinases. POMK is an O-mannose kinase important for dystroglycan receptor function and matriglycan elongation (
      • Yoshida-Moriguchi T.
      • Willer T.
      • Anderson M.E.
      • Venzke D.
      • Whyte T.
      • Muntoni F.
      • Lee H.
      • Nelson S.F.
      • Yu L.
      • Campbell K.P.
      SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function.
      ,
      • Walimbe A.S.
      • Okuma H.
      • Joseph S.
      • Yang T.
      • Yonekawa T.
      • Hord J.M.
      • Venzke D.
      • Anderson M.E.
      • Torelli S.
      • Manzur A.
      • Devereaux M.
      • Cuellar M.
      • Prouty S.
      • Ocampo Landa S.
      • Yu L.
      • et al.
      POMK regulates dystroglycan function via LARGE1-mediated elongation of matriglycan.
      ). VLK is the first secreted tyrosine kinase identified, and it phosphorylates a broad range of secreted and ER-resident substrates (
      • Bordoli M.R.
      • Yum J.
      • Breitkopf S.B.
      • Thon J.N.
      • Italiano Jr., J.E.
      • Xiao J.
      • Worby C.
      • Wong S.K.
      • Lin G.
      • Edenius M.
      • Keller T.L.
      • Asara J.M.
      • Dixon J.E.
      • Yeo C.Y.
      • Whitman M.
      A secreted tyrosine kinase acts in the extracellular environment.
      ). A PSI-BLAST search using VLK as a query produces another small family of potential secreted kinases that includes Fam69A, Fam69B, Fam69C, DIA1, and DIA1R. Very little is known about these proteins (
      • Hareza A.
      • Bakun M.
      • Świderska B.
      • Dudkiewicz M.
      • Koscielny A.
      • Bajur A.
      • Jaworski J.
      • Dadlez M.
      • Pawłowski K.
      Phosphoproteomic insights into processes influenced by the kinase-like protein DIA1/C3orf58.
      ,
      • Dudkiewicz M.
      • Lenart A.
      • Pawłowski K.
      A novel predicted calcium-regulated kinase family implicated in neurological disorders.
      ,
      • Tennant-Eyles A.J.
      • Moffitt H.
      • Whitehouse C.A.
      • Roberts R.G.
      Characterisation of the FAM69 family of cysteine-rich endoplasmic reticulum proteins.
      ).

      Fj family of atypical kinases

      As alluded to in the introduction, the study of extracellular kinases was spearheaded by Ken Irvine’s laboratory when they published the first example of a secreted kinase, the fly protein Fj, which they went on to show phosphorylated unusual cadherin domains (
      • Ishikawa H.O.
      • Takeuchi H.
      • Haltiwanger R.S.
      • Irvine K.D.
      Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains.
      ). The murine equivalent of Fj, four-jointed box 1 (FJX1) is involved in forming appropriate dendrite arbor morphology in the hippocampus (
      • Probst B.
      • Rock R.
      • Gessler M.
      • Vortkamp A.
      • Püschel A.W.
      The rodent four-jointed ortholog Fjx1 regulates dendrite extension.
      ), and recently, human FJX1 has been shown to increase the invasive potential of nasopharyngeal cancer cells (
      • Chai S.J.
      • Ahmad Zabidi M.M.
      • Gan S.P.
      • Rajadurai P.
      • Lim P.V.H.
      • Ng C.C.
      • Yap L.F.
      • Teo S.H.
      • Lim K.P.
      • Patel V.
      • Cheong S.C.
      An oncogenic role for four-jointed box 1 (FJX1) in nasopharyngeal carcinoma.
      ,
      • Chai S.J.
      • Yap Y.Y.
      • Foo Y.C.
      • Yap L.F.
      • Ponniah S.
      • Teo S.H.
      • Cheong S.C.
      • Patel V.
      • Lim K.P.
      Identification of four-jointed box 1 (FJX1)-specific peptides for immunotherapy of nasopharyngeal carcinoma.
      ). In addition to FJX1 and Fam20A, B, and C, this small family contains two additional members, Fam198A and Fam198B. To date, neither Fam198A nor B has been ascribed kinase activity, and very little is known about their cellular functions (
      • Hsu C.Y.
      • Chang G.C.
      • Chen Y.J.
      • Hsu Y.C.
      • Hsiao Y.J.
      • Su K.Y.
      • Chen H.Y.
      • Lin C.Y.
      • Chen J.S.
      • Chen Y.J.
      • Hong Q.S.
      • Ku W.H.
      • Wu C.Y.
      • Ho B.C.
      • Chiang C.C.
      • et al.
      FAM198B is associated with prolonged survival and inhibits metastasis in lung adenocarcinoma via blockage of ERK-mediated MMP-1 expression.
      ,
      • Wei Z.
      • Liu T.
      • Lei J.
      • Wu Y.
      • Wang S.
      • Liao K.
      Fam198a, a member of secreted kinase, secrets through caveolae biogenesis pathway.
      ).

      Fam20B, the secreted xylose kinase

      Vertebrates exhibit three members of the Fam20 family of proteins (Fam20A,B, and C), whereas early invertebrates such as hydra and sponge have a single homolog of Fam20 whose activity resembles the human Fam20B-like protein (Fig. 1) (
      • Zhang H.
      • Zhu Q.
      • Cui J.
      • Wang Y.
      • Chen M.J.
      • Guo X.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Xiao J.
      Structure and evolution of the Fam20 kinases.
      ). Within the Fam20 family of secretory kinases, Fam20B was identified as a xylosylkinase kinase that phosphorylates xylose residues within the conserved tetrasaccharide linkages of proteoglycans (
      • Koike T.
      • Izumikawa T.
      • Tamura J.
      • Kitagawa H.
      FAM20B is a kinase that phosphorylates xylose in the glycosaminoglycan-protein linkage region.
      ). Interestingly, the xylose phosphorylation on the proteoglycan tetrasaccharide linkage was first identified in hydra (
      • Yamada S.
      • Morimoto H.
      • Fujisawa T.
      • Sugahara K.
      Glycosaminoglycans in Hydra magnipapillata (Hydrozoa, Cnidaria): Demonstration of chondroitin in the developing nematocyst, the sting organelle, and structural characterization of glycosaminoglycans.
      ), and further biochemical investigation revealed that hydra Fam20 and sponge Fam20 lacked protein kinase activity but exhibited robust xylosylkinase activity (
      • Zhang H.
      • Zhu Q.
      • Cui J.
      • Wang Y.
      • Chen M.J.
      • Guo X.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Xiao J.
      Structure and evolution of the Fam20 kinases.
      ). In fact, Fam20B is thought to be the first ancestral template protein for the Fam20 family of kinases and the function of xylose phosphorylation is conserved through the animal phylum from sponges to humans (
      • Zhang H.
      • Zhu Q.
      • Cui J.
      • Wang Y.
      • Chen M.J.
      • Guo X.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Xiao J.
      Structure and evolution of the Fam20 kinases.
      ). This evolutionary relationship is apparent in available structures. The ATP-binding sites of Fam20B and Fam20C are highly conserved (Fig. 2, A and B). However, Fam20B has a unique saccharide binding site not present in Fam20C or Fam20A (Fig. 2, A and C) (
      • Zhang H.
      • Zhu Q.
      • Cui J.
      • Wang Y.
      • Chen M.J.
      • Guo X.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Xiao J.
      Structure and evolution of the Fam20 kinases.
      ). Fam20C homologs are characterized by an occluded substrate binding pocket that cannot accommodate bulky saccharide substrate due to steric clashes. This occlusion results from slight structural rearrangements arising from distal residue substitutions that position a flexible loop within the binding pocket (Fig. 2D) (
      • Zhang H.
      • Zhu Q.
      • Cui J.
      • Wang Y.
      • Chen M.J.
      • Guo X.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Xiao J.
      Structure and evolution of the Fam20 kinases.
      ). The Fam20B-mediated xylose phosphorylation robustly stimulates galactosyltransferase II (GalT-II) activity leading to further addition of galactose to the tetrasaccharide linkages and accelerated proteoglycan chain extension (Fig. 5) (
      • Wen J.
      • Xiao J.
      • Rahdar M.
      • Choudhury B.P.
      • Cui J.
      • Taylor G.S.
      • Esko J.D.
      • Dixon J.E.
      Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.
      ). Furthermore, EXTL2 (Exostosin-Like Glycosyltransferase 2) polymerase utilizes the xylose phosphorylation to transfer a GlcNAc residue to the tetrasaccharide linkage region leading to termination of proteoglycan chain elongation (
      • Nadanaka S.
      • Zhou S.
      • Kagiyama S.
      • Shoji N.
      • Sugahara K.
      • Sugihara K.
      • Asano M.
      • Kitagawa H.
      EXTL2, a member of the EXT family of tumor suppressors, controls glycosaminoglycan biosynthesis in a xylose kinase-dependent manner.
      ). Intriguingly, depletion of Fam20B leads to immature proteoglycan formation, a phenotype quite reminiscent of Ehlers–Danlos syndrome, a rare inherited condition that affects connective tissue owing to GalT-II mutations (
      • Wen J.
      • Xiao J.
      • Rahdar M.
      • Choudhury B.P.
      • Cui J.
      • Taylor G.S.
      • Esko J.D.
      • Dixon J.E.
      Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.
      ). Thus, Fam20B plays an evolutionarily conserved quality-control role for proteoglycan biosynthesis and is arguably the ancestral Fam20.
      Figure thumbnail gr1
      Figure 1Fam20 is conserved in animal kingdom. Fam20B glycan kinase is the ancestral Fam20 kinase with a conserved role across the entire animal kingdom (nematode exception). Fam20C is first observed in nematodes with gene duplications observed in some organisms. In mammals only one copy of Fam20C is observed. Fam20A is observed in vertebrates only. (UniProt/UniParc accession IDs are provided).
      Figure thumbnail gr2
      Figure 2Structure of FAM20B, the glycan kinase. A, structure of Hydra magnipapillata FAM20B (hmFAM20B, PDB ID: 5xoo, chain A, white) with bound adenosine (ADN) and Galβ1-4Xylβ1 substrate. N and C lobes indicated approximately. B, FAM20B ATP-binding site (PDB ID:5xoo, chain A, white, ADN:adenosine) is highly conserved with C. elegans FAM20C ATP-binding site (PDB ID:4kqb, chain A, goldenrod, ADP, adenosine diphosphate). Similar residues labeled (FAM20B:black, FAM20C:orange). C, FAM20B saccharide binding site containing Galβ1-4Xylβ1 substrate (gray) (PDB ID:5xoo, chain A). D, superimposed FAM20B (PDB ID:5xoo, chain A, white) with C. elegans FAM20C (PDB ID:4kqb, chain A) at saccharide binding site. Arrow indicates flexible loop occluding saccharide binding. E, gene diagram depicting disease mutations. fs, frame shift.
      Whole-body genetic depletion of Fam20B in mice was embryonic lethal at E13.5 with the embryos exhibiting severe development defects and significant organ hypoplasia (
      • Vogel P.
      • Hansen G.M.
      • Read R.W.
      • Vance R.B.
      • Thiel M.
      • Liu J.
      • Wronski T.J.
      • Smith D.D.
      • Jeter-Jones S.
      • Brommage R.
      Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice.
      ). These observations were consistent with studies in zebrafish wherein loss-of-function mutants of Fam20B led to aberrant cartilage matrix organization and early stages of chondrocyte hypertrophy leading to skeletal defects (
      • Eames B.F.
      • Yan Y.L.
      • Swartz M.E.
      • Levic D.S.
      • Knapik E.W.
      • Postlethwait J.H.
      • Kimmel C.B.
      Mutations in fam20b and xylt1 reveal that cartilage matrix controls timing of endochondral ossification by inhibiting chondrocyte maturation.
      ). These initial in vivo observations were further echoed when tissue-specific depletion of Fam20B in mice led to the development of supernumerary teeth (
      • Tian Y.
      • Ma P.
      • Liu C.
      • Yang X.
      • Crawford D.M.
      • Yan W.
      • Bai D.
      • Qin C.
      • Wang X.
      Inactivation of Fam20B in the dental epithelium of mice leads to supernumerary incisors.
      ,
      • Wu J.
      • Tian Y.
      • Han L.
      • Liu C.
      • Sun T.
      • Li L.
      • Yu Y.
      • Lamichhane B.
      • D'Souza R.N.
      • Millar S.E.
      • Krumlauf R.
      • Ornitz D.M.
      • Feng J.Q.
      • Klein O.
      • Zhao H.
      • et al.
      FAM20B-catalyzed glycosaminoglycans control murine tooth number by restricting FGFR2b signaling.
      ), chondrosarcoma with major postnatal ossification defects (
      • Ma P.
      • Yan W.
      • Tian Y.
      • Wang J.
      • Feng J.Q.
      • Qin C.
      • Cheng Y.S.
      • Wang X.
      Inactivation of Fam20B in joint cartilage leads to chondrosarcoma and postnatal ossification defects.
      ), and severe craniofacial defects (
      • Liu X.
      • Li N.
      • Zhang H.
      • Liu J.
      • Zhou N.
      • Ran C.
      • Chen X.
      • Lu Y.
      • Wang X.
      • Qin C.
      • Xiao J.
      • Liu C.
      Inactivation of Fam20b in the neural crest-derived mesenchyme of mouse causes multiple craniofacial defects.
      ). Thus, the overarching role of Fam20B in proteoglycan biosynthesis likely contributes to the skeletal and developmental defects observed upon Fam20B depletion in tissue-specific in vivo models. In humans, two lethal compound heterozygous variants in Fam20B have been identified in a girl who died soon after birth (Fig. 2E) (
      • Kuroda Y.
      • Murakami H.
      • Enomoto Y.
      • Tsurusaki Y.
      • Takahashi K.
      • Mitsuzuka K.
      • Ishimoto H.
      • Nishimura G.
      • Kurosawa K.
      A novel gene (FAM20B encoding glycosaminoglycan xylosylkinase) for neonatal short limb dysplasia resembling Desbuquois dysplasia.
      ). The genetic alterations reported were T59Afs and N347Mfs and the patient exhibited severe organ hypoplasia, skeletal defects, and respiratory failure (
      • Kuroda Y.
      • Murakami H.
      • Enomoto Y.
      • Tsurusaki Y.
      • Takahashi K.
      • Mitsuzuka K.
      • Ishimoto H.
      • Nishimura G.
      • Kurosawa K.
      A novel gene (FAM20B encoding glycosaminoglycan xylosylkinase) for neonatal short limb dysplasia resembling Desbuquois dysplasia.
      ). The amino terminal T59A frameshift leads to hypomorphic gene function and essential loss of one allele of Fam20B. The carboxy-terminal alteration, N347M frameshift, results in disruption of more than 15% of the protein sequence and results in the loss of C389, which forms a disulfide bond with C332 and likely contributes to the global stability of the protein. The N347M frameshift, therefore, results in a destabilized Fam20B and also represents a functionally inactive variant. Intriguingly, osteoarthiritis and osteochondropathy patients with decreased proteoglycans and chondrocyte numbers exhibited marked reduction of Fam20B, GalT-II, and EXTL2 protein levels in knee cartilage biopsy samples (
      • Lei J.
      • Deng H.
      • Ran Y.
      • Lv Y.
      • Amhare A.F.
      • Wang L.
      • Guo X.
      • Han J.
      • Lammi M.J.
      Altered expression of aggrecan, FAM20B, B3GALT6, and EXTL2 in patients with osteoarthritis and Kashin-beck disease.
      ). This suggests that Fam20B could be a predictive marker for specific bone diseases.

      Fam20C, the secreted Golgi casein kinase

      As stated previously, the story of milk casein as a phosphoprotein started in the late 19th century when Olof Hammarsten reported the presence of phosphorus in casein (
      • Hammarsten O.
      Zur Frage ob das Caseïn ein einheitlicher Stoff sei.
      ). Fifty years later, Fritz Lipmann identified that the phosphorus was covalently bound to casein as phosphoseryl groups (
      • Lipmann F.
      Über die Bindung der Phosphorsäure in Phosphorproteinen. I.
      ). Eventually, the sequences surrounding those phosphoseryl groups in casein were identified as SxE/pS, which prompted the idea that SxE/pS sequence was the preferential motif for enzymes phosphorylating casein (
      • Mercier J.C.
      Phosphorylation of caseins, present evidence for an amino-acid triplet code post-translationally recognized by specific kinases.
      ,
      • Mercier J.C.
      • Grosclaude F.
      • Ribadeau-Dumas B.
      Primary structure of bovine s1 casein. Complete sequence.
      ) within the secretory pathway (
      • Bingham E.W.
      • Farrell Jr., H.M.
      • Basch J.J.
      Phosphorylation of casein. Role of the Golgi apparatus.
      ). In subsequent years, two cytoplasmic kinases were shown to robustly phosphorylate casein in vitro and because of this ability were designated casein kinase 1 and 2 (
      • Cozza G.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      “Genuine” casein kinase (Fam20C): The mother of the phosphosecretome.
      ). This was despite the fact that they would never come into contact with casein because they were localized to the cytoplasm and nucleus while casein, a secreted protein, resided in the secretory pathway and extracellularly. The bona fide “Golgi casein kinase” activity was initially observed in lactating mammary glands (
      • Mercier J.C.
      Phosphorylation of caseins, present evidence for an amino-acid triplet code post-translationally recognized by specific kinases.
      ,
      • Bingham E.W.
      • Farrell Jr., H.M.
      • Basch J.J.
      Phosphorylation of casein. Role of the Golgi apparatus.
      ,
      • Moore A.
      • Boulton A.P.
      • Heid H.W.
      • Jarasch E.D.
      • Craig R.K.
      Purification and tissue-specific expression of casein kinase from the lactating Guinea-pig mammary gland.
      ) and partially purified from milk (
      • Duncan J.S.
      • Wilkinson M.C.
      • Burgoyne R.D.
      Purification of Golgi casein kinase from bovine milk.
      ). Lorenzo Pinna and colleagues extensively characterized the activity of the partially purified protein from Golgi fractions and further reported that the kinase was highly resistant to the majority of the well-established kinase inhibitors including staurosporine (
      • Meggio F.
      • Boulton A.P.
      • Marchiori F.
      • Borin G.
      • Lennon D.P.
      • Calderan A.
      • Pinna L.A.
      Substrate-specificity determinants for a membrane-bound casein kinase of lactating mammary gland. A study with synthetic peptides.
      ,
      • Lasa M.
      • Marin O.
      • Pinna L.A.
      Rat liver Golgi apparatus contains a protein kinase similar to the casein kinase of lactating mammary gland.
      ,
      • Brunati A.M.
      • Marin O.
      • Bisinella A.
      • Salviati A.
      • Pinna L.A.
      Novel consensus sequence for the Golgi apparatus casein kinase, revealed using proline-rich protein-1 (PRP1)-derived peptide substrates.
      ,
      • Tibaldi E.
      • Arrigoni G.
      • Brunati A.M.
      • James P.
      • Pinna L.A.
      Analysis of a sub-proteome which co-purifies with and is phosphorylated by the Golgi casein kinase.
      ,
      • Cozza G.
      • Salvi M.
      • Banerjee S.
      • Tibaldi E.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins.
      ). In 2012, this elusive activity was identified molecularly when Fam20C was experimentally recognized to be the Golgi casein kinase capable of phosphorylating casein in vivo (
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ). Although atypical, crystallography studies on the nematode-ortholog of Fam20C (
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ) revealed that the kinase exhibited the canonical N- and C-lobed kinase structure with a well-defined ATP-binding active-site pocket (Fig. 3A) . The breadth of Fam20C’s activity was alluded to when phosphoproteomic studies of human plasma, serum, and cerebrospinal fluid demonstrated that more than two-thirds of secreted phosphorylated proteins were phosphorylated on SxE/pS motifs (
      • Carrascal M.
      • Gay M.
      • Ovelleiro D.
      • Casas V.
      • Gelpí E.
      • Abian J.
      Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.
      ,
      • Bahl J.M.
      • Jensen S.S.
      • Larsen M.R.
      • Heegaard N.H.
      Characterization of the human cerebrospinal fluid phosphoproteome by titanium dioxide affinity chromatography and mass spectrometry.
      ,
      • Zhou W.
      • Ross M.M.
      • Tessitore A.
      • Ornstein D.
      • Vanmeter A.
      • Liotta L.A.
      • Petricoin 3rd, E.F.
      An initial characterization of the serum phosphoproteome.
      ). In fact, phosphoproteomic analysis of secreted neuropeptides in the nervous and endocrine system revealed that the predominant phospho-motif was SxE (
      • Lietz C.B.
      • Toneff T.
      • Mosier C.
      • Podvin S.
      • O'Donoghue A.J.
      • Hook V.
      Phosphopeptidomics reveals differential phosphorylation states and novel SxE phosphosite motifs of neuropeptides in dense core secretory vesicles.
      ). This was solidified by studies in which Fam20C was ablated in several tissue culture cell lines and the culture media was analyzed for secreted phosphoproteins (
      • Tagliabracci V.S.
      • Wiley S.E.
      • Guo X.
      • Kinch L.N.
      • Durrant E.
      • Wen J.
      • Xiao J.
      • Cui J.
      • Nguyen K.B.
      • Engel J.L.
      • Coon J.J.
      • Grishin N.
      • Pinna L.A.
      • Pagliarini D.J.
      • Dixon J.E.
      A single kinase generates the majority of the secreted phosphoproteome.
      ). Cumulatively, this work resulted in affirming that Fam20C is the kinase responsible for phosphorylating the majority of secreted proteins and broadened Fam20C’s substrate preference to include phosphorylation sites other than SxE/pS sites (
      • Tagliabracci V.S.
      • Wiley S.E.
      • Guo X.
      • Kinch L.N.
      • Durrant E.
      • Wen J.
      • Xiao J.
      • Cui J.
      • Nguyen K.B.
      • Engel J.L.
      • Coon J.J.
      • Grishin N.
      • Pinna L.A.
      • Pagliarini D.J.
      • Dixon J.E.
      A single kinase generates the majority of the secreted phosphoproteome.
      ). For instance, a recent study reported that specific threonine residues on the neuroendocrine chaperone 7B2 were phosphorylated by Fam20C (
      • Ramos-Molina B.
      • Lindberg I.
      Phosphorylation and alternative splicing of 7B2 reduce prohormone convertase 2 activation.
      ). Surprisingly, there were nonoverlapping substrates between the secreted phosphoproteome from the different cell lines indicating that individual cell populations have different milieux of secreted proteins.
      Figure thumbnail gr3
      Figure 3Structure of Fam20C, the secreted protein kinase. A, structure of C. elegans FAM20C (ceFAM20C, PDB ID:4kqb, chain A, goldenrod). N and C lobe indicated approximately. ATP-binding site diagram of important residues. Parenthetical residues represent structurally equivalent residues in Homo sapiens FAM20C. B, heterotetramer of Danio rerio FAM20C (drFAM20C, goldenrod) and Homo sapiens FAM20A (hFAM20A, cyan) (PDB ID:5yh2; chains A–D). Heterodimer interface and heterotetramer interfaces indicated. C, heterodimer of Homo sapiens Fam20C (hFAM20C, goldenrod1) and Homo sapiens FAM20A (hFAM20A, cyan) (PDB ID:5yh3, chains A and C). Residues important to the heterodimer interface indicated. N and C lobe indicated approximately. D, gene diagram depicting disease mutations (fs, frame shift; X, STOP/termination). E, cartoon depiction of kinase indicated positions of mutated residues when resolved (mutations as red spheres, PDB ID:5yh3, chain C). Residue labels color coded to indicate mutation type (red: missense mutation, orange: frameshift, and pink: STOP/termination). N and C lobes indicated approximately.
      Fam20C has a strong cofactor preference for Mn2+ and Co2+ ions over the canonical Mg2+ ion for its kinase activity (
      • Lasa M.
      • Marin O.
      • Pinna L.A.
      Rat liver Golgi apparatus contains a protein kinase similar to the casein kinase of lactating mammary gland.
      ) although the physiological levels of Mg2+ in cells (around 1 mM) are 104 fold higher than Mn2+ (about 100 nM) (
      • Cozza G.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      “Genuine” casein kinase (Fam20C): The mother of the phosphosecretome.
      ). Lorenzo Pinna and colleagues argued that under physiological circumstances, specific signaling components may play a role in promoting Fam20C to utilize Mg2+ over Mn2+ in the secretory pathway (
      • Cozza G.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      “Genuine” casein kinase (Fam20C): The mother of the phosphosecretome.
      ). The group reported that sphingosine and sphingosine-1-phosphate significantly improved the ability of Fam20C to utilize Mg2+ as a cofactor (
      • Cozza G.
      • Salvi M.
      • Banerjee S.
      • Tibaldi E.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins.
      ,
      • Cozza G.
      • Salvi M.
      • Tagliabracci V.S.
      • Pinna L.A.
      Fam20C is under the control of sphingolipid signaling in human cell lines.
      ). Indeed, sphingosine addition led to an eightfold higher activity of Fam20C in vitro with a threefold increase in Vmax and a consequent threefold decrease in Km (
      • Cozza G.
      • Salvi M.
      • Banerjee S.
      • Tibaldi E.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins.
      ,
      • Cozza G.
      • Salvi M.
      • Tagliabracci V.S.
      • Pinna L.A.
      Fam20C is under the control of sphingolipid signaling in human cell lines.
      ). However, ceramide, the precursor of sphingosine, had no effect on Fam20C activity, thus suggesting sphingosine as a specific activator of Fam20C (
      • Cozza G.
      • Salvi M.
      • Banerjee S.
      • Tibaldi E.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins.
      ,
      • Cozza G.
      • Salvi M.
      • Tagliabracci V.S.
      • Pinna L.A.
      Fam20C is under the control of sphingolipid signaling in human cell lines.
      ). Interestingly, the activity of Fam20C is dynamically controlled by its binding partner Fam20A (Fig. 3, B and C). Fam20A and Fam20C together form a heterodimeric complex (Fig. 3C), which dramatically promotes the activity of Fam20C to phosphorylate its substrates (
      • Cui J.
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Rahdar M.
      • Dixon J.E.
      A secretory kinase complex regulates extracellular protein phosphorylation.
      ,
      • Zhang H.
      • Zhu Q.
      • Cui J.
      • Wang Y.
      • Chen M.J.
      • Guo X.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Xiao J.
      Structure and evolution of the Fam20 kinases.
      ). Two heterodimers can further associate to form a heterotetrameric complex (Fig. 3B), but it remains an open question as to which form exists in vivo. This uncommon allosteric mode of pseudokinase-mediated activation of Fam20C is further explained below in the Fam20A section. Finally, functional annotations of Fam20C substrates suggest that Fam20C will play important roles in many physiological processes and disease states.

      FAM20C substrates in nutrition and mineralization

      The gene encoding casein resides on chromosome 4 surrounded by other genes encoding proteins that contain multiple SxE motifs. Casein accounts for approximately 80% of the total protein in bovine milk where it interacts with calcium phosphate forming colloidal structures called casein micelles, thereby providing nutrients including calcium and phosphate for growth of bones and teeth to mammalian infants (
      • Fang Z.H.
      • Visker M.
      • Miranda G.
      • Delacroix-Buchet A.
      • Bovenhuis H.
      • Martin P.
      The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways.
      ). The consequences of casein phosphorylation have been intensively studied with regard to cheese manufacturing where it is suggested to affect milk technological properties by stabilizing calcium phosphate nanoclusters and promoting micellar growth (
      • Fang Z.H.
      • Visker M.
      • Miranda G.
      • Delacroix-Buchet A.
      • Bovenhuis H.
      • Martin P.
      The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways.
      ,
      • Anema S.G.
      • de Kruif C.G.
      Protein composition of different sized casein micelles in milk after the binding of lactoferrin or lysozyme.
      ,
      • Holt C.
      • Carver J.A.
      • Ecroyd H.
      • Thorn D.C.
      Invited review: Caseins and the casein micelle: Their biological functions, structures, and behavior in foods.
      ).
      In addition, chromosome 4 harbors another gene cluster encoding the small integrin binding ligand-N-linked glycoproteins (SIBLINGs). These genes are known to regulate bone and tooth development and encode osteopontin, dentin matrix protein-1 (DMP1), matrix extracellular phosphoglycoprotein, bone sialoprotein, and dentin sialophosphoprotein, all of which are involved in binding calcium and all of which are Fam20C substrates (
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ). In fact, Fam20C phosphorylates DMP1 in osteoblasts and young osteoclasts, which leads to the secretion of phospho-DMP1 into the pericanalicular matrix of mineralized bone (
      • Oya K.
      • Ishida K.
      • Nishida T.
      • Sato S.
      • Kishino M.
      • Hirose K.
      • Ogawa Y.
      • Ikebe K.
      • Takeshige F.
      • Yasuda H.
      • Komori T.
      • Toyosawa S.
      Immunohistochemical analysis of dentin matrix protein 1 (Dmp1) phosphorylation by Fam20C in bone: Implications for the induction of biomineralization.
      ). Fam20C is further thought to indirectly promote DMP1 transcription (
      • Kinoshita Y.
      • Hori M.
      • Taguchi M.
      • Fukumoto S.
      Functional analysis of mutant FAM20C in Raine syndrome with FGF23-related hypophosphatemia.
      ). In addition, Fam20C phosphorylates multiple sites on osteopontin and promotes its secretion (
      • Tibaldi E.
      • Brocca A.
      • Sticca A.
      • Gola E.
      • Pizzi M.
      • Bordin L.
      • Pagano M.A.
      • Mazzorana M.
      • Donà G.
      • Violi P.
      • Marin O.
      • Romano A.
      • Angeli P.
      • Carraro A.
      • Brunati A.M.
      Fam20C-mediated phosphorylation of osteopontin is critical for its secretion but dispensable for its action as a cytokine in the activation of hepatic stellate cells in liver fibrogenesis.
      ) but inhibits its binding to αvβ3 integrin (
      • Schytte G.N.
      • Christensen B.
      • Bregenov I.
      • Kjøge K.
      • Scavenius C.
      • Petersen S.V.
      • Enghild J.J.
      • Sørensen E.S.
      FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvβ3 integrin.
      ). These negatively charged phosphorylated substrates allude to Fam20C’s involvement in Ca2+ regulation in many varied and diverse processes including nutrition and the formation of mineralized tissues. Indeed, a large body of literature, focusing on conditional tissue-specific knockout mice and cell models, reports the roles of Fam20C in promoting biomineralization including the growth and development of osteoblasts, osteoclasts, bone, dentin, and enamel (Fig. 5) (
      • Du E.X.
      • Wang X.F.
      • Yang W.C.
      • Kaback D.
      • Yee S.P.
      • Qin C.L.
      • George A.
      • Hao J.J.
      Characterization of Fam20C expression in odontogenesis and osteogenesis using transgenic mice.
      ,
      • Liu C.
      • Zhang H.
      • Jani P.
      • Wang X.
      • Lu Y.
      • Li N.
      • Xiao J.
      • Qin C.
      FAM20C regulates osteoblast behaviors and intracellular signaling pathways in a cell-autonomous manner.
      ,
      • Liu C.
      • Zhou N.
      • Wang Y.
      • Zhang H.
      • Jani P.
      • Wang X.
      • Lu Y.
      • Li N.
      • Xiao J.
      • Qin C.
      Abrogation of Fam20c altered cell behaviors and BMP signaling of immortalized dental mesenchymal cells.
      ,
      • Liu P.
      • Zhang H.
      • Liu C.
      • Wang X.
      • Chen L.
      • Qin C.
      Inactivation of Fam20C in cells expressing type I collagen causes periodontal disease in mice.
      ,
      • Ma P.
      • Yan W.
      • Tian Y.
      • He J.
      • Brookes S.J.
      • Wang X.
      The importance of serine phosphorylation of ameloblastin on enamel formation.
      ,
      • Qin Z.
      • Wang P.
      • Li X.
      • Zhang S.
      • Tian M.
      • Dai Y.
      • Fu L.
      Systematic network-based discovery of a Fam20C inhibitor (FL-1607) with apoptosis modulation in triple-negative breast cancer.
      ,
      • Wang S.K.
      • Reid B.M.
      • Dugan S.L.
      • Roggenbuck J.A.
      • Read L.
      • Aref P.
      • Taheri A.P.
      • Yeganeh M.Z.
      • Simmer J.P.
      • Hu J.C.
      FAM20A mutations associated with enamel renal syndrome.
      ,
      • Wang S.K.
      • Samann A.C.
      • Hu J.C.
      • Simmer J.P.
      FAM20C functions intracellularly within both ameloblasts and odontoblasts in vivo.
      ,
      • Wang X.
      • Hao J.
      • Xie Y.
      • Sun Y.
      • Hernandez B.
      • Yamoah A.K.
      • Prasad M.
      • Zhu Q.
      • Feng J.Q.
      • Qin C.
      Expression of FAM20C in the osteogenesis and odontogenesis of mouse.
      ,
      • Wang X.
      • Jung J.
      • Liu Y.
      • Yuan B.
      • Lu Y.
      • Feng J.Q.
      • Qin C.
      The specific role of FAM20C in amelogenesis.
      ,
      • Wang X.
      • Wang S.
      • Li C.
      • Gao T.
      • Liu Y.
      • Rangiani A.
      • Sun Y.
      • Hao J.
      • George A.
      • Lu Y.
      • Groppe J.
      • Yuan B.
      • Feng J.Q.
      • Qin C.
      Inactivation of a novel FGF23 regulator, FAM20C, leads to hypophosphatemic rickets in mice.
      ,
      • Wang X.
      • Wang S.
      • Lu Y.
      • Gibson M.P.
      • Liu Y.
      • Yuan B.
      • Feng J.Q.
      • Qin C.
      FAM20C plays an essential role in the formation of murine teeth.
      ,
      • Yan W.J.
      • Ma P.
      • Tian Y.
      • Wang J.Y.
      • Qin C.L.
      • Feng J.Q.
      • Wang X.F.
      The importance of a potential phosphorylation site in enamelin on enamel formation.
      ).

      Fam20C substrates promoting secretion and ER homeostasis

      Phosphoproteomic analysis of pancreatic β -islet cells from type 2 diabetic obese (T2D) mice revealed 39 potential phosphosites conforming to the SxE motif (
      • Kang T.
      • Boland B.B.
      • Alarcon C.
      • Grimsby J.S.
      • Rhodes C.J.
      • Larsen M.R.
      Proteomic analysis of restored insulin production and trafficking in obese diabetic mouse pancreatic islets following euglycemia.
      ). The study reported that Fam20C levels went up in the cells of T2D mice, thereby promoting secretion of immature proinsulin under hyperglycaemic conditions (
      • Kang T.
      • Boland B.B.
      • Alarcon C.
      • Grimsby J.S.
      • Rhodes C.J.
      • Larsen M.R.
      Proteomic analysis of restored insulin production and trafficking in obese diabetic mouse pancreatic islets following euglycemia.
      ). Upon restoring euglycaemia, the levels of Fam20C and 11 corresponding SxE phosphosites were brought back to basal level (
      • Kang T.
      • Boland B.B.
      • Alarcon C.
      • Grimsby J.S.
      • Rhodes C.J.
      • Larsen M.R.
      Proteomic analysis of restored insulin production and trafficking in obese diabetic mouse pancreatic islets following euglycemia.
      ). This study suggests that Fam20C might play an important role in the control of insulin section from the β -islet cells of pancreas. In fact, recent studies suggest Fam20C plays a pivotal role in ER homeostasis, which promotes proper section, including phosphorylation of proteins sequestered within the secretory pathway (Fig. 5). Recent works report that Fam20C phosphorylation of ER oxidoreductin 1α (Ero1α) on Ser145 (SxE site) is important for regulating ER redox homeostasis and oxidative protein folding (
      • Zhang J.
      • Zhu Q.
      • Wang X.
      • Yu J.
      • Chen X.
      • Wang J.
      • Wang X.
      • Xiao J.
      • Wang C.C.
      • Wang L.
      Secretory kinase Fam20C tunes endoplasmic reticulum redox state via phosphorylation of Ero1α.
      ). This Ero1α phosphorylation is induced following secretion-demanding conditions such as lactation and interestingly, this posttranslational event occurs in the Golgi apparatus, and Ero1α is retrograde-transported to the ER mediated by ERp44 (
      • Zhang J.
      • Zhu Q.
      • Wang X.
      • Yu J.
      • Chen X.
      • Wang J.
      • Wang X.
      • Xiao J.
      • Wang C.C.
      • Wang L.
      Secretory kinase Fam20C tunes endoplasmic reticulum redox state via phosphorylation of Ero1α.
      ). Furthermore, Fam20C maintains ER proteostasis and protects against ER stress-induced cell death (
      • Yu J.
      • Li T.
      • Liu Y.
      • Wang X.
      • Zhang J.
      • Wang X.
      • Shi G.
      • Lou J.
      • Wang L.
      • Wang C.C.
      • Wang L.
      Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress.
      ). Protein disulfide isomerase (PDI) is a highly abundant ER-resident enzyme playing critical roles as both a thiol-disulfide oxidoreductase and a molecular chaperone, which prevents protein misfolding in the ER (
      • Hatahet F.
      • Ruddock L.W.
      Protein disulfide isomerase: A critical evaluation of its function in disulfide bond formation.
      ,
      • Wang L.
      • Wang X.
      • Wang C.C.
      Protein disulfide-isomerase, a folding catalyst and a redox-regulated chaperone.
      ). Fam20C phosphorylates PDI on Ser357 upon ER stress and promotes the activity of PDI to maintain ER proteostasis (
      • Yu J.
      • Li T.
      • Liu Y.
      • Wang X.
      • Zhang J.
      • Wang X.
      • Shi G.
      • Lou J.
      • Wang L.
      • Wang C.C.
      • Wang L.
      Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress.
      ). Indeed, loss of Ser357 (Ser359 in mouse) leads to acute liver damage in mice challenged with proteotoxic stress (
      • Yu J.
      • Li T.
      • Liu Y.
      • Wang X.
      • Zhang J.
      • Wang X.
      • Shi G.
      • Lou J.
      • Wang L.
      • Wang C.C.
      • Wang L.
      Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress.
      ). Interestingly, recent studies show that Fam20C phosphorylation is required for the secretion of certain proteins. For example, Fam20C phosphorylates calcium binding protein 45 kDa (Cab45), a Golgi protein, regulating the sorting and secretion of proteins (
      • Hecht T.K.
      • Blank B.
      • Steger M.
      • Lopez V.
      • Beck G.
      • Ramazanov B.
      • Mann M.
      • Tagliabracci V.
      • von Blume J.
      Fam20C regulates protein secretion by Cab45 phosphorylation.
      ). This phosphorylation regulates Cab45 oligomerization independent of its Ca2+ binding ability and facilitates translocation of Cab45 into trans Golgi network-derived vesicles, thus accelerating vesicle budding (
      • Hecht T.K.
      • Blank B.
      • Steger M.
      • Lopez V.
      • Beck G.
      • Ramazanov B.
      • Mann M.
      • Tagliabracci V.
      • von Blume J.
      Fam20C regulates protein secretion by Cab45 phosphorylation.
      ). Furthermore, the Cab45 phosphorylation enhances secretion of its client proteins, including lysozyme C (
      • Hecht T.K.
      • Blank B.
      • Steger M.
      • Lopez V.
      • Beck G.
      • Ramazanov B.
      • Mann M.
      • Tagliabracci V.
      • von Blume J.
      Fam20C regulates protein secretion by Cab45 phosphorylation.
      ). Similarly, Fam20C phosphorylation has been shown to be important for the secretion of osteopontin (
      • Tibaldi E.
      • Brocca A.
      • Sticca A.
      • Gola E.
      • Pizzi M.
      • Bordin L.
      • Pagano M.A.
      • Mazzorana M.
      • Donà G.
      • Violi P.
      • Marin O.
      • Romano A.
      • Angeli P.
      • Carraro A.
      • Brunati A.M.
      Fam20C-mediated phosphorylation of osteopontin is critical for its secretion but dispensable for its action as a cytokine in the activation of hepatic stellate cells in liver fibrogenesis.
      ).

      Fam20C substrates in blood

      Phosphoproteomic analyses of plasma and serum revealed that the majority of phosphorylated sites identified adhered to the SxE/pS motif (
      • Carrascal M.
      • Gay M.
      • Ovelleiro D.
      • Casas V.
      • Gelpí E.
      • Abian J.
      Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.
      ,
      • Zhou W.
      • Ross M.M.
      • Tessitore A.
      • Ornstein D.
      • Vanmeter A.
      • Liotta L.A.
      • Petricoin 3rd, E.F.
      An initial characterization of the serum phosphoproteome.
      ), thus triggering the hypothesis that the majority of the extracellular plasma/serum phosphoproteins could be Fam20C substrates. Multiple proteins with well-established roles in blood coagulation, phosphate homeostasis, and complement pathways have been identified in phosphoproteomic studies by comparing the phosphoproteome of wild-type cells with cells lacking Fam20C (
      • Tagliabracci V.S.
      • Wiley S.E.
      • Guo X.
      • Kinch L.N.
      • Durrant E.
      • Wen J.
      • Xiao J.
      • Cui J.
      • Nguyen K.B.
      • Engel J.L.
      • Coon J.J.
      • Grishin N.
      • Pinna L.A.
      • Pagliarini D.J.
      • Dixon J.E.
      A single kinase generates the majority of the secreted phosphoproteome.
      ,
      • Tagliabracci V.S.
      • Engel J.L.
      • Wiley S.E.
      • Xiao J.
      • Gonzalez D.J.
      • Appaiah H.N.
      • Koller A.
      • Nizet V.
      • White K.E.
      • Dixon J.E.
      Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis.
      ) (Fig. 5). The major vertebrate clotting factor fibrinogen (alpha and gamma chains) was identified as a potential substrate of Fam20C in these phosphoproteomic screens (
      • Tagliabracci V.S.
      • Wiley S.E.
      • Guo X.
      • Kinch L.N.
      • Durrant E.
      • Wen J.
      • Xiao J.
      • Cui J.
      • Nguyen K.B.
      • Engel J.L.
      • Coon J.J.
      • Grishin N.
      • Pinna L.A.
      • Pagliarini D.J.
      • Dixon J.E.
      A single kinase generates the majority of the secreted phosphoproteome.
      ). Phosphorus was found in fibrinogen as early as 1962 and the amino acid sequence revealed the sites to be SxE (
      • Blombaeck B.
      • Blombaeck M.
      • Edman P.
      • Hessel B.
      Amino-acid sequence and the occurrence of phosphorus in human fibrinopeptides.
      ). During tissue and vascular injury, fibrinogen is cleaved by thrombin to fibrin peptides, which form a fibrin-based blood clot and stop bleeding (
      • de Maat M.
      Regulation and Modulation of the Plasma Fibrinogen Level.
      ). It has been reported that phosphorylated fibrinogen binds better to thrombin, thus releasing more fibrin peptides and promoting faster coagulation (
      • Hanna L.S.
      • Scheraga H.A.
      • Francis C.W.
      • Marder V.J.
      Comparison of structures of various human fibrinogens and a derivative thereof by a study of the kinetics of release of fibrinopeptides.
      ,
      • Regañón E.
      • Vila V.
      • Aznar J.
      • Laiz B.
      Human fibrinogen heterogeneity. A study of limited fibrinogen degradation.
      ). Fam20C has been found to directly phosphorylate fibrinogen alpha and gamma chains in vitro (
      • Tagliabracci V.S.
      • Wiley S.E.
      • Guo X.
      • Kinch L.N.
      • Durrant E.
      • Wen J.
      • Xiao J.
      • Cui J.
      • Nguyen K.B.
      • Engel J.L.
      • Coon J.J.
      • Grishin N.
      • Pinna L.A.
      • Pagliarini D.J.
      • Dixon J.E.
      A single kinase generates the majority of the secreted phosphoproteome.
      ), and further work is needed to define the physiological roles of the phosphorylation events. On a similar note, Fam20C phosphorylates the A2 domain of von Willebrand factor (vWF) on two SxE sites, pSer1517 and pSer1613 (
      • Da Q.
      • Han H.
      • Valladolid C.
      • Fernández M.
      • Khatlani T.
      • Pradhan S.
      • Nolasco J.
      • Matsunami R.K.
      • Engler D.A.
      • Cruz M.A.
      • Vijayan K.V.
      In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion.
      ). The modifications promote platelet adhesion to sites of vascular injury and helps in coagulation (
      • Da Q.
      • Han H.
      • Valladolid C.
      • Fernández M.
      • Khatlani T.
      • Pradhan S.
      • Nolasco J.
      • Matsunami R.K.
      • Engler D.A.
      • Cruz M.A.
      • Vijayan K.V.
      In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion.
      ). Among the other serum/plasma proteins identified as Fam20C substrates are collagen and the complement components C3 and C4 (
      • Tagliabracci V.S.
      • Wiley S.E.
      • Guo X.
      • Kinch L.N.
      • Durrant E.
      • Wen J.
      • Xiao J.
      • Cui J.
      • Nguyen K.B.
      • Engel J.L.
      • Coon J.J.
      • Grishin N.
      • Pinna L.A.
      • Pagliarini D.J.
      • Dixon J.E.
      A single kinase generates the majority of the secreted phosphoproteome.
      ) wherein collagen and C3 have been reported to be phosphorylated previously (
      • Qiu Y.
      • Poppleton E.
      • Mekkat A.
      • Yu H.
      • Banerjee S.
      • Wiley S.E.
      • Dixon J.E.
      • Kaplan D.L.
      • Lin Y.S.
      • Brodsky B.
      Enzymatic phosphorylation of ser in a type I collagen peptide.
      ,
      • Nilsson Ekdahl K.
      • Nilsson B.
      Phosphorylation of complement component C3 after synthesis in U937 cells by a putative protein kinase, casein kinase 2, which is regulated by CD11b: Evidence that membrane-bound proteases preferentially cleave phosphorylated C3.
      ). Further work is needed to establish the role of Fam20C and phosphorylation of its key substrates in the blood coagulation pathway.
      Another well-characterized substrate of Fam20C in serum is fibroblast growth factor-23 (FGF23), a bone-derived hormone that regulates serum phosphate levels (
      • Tagliabracci V.S.
      • Engel J.L.
      • Wiley S.E.
      • Xiao J.
      • Gonzalez D.J.
      • Appaiah H.N.
      • Koller A.
      • Nizet V.
      • White K.E.
      • Dixon J.E.
      Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis.
      ,
      • Bhattacharyya N.
      • Chong W.H.
      • Gafni R.I.
      • Collins M.T.
      Fibroblast growth factor 23: State of the field and future directions.
      ). Mice with Fam20C deletion exhibit an increase in bioactive serum FGF23 leading to the development of hypophosphatemic rickets and skeletal defects (
      • Vogel P.
      • Hansen G.M.
      • Read R.W.
      • Vance R.B.
      • Thiel M.
      • Liu J.
      • Wronski T.J.
      • Smith D.D.
      • Jeter-Jones S.
      • Brommage R.
      Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice.
      ,
      • Wang X.
      • Wang S.
      • Li C.
      • Gao T.
      • Liu Y.
      • Rangiani A.
      • Sun Y.
      • Hao J.
      • George A.
      • Lu Y.
      • Groppe J.
      • Yuan B.
      • Feng J.Q.
      • Qin C.
      Inactivation of a novel FGF23 regulator, FAM20C, leads to hypophosphatemic rickets in mice.
      ), which can be partially reversed by feeding the mice a high-phosphate-containing diet (
      • Zhang H.
      • Li L.
      • Kesterke M.J.
      • Lu Y.
      • Qin C.
      High-phosphate diet improved the skeletal development of Fam20c-deficient mice.
      ). In fact, within the Golgi, Fam20C phosphorylates FGF23 on Ser180 (SxE site), which inhibits its O-glycosylation and subsequently promotes proteolysis and inactivation of the hormone (
      • Tagliabracci V.S.
      • Engel J.L.
      • Wiley S.E.
      • Xiao J.
      • Gonzalez D.J.
      • Appaiah H.N.
      • Koller A.
      • Nizet V.
      • White K.E.
      • Dixon J.E.
      Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis.
      ). Intriguingly, proteolysis-resistant missense alterations adjacent to Ser180 (R176Q, R179W, and R179Q) activate FGF23 leading to hypophosphatemic rickets (
      • Consortium A.
      Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23.
      ). Furthermore, knockdown of Fam20C in cells promotes FGF23 mRNA expression (
      • Kinoshita Y.
      • Hori M.
      • Taguchi M.
      • Fukumoto S.
      Functional analysis of mutant FAM20C in Raine syndrome with FGF23-related hypophosphatemia.
      ), and elevated levels of serum FGF23 contribute to cardiovascular complications and increased mortality in patients with chronic kidney disease (
      • Coresh J.
      • Selvin E.
      • Stevens L.A.
      • Manzi J.
      • Kusek J.W.
      • Eggers P.
      • Van Lente F.
      • Levey A.S.
      Prevalence of chronic kidney disease in the United States.
      ).

      Fam20C substrates in heart

      Besides FGF23, which contributes directly to cardiovascular problems in patients, various other substrates of Fam20C have been implicated in heart disease (Fig. 5). PCSK9 (proprotein convertase subtilisin-kexin 9) patient genetic variations altering SxE sites correlate with LDL-cholesterol dysregulation, a risk factor for heart disease (
      • Ben Djoudi Ouadda A.
      • Gauthier M.S.
      • Susan-Resiga D.
      • Girard E.
      • Essalmani R.
      • Black M.
      • Marcinkiewicz J.
      • Forget D.
      • Hamelin J.
      • Evagelidis A.
      • Ly K.
      • Day R.
      • Galarneau L.
      • Corbin F.
      • Coulombe B.
      • et al.
      Ser-phosphorylation of PCSK9 (proprotein convertase subtilisin-kexin 9) by Fam20C (family with sequence similarity 20, member C) kinase enhances its ability to degrade the LDLR (low-density lipoprotein receptor).
      ). Importantly, Fam20C-mediated phosphorylation of PCSK9 improves PCSK9 secretion and enhances the degradation of the low-density lipoprotein receptor (LDLR) in endosomes/lysosomes (
      • Ben Djoudi Ouadda A.
      • Gauthier M.S.
      • Susan-Resiga D.
      • Girard E.
      • Essalmani R.
      • Black M.
      • Marcinkiewicz J.
      • Forget D.
      • Hamelin J.
      • Evagelidis A.
      • Ly K.
      • Day R.
      • Galarneau L.
      • Corbin F.
      • Coulombe B.
      • et al.
      Ser-phosphorylation of PCSK9 (proprotein convertase subtilisin-kexin 9) by Fam20C (family with sequence similarity 20, member C) kinase enhances its ability to degrade the LDLR (low-density lipoprotein receptor).
      ). On a similar note, PCSK7 is phosphorylated by Fam20C on Ser505 (SxE site) leading to higher triglyceride uptake into adipocytes (
      • Ashraf Y.
      • Duval S.
      • Sachan V.
      • Essalmani R.
      • Susan-Resiga D.
      • Roubtsova A.
      • Hamelin J.
      • Gerhardy S.
      • Kirchhofer D.
      • Tagliabracci V.S.
      • Prat A.
      • Kiss R.S.
      • Seidah N.G.
      Proprotein convertase 7 (PCSK7) reduces apoA-V levels.
      ). Interestingly, exome sequencing revealed a low frequency coding variant PCSK7, R504H, correlated with 30% lower plasma triglyceride levels in individuals harboring this change (
      • Ashraf Y.
      • Duval S.
      • Sachan V.
      • Essalmani R.
      • Susan-Resiga D.
      • Roubtsova A.
      • Hamelin J.
      • Gerhardy S.
      • Kirchhofer D.
      • Tagliabracci V.S.
      • Prat A.
      • Kiss R.S.
      • Seidah N.G.
      Proprotein convertase 7 (PCSK7) reduces apoA-V levels.
      ). Further biochemical analyses revealed that the R504H substitution enhanced phosphorylation of the adjacent S505 possibly promoting higher triglyceride uptake (
      • Ashraf Y.
      • Duval S.
      • Sachan V.
      • Essalmani R.
      • Susan-Resiga D.
      • Roubtsova A.
      • Hamelin J.
      • Gerhardy S.
      • Kirchhofer D.
      • Tagliabracci V.S.
      • Prat A.
      • Kiss R.S.
      • Seidah N.G.
      Proprotein convertase 7 (PCSK7) reduces apoA-V levels.
      ).
      Cardiac function, contraction and relaxation, is brought about by a complex interplay of multiple proteins and posttranslational modifications playing essential roles in regulating intracellular calcium (Ca2+) handling (
      • Bers D.M.
      Cardiac sarcoplasmic reticulum calcium leak: Basis and roles in cardiac dysfunction.
      ). The sarcoplasmic reticulum (SR) of cardiac muscle is the Ca2+ storage organelle, and Ca2+ is shuttled between the SR and cytosol via various SR resident receptors during contractions and relaxations of the heart (
      • Bers D.M.
      Cardiac excitation-contraction coupling.
      ). Fam20C resides in the SR of cardiac muscle and phosphorylates multiple major Ca2+ handling machinery proteins including histidine-rich Ca-binding protein (HRC), Stim1, calsequestrin 2, sarcalumenin, triadin, calumenin, and calreticulin (
      • Pollak A.J.
      • Haghighi K.
      • Kunduri S.
      • Arvanitis D.A.
      • Bidwell P.A.
      • Liu G.-S.
      • Singh V.P.
      • Gonzalez D.J.
      • Sanoudou D.
      • Wiley S.E.
      • Dixon J.E.
      • Kranias E.G.
      Phosphorylation of serine96 of histidine-rich calcium- binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia.
      ,
      • Pollak A.J.
      • Liu C.
      • Gudlur A.
      • Mayfield J.E.
      • Dalton N.D.
      • Gu Y.
      • Chen J.
      • Heller Brown J.
      • Hogan P.G.
      • Wiley S.E.
      • Peterson K.L.
      • Dixon J.E.
      A secretory pathway kinase regulates sarcoplasmic reticulum Ca(2+) homeostasis and protects against heart failure.
      ). These proteins play essential roles mediating SR Ca2+ storage, uptake, and release (
      • Pollak A.J.
      • Liu C.
      • Gudlur A.
      • Mayfield J.E.
      • Dalton N.D.
      • Gu Y.
      • Chen J.
      • Heller Brown J.
      • Hogan P.G.
      • Wiley S.E.
      • Peterson K.L.
      • Dixon J.E.
      A secretory pathway kinase regulates sarcoplasmic reticulum Ca(2+) homeostasis and protects against heart failure.
      ,
      • Arvanitis D.A.
      • Vafiadaki E.
      • Sanoudou D.
      • Kranias E.G.
      Histidine-rich calcium binding protein: The new regulator of sarcoplasmic reticulum calcium cycling.
      ). For example, Fam20C-mediated phosphorylation of calsequestrin 2, the major Ca2+ binding protein in the SR, dramatically alters the ability of calsequestrin 2 to oligomerize, which is critical to its function (
      • Pollak A.J.
      • Liu C.
      • Gudlur A.
      • Mayfield J.E.
      • Dalton N.D.
      • Gu Y.
      • Chen J.
      • Heller Brown J.
      • Hogan P.G.
      • Wiley S.E.
      • Peterson K.L.
      • Dixon J.E.
      A secretory pathway kinase regulates sarcoplasmic reticulum Ca(2+) homeostasis and protects against heart failure.
      ). Stim1, the luminal ER/SR Ca2+ sensor responsible for store-operated Ca2+ entry in a variety of cell types, is also dramatically regulated by Fam20C phosphorylation, providing the most compelling evidence of Fam20C-mediated Ca2+ regulation. In addition, a recently discovered Stim1-S88G substitution (within an SxE site) was found in a patient with heart disease and the substitution, which precludes Fam20C phosphorylation, was shown to alter Ca2+ signaling (
      • Pollak A.J.
      • Liu C.
      • Gudlur A.
      • Mayfield J.E.
      • Dalton N.D.
      • Gu Y.
      • Chen J.
      • Heller Brown J.
      • Hogan P.G.
      • Wiley S.E.
      • Peterson K.L.
      • Dixon J.E.
      A secretory pathway kinase regulates sarcoplasmic reticulum Ca(2+) homeostasis and protects against heart failure.
      ,
      • Harris E.
      • Burki U.
      • Marini-Bettolo C.
      • Neri M.
      • Scotton C.
      • Hudson J.
      • Bertoli M.
      • Evangelista T.
      • Vroling B.
      • Polvikoski T.
      • Roberts M.
      • Töpf A.
      • Bushby K.
      • McArthur D.
      • Lochmüller H.
      • et al.
      Complex phenotypes associated with STIM1 mutations in both coiled coil and EF-hand domains.
      ).
      Interestingly, cardiomyocyte-specific Fam20C knockout mice (cKO) exhibited signs of heart failure upon aging or induced pressure overload by transverse aortic constriction (
      • Pollak A.J.
      • Liu C.
      • Gudlur A.
      • Mayfield J.E.
      • Dalton N.D.
      • Gu Y.
      • Chen J.
      • Heller Brown J.
      • Hogan P.G.
      • Wiley S.E.
      • Peterson K.L.
      • Dixon J.E.
      A secretory pathway kinase regulates sarcoplasmic reticulum Ca(2+) homeostasis and protects against heart failure.
      ). At 9 months of age, cKO mice exhibited a significant increase in left ventricle chamber size with distinct features of heart fibrosis and dilated cardiomyopathy (
      • Pollak A.J.
      • Liu C.
      • Gudlur A.
      • Mayfield J.E.
      • Dalton N.D.
      • Gu Y.
      • Chen J.
      • Heller Brown J.
      • Hogan P.G.
      • Wiley S.E.
      • Peterson K.L.
      • Dixon J.E.
      A secretory pathway kinase regulates sarcoplasmic reticulum Ca(2+) homeostasis and protects against heart failure.
      ). The heart failure phenotype in cKO mice is thought to be brought about by dramatic SR Ca2+ handling defects since isolated cardiomyocytes from aged cKO mice exhibited severe Ca2+ cycling defects and delayed relaxation (
      • Pollak A.J.
      • Liu C.
      • Gudlur A.
      • Mayfield J.E.
      • Dalton N.D.
      • Gu Y.
      • Chen J.
      • Heller Brown J.
      • Hogan P.G.
      • Wiley S.E.
      • Peterson K.L.
      • Dixon J.E.
      A secretory pathway kinase regulates sarcoplasmic reticulum Ca(2+) homeostasis and protects against heart failure.
      ).
      Dilated cardiomyopathy (DCM) is an underlying heart defect and is associated with sudden death in over 50% of the cases (
      • Zipes D.P.
      • Wellens H.J.J.
      Sudden cardiac death.
      ). Aged cKO mice exhibit clear signs of DCM, and although multiple substrates have been reported for Fam20C in SR, HRC has been widely implicated in DCM (
      • Arvanitis D.A.
      • Vafiadaki E.
      • Sanoudou D.
      • Kranias E.G.
      Histidine-rich calcium binding protein: The new regulator of sarcoplasmic reticulum calcium cycling.
      ). HRC is an essential Ca2+ handling protein, and its depletion leads to enhanced cardiomyocyte aftercontractions upon stress (
      • Park C.S.
      • Chen S.
      • Lee H.
      • Cha H.
      • Oh J.G.
      • Hong S.
      • Han P.
      • Ginsburg K.S.
      • Jin S.
      • Park I.
      • Singh V.P.
      • Wang H.S.
      • Franzini-Armstrong C.
      • Park W.J.
      • Bers D.M.
      • et al.
      Targeted ablation of the histidine-rich Ca(2+)-binding protein (HRC) gene is associated with abnormal SR Ca(2+)-cycling and severe pathology under pressure-overload stress.
      ). Failing human hearts exhibit lower protein levels of HRC, and multiple genetic variants of HRC have been reported in human DCM cases (
      • Arvanitis D.A.
      • Vafiadaki E.
      • Sanoudou D.
      • Kranias E.G.
      Histidine-rich calcium binding protein: The new regulator of sarcoplasmic reticulum calcium cycling.
      ). Fam20C-mediated phosphorylation of HRC is thought to control Ca2+ leak and enhance SR Ca2+ transport, thereby maintaining ambient signaling (
      • Pollak A.J.
      • Haghighi K.
      • Kunduri S.
      • Arvanitis D.A.
      • Bidwell P.A.
      • Liu G.-S.
      • Singh V.P.
      • Gonzalez D.J.
      • Sanoudou D.
      • Wiley S.E.
      • Dixon J.E.
      • Kranias E.G.
      Phosphorylation of serine96 of histidine-rich calcium- binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia.
      ). The site of phosphorylation on human HRC is S96, which is a canonical SxE phosphorylation site (
      • Pollak A.J.
      • Haghighi K.
      • Kunduri S.
      • Arvanitis D.A.
      • Bidwell P.A.
      • Liu G.-S.
      • Singh V.P.
      • Gonzalez D.J.
      • Sanoudou D.
      • Wiley S.E.
      • Dixon J.E.
      • Kranias E.G.
      Phosphorylation of serine96 of histidine-rich calcium- binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia.
      ). Remarkably, S96A is a common human genetic variant of HRC, and patients with the homozygous Ala/Ala variant exhibit fourfold increased risk of lethal ventricular arrhythmias in idiopathic DCM compared with normal Ser/Ser patients and twofold increased risk when compared with heterozygous individuals (
      • Arvanitis D.A.
      • Vafiadaki E.
      • Sanoudou D.
      • Kranias E.G.
      Histidine-rich calcium binding protein: The new regulator of sarcoplasmic reticulum calcium cycling.
      ). Furthermore, preliminary genetic analysis indicates that roughly 60% of participants had at least one copy of S96A suggesting that this condition has extremely broad implications for heart disease (
      • Arvanitis D.A.
      • Vafiadaki E.
      • Sanoudou D.
      • Kranias E.G.
      Histidine-rich calcium binding protein: The new regulator of sarcoplasmic reticulum calcium cycling.
      ). The intriguing dosage-dependent manner of DCM lethality in the nonphosphorylatable S96A genetic variant of HRC suggests that pS96 HRC phosphorylation by Fam20C is likely an important molecular event in cardioprotection.

      Fam20C genetic alterations in disease

      Biallelic loss-of-function genetic alterations in the Fam20C gene lead to the development of an autosomal recessive disorder called Raine syndrome (OMIM #259775) Figure 3D (
      • Simpson M.A.
      • Hsu R.
      • Keir L.S.
      • Hao J.
      • Sivapalan G.
      • Ernst L.M.
      • Zackai E.H.
      • Al-Gazali L.I.
      • Hulskamp G.
      • Kingston H.M.
      • Prescott T.E.
      • Ion A.
      • Patton M.A.
      • Murday V.
      • George A.
      • et al.
      Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development.
      ,
      • Simpson M.A.
      • Scheuerle A.
      • Hurst J.
      • Patton M.A.
      • Stewart H.
      • Crosby A.H.
      Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia.
      ,
      • Fradin M.
      • Stoetzel C.
      • Muller J.
      • Koob M.
      • Christmann D.
      • Debry C.
      • Kohler M.
      • Isnard M.
      • Astruc D.
      • Desprez P.
      • Zorres C.
      • Flori E.
      • Dollfus H.
      • Doray B.
      Osteosclerotic bone dysplasia in siblings with a Fam20C mutation.
      ). In 1985, two infant sisters with neonatal lethality were reported to exhibit a unique, autosomal recessive case of congenital sclerosing osteomalacia with cerebral calcification (
      • Whyte M.P.
      • McAlister W.H.
      • Kim G.S.
      • Sly W.S.
      • Pierpont M.E.
      • Brown D.M.
      • Fallon M.D.
      Congenital sclerosing osteomalacia with cerebral calcification: A new, recessively inherited, syndrome which radiographically mimics carbonic anhydrase II deficiency. (Abstract).
      ). It was not until 2016 that their archival DNA was sequenced to reveal a Fam20C genetic alteration in a key conserved region (
      • Whyte M.P.
      • McAlister W.H.
      • Fallon M.D.
      • Pierpont M.E.
      • Bijanki V.N.
      • Duan S.
      • Otaify G.A.
      • Sly W.S.
      • Mumm S.
      Raine syndrome (OMIM #259775), caused by FAM20C mutation, is congenital sclerosing osteomalacia with cerebral calcification (OMIM 259660).
      ). These patients may have been arguably the first documented cases of Raine syndrome harboring genetic alterations in Fam20C. The name “Raine syndrome” was coined in 1989 when Raine and colleagues comprehensively reported this lethal osteosclerotic bone dysplasia (
      • Raine J.
      • Winter R.M.
      • Davey A.
      • Tucker S.M.
      Unknown syndrome: Microcephaly, hypoplastic nose, exophthalmos, gum hyperplasia, cleft palate, low set ears, and osteosclerosis.
      ) while links with Fam20C alterations were established by Simpson and colleagues in 2007 (
      • Simpson M.A.
      • Hsu R.
      • Keir L.S.
      • Hao J.
      • Sivapalan G.
      • Ernst L.M.
      • Zackai E.H.
      • Al-Gazali L.I.
      • Hulskamp G.
      • Kingston H.M.
      • Prescott T.E.
      • Ion A.
      • Patton M.A.
      • Murday V.
      • George A.
      • et al.
      Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development.
      ). The cases presented often exhibit neonatal-lethality with extreme skeletal deformities, ectopic calcification, and organ hypoplasia (
      • Simpson M.A.
      • Hsu R.
      • Keir L.S.
      • Hao J.
      • Sivapalan G.
      • Ernst L.M.
      • Zackai E.H.
      • Al-Gazali L.I.
      • Hulskamp G.
      • Kingston H.M.
      • Prescott T.E.
      • Ion A.
      • Patton M.A.
      • Murday V.
      • George A.
      • et al.
      Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development.
      ). Some nonlethal cases have also been reported with patients exhibiting hypophosphatemia, altered facial and skeletal features (
      • Simpson M.A.
      • Scheuerle A.
      • Hurst J.
      • Patton M.A.
      • Stewart H.
      • Crosby A.H.
      Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia.
      ). Over 40 cases of Raine syndrome have been reported worldwide and DNA sequencing revealed that all these patients carried various alterations in the Fam20C gene, which are likely the driving cause of disease (
      • Simpson M.A.
      • Hsu R.
      • Keir L.S.
      • Hao J.
      • Sivapalan G.
      • Ernst L.M.
      • Zackai E.H.
      • Al-Gazali L.I.
      • Hulskamp G.
      • Kingston H.M.
      • Prescott T.E.
      • Ion A.
      • Patton M.A.
      • Murday V.
      • George A.
      • et al.
      Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development.
      ,
      • Simpson M.A.
      • Scheuerle A.
      • Hurst J.
      • Patton M.A.
      • Stewart H.
      • Crosby A.H.
      Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia.
      ,
      • Fradin M.
      • Stoetzel C.
      • Muller J.
      • Koob M.
      • Christmann D.
      • Debry C.
      • Kohler M.
      • Isnard M.
      • Astruc D.
      • Desprez P.
      • Zorres C.
      • Flori E.
      • Dollfus H.
      • Doray B.
      Osteosclerotic bone dysplasia in siblings with a Fam20C mutation.
      ). About 25 unique alterations have been reported for Fam20C in disease, which affect stability, secretion, activity, and integrity of Fam20C protein (Fig. 3D) (
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ). Intriguingly, a direct correlation has been observed between Fam20C activity and disease lethality, wherein, complete deletion leads to neonatal lethality, whereas residual activity is sufficient to keep the individual alive beyond birth to preteen and even teenage years. Two teenagers with hypophosphatemia and rickets exhibited a compound heterozygous Fam20C genetic alteration where one copy of the Fam20C gene contained a T268M substitution (
      • Rafaelsen S.H.
      • Raeder H.
      • Fagerheim A.K.
      • Knappskog P.
      • Carpenter T.O.
      • Johansson S.
      • Bjerknes R.
      Exome sequencing reveals FAM20c mutations associated with fibroblast growth factor 23-related hypophosphatemia, dental anomalies, and ectopic calcification.
      ). Fam20C T268M purified in vitro preserved only 10% of wild-type kinase activity (
      • Cozza G.
      • Salvi M.
      • Banerjee S.
      • Tibaldi E.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins.
      ). Interestingly, FDA-approved multiple sclerosis drug and sphingosine analog, fingolimod, potently activated Fam20C in vitro (
      • Cozza G.
      • Salvi M.
      • Banerjee S.
      • Tibaldi E.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins.
      ). Fingolimod also led to higher activity of Fam20C T268M in vitro (
      • Cozza G.
      • Salvi M.
      • Banerjee S.
      • Tibaldi E.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins.
      ). This suggests that fingolimod may be utilized in partially alleviating the loss of activity of Fam20C in nonlethal Raine syndrome patient cases. Furthermore, a similar amino acid replacement Ser to Thr (S410T) in a patient exhibited very mild symptoms (
      • Sheth J.
      • Bhavsar R.
      • Gandhi A.
      • Sheth F.
      • Pancholi D.
      A case of Raine syndrome presenting with facial dysmorphy and review of literature.
      ). In fact, a canine model of nonlethal Raine syndrome has been reported exhibiting a minimally disruptive Ala to Val substitution in the Fam20C kinase domain (
      • Hytönen M.K.
      • Arumilli M.
      • Lappalainen A.K.
      • Owczarek-Lipska M.
      • Jagannathan V.
      • Hundi S.
      • Salmela E.
      • Venta P.
      • Sarkiala E.
      • Jokinen T.
      • Gorgas D.
      • Kere J.
      • Nieminen P.
      • Drögemüller C.
      • Lohi H.
      Molecular characterization of three canine models of human rare bone diseases: Caffey, van den Ende-Gupta, and Raine syndromes.
      ). Most alterations reported alter the protein sequence of Fam20C in key conserved regions, whereas large chromosomal rearrangements (
      • Simpson M.A.
      • Hsu R.
      • Keir L.S.
      • Hao J.
      • Sivapalan G.
      • Ernst L.M.
      • Zackai E.H.
      • Al-Gazali L.I.
      • Hulskamp G.
      • Kingston H.M.
      • Prescott T.E.
      • Ion A.
      • Patton M.A.
      • Murday V.
      • George A.
      • et al.
      Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development.
      ) and splice-site alterations also result in Fam20C deletions and disease manifestations (
      • Acevedo A.C.
      • Poulter J.A.
      • Alves P.G.
      • de Lima C.L.
      • Castro L.C.
      • Yamaguti P.M.
      • Paula L.M.
      • Parry D.A.
      • Logan C.V.
      • Smith C.E.
      • Johnson C.A.
      • Inglehearn C.F.
      • Mighell A.J.
      Variability of systemic and oro-dental phenotype in two families with non-lethal Raine syndrome with FAM20C mutations.
      ,
      • Eltan M.
      • Alavanda C.
      • Yavas Abali Z.
      • Ergenekon P.
      • Yalındag Ozturk N.
      • Sakar M.
      • Dagcinar A.
      • Kirkgoz T.
      • Kaygusuz S.B.
      • Gokdemir Y.
      • Elcioglu H.N.
      • Guran T.
      • Bereket A.
      • Ata P.
      • Turan S.
      A rare cause of hypophosphatemia: Raine syndrome changing clinical features with age.
      ). The reported Fam20C disease alterations in humans with the exception of splice-site mutations have been listed in Table 1 and Figure 3, D and E with corresponding information on inheritance, lethality, and effect on Fam20C protein/kinase activity.
      Table 1Fam20C genetic alterations in human disease
      Amino acidInheritanceDiseasePossible effectReference
      G153RfsCHLethal, Raine syndromeLoss of kinase domain, alters 74% of protein sequence(
      • Hernández-Zavala A.
      • Cortés-Camacho F.
      • Palma Lara I.
      • Godinez-Aguilar R.
      • Espinosa-García A.M.
      • Pérez-Durán J.
      • Villanueva-Ocampo P.
      • Ugarte-Briones C.
      • Serrano-Bello C.A.
      • Sanchez-Santiago P.
      • Bonilla-Delgado J.
      • Yañez-López M.A.
      • Victoria-Acosta G.
      • López-Ornelas A.
      • García Alonso-Themann P.
      • et al.
      Two novel FAM20C variants in A family with Raine syndrome.
      )
      S159PfsCHLethal, Raine syndromeLoss of kinase domain, alters 73% of protein sequence(
      • Hernández-Zavala A.
      • Cortés-Camacho F.
      • Palma Lara I.
      • Godinez-Aguilar R.
      • Espinosa-García A.M.
      • Pérez-Durán J.
      • Villanueva-Ocampo P.
      • Ugarte-Briones C.
      • Serrano-Bello C.A.
      • Sanchez-Santiago P.
      • Bonilla-Delgado J.
      • Yañez-López M.A.
      • Victoria-Acosta G.
      • López-Ornelas A.
      • García Alonso-Themann P.
      • et al.
      Two novel FAM20C variants in A family with Raine syndrome.
      )
      E166XHomoLethal, Raine syndromeLoss of kinase domain, removes 72% of protein sequence(
      • Mameli C.
      • Zichichi G.
      • Mahmood N.
      • Elalaoui S.C.
      • Mirza A.
      • Dharmaraj P.
      • Burrone M.
      • Cattaneo E.
      • Sheth J.
      • Gandhi A.
      • Kochar G.S.
      • Alkuraya F.S.
      • Kabra M.
      • Mercurio G.
      • Zuccotti G.
      Natural history of non-lethal Raine syndrome during childhood.
      )
      W202CfsHomoNonlethal, mild Raine syndromeDisruption of kinase domain, alters 65% of protein sequence(
      • Acevedo A.C.
      • Poulter J.A.
      • Alves P.G.
      • de Lima C.L.
      • Castro L.C.
      • Yamaguti P.M.
      • Paula L.M.
      • Parry D.A.
      • Logan C.V.
      • Smith C.E.
      • Johnson C.A.
      • Inglehearn C.F.
      • Mighell A.J.
      Variability of systemic and oro-dental phenotype in two families with non-lethal Raine syndrome with FAM20C mutations.
      )
      W226RHomoNonlethal, Amelogenesis imperfectaUnknown, Possibly important for protein folding(
      • Mameli C.
      • Zichichi G.
      • Mahmood N.
      • Elalaoui S.C.
      • Mirza A.
      • Dharmaraj P.
      • Burrone M.
      • Cattaneo E.
      • Sheth J.
      • Gandhi A.
      • Kochar G.S.
      • Alkuraya F.S.
      • Kabra M.
      • Mercurio G.
      • Zuccotti G.
      Natural history of non-lethal Raine syndrome during childhood.
      ,
      • Elalaoui S.C.
      • Al-Sheqaih N.
      • Ratbi I.
      • Urquhart J.E.
      • O'Sullivan J.
      • Bhaskar S.
      • Williams S.S.
      • Elalloussi M.
      • Lyahyai J.
      • Sbihi L.
      • Cherkaoui Jaouad I.
      • Sbihi A.
      • Newman W.G.
      • Sefiani A.
      Non lethal Raine syndrome and differential diagnosis.
      )
      I258NCHNonlethal, severe skeletal deformitiesAffects Fam20C folding and secretion(
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Simpson M.A.
      • Scheuerle A.
      • Hurst J.
      • Patton M.A.
      • Stewart H.
      • Crosby A.H.
      Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia.
      )
      T268MCHNonlethal, mild Raine syndrome, hypophosphatemia90% loss of kinase activity and reduced secretion(
      • Cozza G.
      • Salvi M.
      • Banerjee S.
      • Tibaldi E.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Pinna L.A.
      A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Rafaelsen S.H.
      • Raeder H.
      • Fagerheim A.K.
      • Knappskog P.
      • Carpenter T.O.
      • Johansson S.
      • Bjerknes R.
      Exome sequencing reveals FAM20c mutations associated with fibroblast growth factor 23-related hypophosphatemia, dental anomalies, and ectopic calcification.
      )
      G280RCHNonlethal, severe skeletal deformitiesAffects Fam20C folding and secretion(
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Simpson M.A.
      • Scheuerle A.
      • Hurst J.
      • Patton M.A.
      • Stewart H.
      • Crosby A.H.
      Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia.
      )
      F302LCHNonlethal, hypophosphatemic osteomalacia with osteosclerosisUnknown, possibly important for protein folding and/or Fam20A/C tetramer formation(
      • Rolvien T.
      • Kornak U.
      • Schinke T.
      • Amling M.
      • Oheim R.
      A novel FAM20C mutation causing hypophosphatemic osteomalacia with osteosclerosis (mild Raine syndrome) in an elderly man with spontaneous osteonecrosis of the knee.
      )
      Y305XCHNonlethal, mild Raine syndrome, hypophosphatemiaHypomorphic, removes 48% of protein sequence.(
      • Rafaelsen S.H.
      • Raeder H.
      • Fagerheim A.K.
      • Knappskog P.
      • Carpenter T.O.
      • Johansson S.
      • Bjerknes R.
      Exome sequencing reveals FAM20c mutations associated with fibroblast growth factor 23-related hypophosphatemia, dental anomalies, and ectopic calcification.
      )
      R319KCHLethal, multiple defects including Raine syndromeUnknown(
      • Boissel S.
      • Fallet-Bianco C.
      • Chitayat D.
      • Kremer V.
      • Nassif C.
      • Rypens F.
      • Delrue M.-A.
      • Dal Soglio D.
      • Oligny L.L.
      • Patey N.
      • Flori E.
      • Cloutier M.
      • Dyment D.
      • Campeau P.
      • Karalis A.
      • et al.
      Genomic study of severe fetal anomalies and discovery of GREB1L mutations in renal agenesis.
      )
      P328SHomoLethal, Raine Syndrome; Nonlethal in two siblings, severe retardation and skeletal abnormalitiesAffects Fam20C folding and secretion(
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Fradin M.
      • Stoetzel C.
      • Muller J.
      • Koob M.
      • Christmann D.
      • Debry C.
      • Kohler M.
      • Isnard M.
      • Astruc D.
      • Desprez P.
      • Zorres C.
      • Flori E.
      • Dollfus H.
      • Doray B.
      Osteosclerotic bone dysplasia in siblings with a Fam20C mutation.
      )
      M336RHomoLethal, Raine syndromeAffects N-glycosylation, protein folding, and secretion of Fam20C(
      • Hung C.Y.
      • Rodriguez M.
      • Roberts A.
      • Bauer M.
      • Mihalek I.
      • Bodamer O.
      A novel FAM20C mutation causes a rare form of neonatal lethal Raine syndrome.
      )
      G365DHetLethal, Raine syndromePossible loss of function of Fam20C(
      • Whyte M.P.
      • McAlister W.H.
      • Fallon M.D.
      • Pierpont M.E.
      • Bijanki V.N.
      • Duan S.
      • Otaify G.A.
      • Sly W.S.
      • Mumm S.
      Raine syndrome (OMIM #259775), caused by FAM20C mutation, is congenital sclerosing osteomalacia with cerebral calcification (OMIM 259660).
      )
      Y369fsCHNonlethal, mild Raine syndromeHypomorphic, alters 37% of protein sequence(
      • Mamedova E.
      • Dimitrova D.
      • Przhiyalkovskaya E.
      • Buryakina S.
      • Vasilyev E.
      • Tiulpakov A.
      • Belaya Z.
      Non-lethal raine syndrome in a middle-aged woman caused by a novel FAM20C mutation.
      )
      G379RHomoLethal, Raine SyndromeAffects Fam20C folding and secretion(
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Simpson M.A.
      • Scheuerle A.
      • Hurst J.
      • Patton M.A.
      • Stewart H.
      • Crosby A.H.
      Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia.
      )
      G379ECHLethal, Raine SyndromeAffects Fam20C folding and secretion(
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Simpson M.A.
      • Scheuerle A.
      • Hurst J.
      • Patton M.A.
      • Stewart H.
      • Crosby A.H.
      Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia.
      )
      L388RHomoLethal, Raine SyndromeAffects Fam20C folding and secretion(
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Simpson M.A.
      • Hsu R.
      • Keir L.S.
      • Hao J.
      • Sivapalan G.
      • Ernst L.M.
      • Zackai E.H.
      • Al-Gazali L.I.
      • Hulskamp G.
      • Kingston H.M.
      • Prescott T.E.
      • Ion A.
      • Patton M.A.
      • Murday V.
      • George A.
      • et al.
      Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development.
      )
      W407GHomoNonlethal, craniofacial anomalies, intracranial calcification, developmental delayUnknown, Possibly important for protein folding(
      • Tamai K.
      • Tada K.
      • Takeuchi A.
      • Nakamura M.
      • Marunaka H.
      • Washio Y.
      • Tanaka H.
      • Miya F.
      • Okamoto N.
      • Kageyama M.
      Fetal ultrasonographic findings including cerebral hyperechogenicity in a patient with non-lethal form of Raine syndrome.
      )
      R408WHomoNonlethal, mild Raine Syndrome hypophosphatemiaDiminishes Fam20C activity to 50%(
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Takeyari S.
      • Yamamoto T.
      • Kinoshita Y.
      • Fukumoto S.
      • Glorieux F.H.
      • Michigami T.
      • Hasegawa K.
      • Kitaoka T.
      • Kubota T.
      • Imanishi Y.
      • Shimotsuji T.
      • Ozono K.
      Hypophosphatemic osteomalacia and bone sclerosis caused by a novel homozygous mutation of the FAM20C gene in an elderly man with a mild variant of Raine syndrome.
      )
      R409CHomoLethal, Raine syndromeUnknown(
      • Seidahmed M.Z.
      • Alazami A.M.
      • Abdelbasit O.B.
      • Al Hussein K.
      • Miqdad A.M.
      • Abu-Sa'da O.
      • Mustafa T.
      • Bahjat S.
      • Alkuraya F.S.
      Report of a case of Raine syndrome and literature review.
      )
      S410THomoNonlethal, mild skeletal issuesMinimal structural disruption expected(
      • Sheth J.
      • Bhavsar R.
      • Gandhi A.
      • Sheth F.
      • Pancholi D.
      A case of Raine syndrome presenting with facial dysmorphy and review of literature.
      )
      D451NHomoLethal at preteen, Raine Syndrome. Nonlethal cases reportedDisrupt salt-bridge in catalytic segment and affects Fam20C secretion(
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Simpson M.A.
      • Scheuerle A.
      • Hurst J.
      • Patton M.A.
      • Stewart H.
      • Crosby A.H.
      Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia.
      ,
      • Mameli C.
      • Zichichi G.
      • Mahmood N.
      • Elalaoui S.C.
      • Mirza A.
      • Dharmaraj P.
      • Burrone M.
      • Cattaneo E.
      • Sheth J.
      • Gandhi A.
      • Kochar G.S.
      • Alkuraya F.S.
      • Kabra M.
      • Mercurio G.
      • Zuccotti G.
      Natural history of non-lethal Raine syndrome during childhood.
      )
      R459GCHNonlethal, mild Raine syndromeMutation adjacent to cation interacting Asp(
      • Mamedova E.
      • Dimitrova D.
      • Przhiyalkovskaya E.
      • Buryakina S.
      • Vasilyev E.
      • Tiulpakov A.
      • Belaya Z.
      Non-lethal raine syndrome in a middle-aged woman caused by a novel FAM20C mutation.
      )
      P496LHomoNonlethal, mild Raine symptomsLikely disruption of Fam20C activation loop(
      • Acevedo A.C.
      • Poulter J.A.
      • Alves P.G.
      • de Lima C.L.
      • Castro L.C.
      • Yamaguti P.M.
      • Paula L.M.
      • Parry D.A.
      • Logan C.V.
      • Smith C.E.
      • Johnson C.A.
      • Inglehearn C.F.
      • Mighell A.J.
      Variability of systemic and oro-dental phenotype in two families with non-lethal Raine syndrome with FAM20C mutations.
      )
      R510CCHLethal, multiple defect, Raine syndromeUnknown(
      • Boissel S.
      • Fallet-Bianco C.
      • Chitayat D.
      • Kremer V.
      • Nassif C.
      • Rypens F.
      • Delrue M.-A.
      • Dal Soglio D.
      • Oligny L.L.
      • Patey N.
      • Flori E.
      • Cloutier M.
      • Dyment D.
      • Campeau P.
      • Karalis A.
      • et al.
      Genomic study of severe fetal anomalies and discovery of GREB1L mutations in renal agenesis.
      )
      R549WHomo/CHLethal/Nonlethal, Raine SyndromeAffects Fam20C folding and secretion(
      • Tagliabracci V.S.
      • Engel J.L.
      • Wen J.
      • Wiley S.E.
      • Worby C.A.
      • Kinch L.N.
      • Xiao J.
      • Grishin N.V.
      • Dixon J.E.
      Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.
      ,
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Kim S.-A.
      • Dixon J.E.
      Crystal structure of the Golgi casein kinase.
      ,
      • Simpson M.A.
      • Hsu R.
      • Keir L.S.
      • Hao J.
      • Sivapalan G.
      • Ernst L.M.
      • Zackai E.H.
      • Al-Gazali L.I.
      • Hulskamp G.
      • Kingston H.M.
      • Prescott T.E.
      • Ion A.
      • Patton M.A.
      • Murday V.
      • George A.
      • et al.
      Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development.
      ,
      • Eltan M.
      • Alavanda C.
      • Yavas Abali Z.
      • Ergenekon P.
      • Yalındag Ozturk N.
      • Sakar M.
      • Dagcinar A.
      • Kirkgoz T.
      • Kaygusuz S.B.
      • Gokdemir Y.
      • Elcioglu H.N.
      • Guran T.
      • Bereket A.
      • Ata P.
      • Turan S.
      A rare cause of hypophosphatemia: Raine syndrome changing clinical features with age.
      )
      R558WHomoLethal, Raine SyndromeUnknown(
      • Kochar G.S.
      • Choudhary A.
      • Gadodia A.
      • Gupta N.
      • Simpson M.A.
      • Crosby A.H.
      • Kabra M.
      Raine syndrome: A clinical, radiographic and genetic investigation of a case from the Indian subcontinent.
      )
      45, XY (7;7) (p22;p22)CHLethal, Raine syndromeMicrodeletion(
      • Simpson M.A.
      • Hsu R.
      • Keir L.S.
      • Hao J.
      • Sivapalan G.
      • Ernst L.M.
      • Zackai E.H.
      • Al-Gazali L.I.
      • Hulskamp G.
      • Kingston H.M.
      • Prescott T.E.
      • Ion A.
      • Patton M.A.
      • Murday V.
      • George A.
      • et al.
      Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development.
      )
      46, XY[hg19] 7p22.3 (36480–523731)HomoLethal, Raine syndrome487 kb deletion including FAM20C(
      • Ababneh F.K.
      • AlSwaid A.
      • Youssef T.
      • Al Azzawi M.
      • Crosby A.
      • AlBalwi M.A.
      Hereditary deletion of the entire FAM20C gene in a patient with Raine syndrome.
      )
      CH, Compound heterozygous; fs, Frameshift; Het, Heterozygous; Homo, Homozygous; X, STOP/Termination.
      Clinical presentation is heterogeneous and the classifications presented here reflect the symptomology reported in the literature.

      Fam20A, the secreted pseudokinase

      Unlike Fam20C, which is ubiquitously present in all tissues, Fam20A is preferentially expressed in lactating mammary glands and in enamel and dental matrices (
      • Cui J.
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Rahdar M.
      • Dixon J.E.
      A secretory kinase complex regulates extracellular protein phosphorylation.
      ,
      • Vogel P.
      • Hansen G.M.
      • Read R.W.
      • Vance R.B.
      • Thiel M.
      • Liu J.
      • Wronski T.J.
      • Smith D.D.
      • Jeter-Jones S.
      • Brommage R.
      Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice.
      ). Fam20A forms a functional heterotetrametric complex with Fam20C (Fig. 5) and allosterically increases Fam20C activity, via heterodimerization, toward its substrates (Fig. 3, B and C) (
      • Cui J.
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Rahdar M.
      • Dixon J.E.
      A secretory kinase complex regulates extracellular protein phosphorylation.
      ,
      • Zhang H.
      • Zhu Q.
      • Cui J.
      • Wang Y.
      • Chen M.J.
      • Guo X.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Xiao J.
      Structure and evolution of the Fam20 kinases.
      ). Interestingly, formation of the heterodimer is sufficient to allosterically increase Fam20C activity both in vitro and in cells, and the unique contributions of the heterotetramer are still unknown (
      • Zhang H.
      • Zhu Q.
      • Cui J.
      • Wang Y.
      • Chen M.J.
      • Guo X.
      • Tagliabracci V.S.
      • Dixon J.E.
      • Xiao J.
      Structure and evolution of the Fam20 kinases.
      ). Fam20A is a paralog of Fam20C and is the first secreted pseudokinase identified (Fig. 4, A and B) (
      • Cui J.
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Rahdar M.
      • Dixon J.E.
      A secretory kinase complex regulates extracellular protein phosphorylation.
      ). Pseudokinases are proteins that share sequence homology with kinases but lack kinase activity either due to mutations in normally conserved amino acids that catalyze phosphoryl transfer (
      • Boudeau J.
      • Miranda-Saavedra D.
      • Barton G.J.
      • Alessi D.R.
      Emerging roles of pseudokinases.
      ) or utilize the kinase fold to transfer molecules other than phosphate (
      • Black M.H.
      • Osinski A.
      • Gradowski M.
      • Servage K.A.
      • Pawłowski K.
      • Tomchick D.R.
      • Tagliabracci V.S.
      Bacterial pseudokinase catalyzes protein polyglutamylation to inhibit the SidE-family ubiquitin ligases.
      ,
      • Sreelatha A.
      • Yee S.S.
      • Lopez V.A.
      • Park B.C.
      • Kinch L.N.
      • Pilch S.
      • Servage K.A.
      • Zhang J.
      • Jiou J.
      • Karasiewicz-Urbańska M.
      • Łobocka M.
      • Grishin N.V.
      • Orth K.
      • Kucharczyk R.
      • Pawłowski K.
      • et al.
      Protein AMPylation by an evolutionarily conserved pseudokinase.
      ). A conserved Gln residue in Fam20A replaces a Mn2+ cation coordinating Glu residue of Fam20C, which is essential for catalysis (
      • Cui J.
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Rahdar M.
      • Dixon J.E.
      A secretory kinase complex regulates extracellular protein phosphorylation.
      ). In fact, mutagenesis studies revealed that replacing the Gln to a Glu in Fam20A triggered hydrolysis of ATP and restored kinase activity (
      • Cui J.
      • Xiao J.
      • Tagliabracci V.S.
      • Wen J.
      • Rahdar M.
      • Dixon J.E.
      A secretory kinase complex regulates extracellular protein phosphorylation.
      ). In addition to the lack of an essential residue required for catalysis, Fam20A binds to ATP (Fig. 4, A and C) in a unique conformation (
      • Cui J.
      • Zhu Q.
      • Zhang H.
      • Cianfrocco M.A.
      • Leschziner A.E.
      • Dixon J.E.
      • Xiao J.
      Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.
      ). Structural studies revealed that the ribose moiety of the ATP is “upside down,” and the entire nucleotide is inverted with the phosphate groups pointing at the opposite direction (
      • Cui J.
      • Zhu Q.
      • Zhang H.
      • Cianfrocco M.A.
      • Leschziner A.E.
      • Dixon J.E.
      • Xiao J.
      Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.
      ). Hence, the γ-phosphate is positioned away from the active site and cannot be utilized for transfer. Several hydrophobic residues and hydrogen bonds in the pseudokinase pocket bind the adenine of ATP (Fig. 4, B and C) while the otherwise-hydrolyzable γ-phosphate is surrounded and stabilized by extensive salt bridge and hydrogen bonds (
      • Cui J.
      • Zhu Q.
      • Zhang H.
      • Cianfrocco M.A.
      • Leschziner A.E.
      • Dixon J.E.
      • Xiao J.
      Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.
      ). Furthermore, the “inverted” ATP-binding to Fam20A seems to prefer the absence of metal ions as biochemical studies indicated that the dissociation constant of Fam20A ATP-binding is 50-fold higher in the presence of Mn2+ cation (
      • Cui J.
      • Zhu Q.
      • Zhang H.
      • Cianfrocco M.A.
      • Leschziner A.E.
      • Dixon J.E.
      • Xiao J.
      Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.
      ). Intriguingly, ion-independent ATP-binding of Fam20A remarkably promoted the formation and structural homogeneity of the heterotetrameric Fam20A-Fam20C complex (
      • Cui J.
      • Zhu Q.
      • Zhang H.
      • Cianfrocco M.A.
      • Leschziner A.E.
      • Dixon J.E.
      • Xiao J.
      Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.
      ). Although cation-independent ATP-binding has been reported previously in other pseudokinases (
      • Boudeau J.
      • Miranda-Saavedra D.
      • Barton G.J.
      • Alessi D.R.
      Emerging roles of pseudokinases.
      ,
      • Murphy J.M.
      • Zhang Q.
      • Young S.N.
      • Reese M.L.
      • Bailey F.P.
      • Eyers P.A.
      • Ungureanu D.
      • Hammaren H.
      • Silvennoinen O.
      • Varghese L.N.
      • Chen K.
      • Tripaydonis A.
      • Jura N.
      • Fukuda K.
      • Qin J.
      • et al.
      A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.
      ,
      • Zeqiraj E.
      • Filippi B.M.
      • Deak M.
      • Alessi D.R.
      • van Aalten D.M.
      Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation.
      ), the inverted binding to ATP and the heterotetramer formation in the secretory pathway make Fam20A a unique pseudokinase. Interestingly, subtle structural differences from Fam20C redesign Fam20A’s ability to achieve kinase-independent function (
      • Cui J.
      • Zhu Q.
      • Zhang H.
      • Cianfrocco M.A.
      • Leschziner A.E.
      • Dixon J.E.
      • Xiao J.
      Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.
      ). Fam20A has a unique and highly conserved insertion in the Gly-rich loop, which triggers the formation of two unique disulfide bonds (human Fam20A: Cys209-Cys319 and Cys211-Cys323) (
      • Cui J.
      • Zhu Q.
      • Zhang H.
      • Cianfrocco M.A.
      • Leschziner A.E.
      • Dixon J.E.
      • Xiao J.
      Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.
      ), and truncation of this insertion due to aberrant RNA splicing leads to the development of tooth enamel defects called amelogenesis imperfecta in a patient (
      • Cho S.H.
      • Seymen F.
      • Lee K.E.
      • Lee S.K.
      • Kweon Y.S.
      • Kim K.J.
      • Jung S.E.
      • Song S.J.
      • Yildirim M.
      • Bayram M.
      • Tuna E.B.
      • Gencay K.
      • Kim J.W.
      Novel FAM20A mutations in hypoplastic amelogenesis imperfecta.
      ).
      Figure thumbnail gr4
      Figure 4Structure of Fam20A, the secreted pseudokinase. A, structure of Homo sapiens FAM20A (hFAM20A, PDB ID:5yh3, chain A, cyan). Boxes indicate the pseudokinase active site and ATP-binding site. N and C lobes indicated approximately. B, superimposition of Homo sapiens FAM20A (hFAM20A, PDB ID:5yh3, chain A, cyan) and C. elegans FAM20C (ceFAM20C, PDB ID:4kqb, chain A, goldenrod) active sites. Manganese coordinating residues indicated. Q258 abolishes manganese and ATP-binding. ceFAM20C ATP-binding displayed for reference. C, superimposition of Homo sapiens FAM20A (hFAM20A, PDB ID:5yh3, chain A, cyan) and C. elegans FAM20C (ceFAM20C, PDB ID:4kqb, chain A, goldenrod) bound ATP/adenosine diphosphate (ADP). hFAM20A binds ATP in an inverted fashion. D, gene diagram depicting disease mutations (del, deletion; fs, frame shift; X, STOP/termination). E, cartoon depiction of kinase indicated positions of mutated residues when resolved (mutations as red spheres, PDBID:5yh3, chain C). Residue labels color coded to indicate mutation type (red: missense mutation, orange: frameshift, pink: STOP/termination, and yellow: deletion). N and C lobes indicated approximately.
      Variations in the gene encoding Fam20A result in amelogenesis imperfecta (AI), nephrocalcinosis (NC), and ectopic calcification (EC) (
      • O'Sullivan J.
      • Bitu C.C.
      • Daly S.B.
      • Urquhart J.E.
      • Barron M.J.
      • Bhaskar S.S.
      • Martelli-Junior H.
      • dos Santos Neto P.E.
      • Mansilla M.A.
      • Murray J.C.
      • Coletta R.D.
      • Black G.C.
      • Dixon M.J.
      Whole-exome sequencing identifies FAM20A mutations as a cause of amelogenesis imperfecta and gingival hyperplasia syndrome.
      ). Similar observations were echoed from whole-body and tissue-specific genetic depletion of Fam20A in mice, which exhibited clear phenotypes of AI and dental defects (
      • Vogel P.
      • Hansen G.M.
      • Read R.W.
      • Vance R.B.
      • Thiel M.
      • Liu J.
      • Wronski T.J.
      • Smith D.D.
      • Jeter-Jones S.
      • Brommage R.
      Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice.
      ,
      • Li L.L.
      • Liu P.H.
      • Xie X.H.
      • Ma S.
      • Liu C.
      • Chen L.
      • Qin C.L.
      Loss of epithelial FAM20A in mice causes amelogenesis imperfecta, tooth eruption delay and gingival overgrowth.
      ). An exhaustive list of Fam20A patient variations with corresponding clinical information has been reported by Nitayavardhana and colleagues in 2020 (
      • Nitayavardhana I.
      • Theerapanon T.
      • Srichomthong C.
      • Piwluang S.
      • Wichadakul D.
      • Porntaveetus T.
      • Shotelersuk V.
      Four novel mutations of FAM20A in amelogenesis imperfecta type IG and review of literature for its genotype and phenotype spectra.
      ). To date, about 40 different disease-causing genetic alterations have been reported in Fam20A in 70 patients of 50 independent families (Fig. 4D) (
      • Nitayavardhana I.
      • Theerapanon T.
      • Srichomthong C.
      • Piwluang S.
      • Wichadakul D.
      • Porntaveetus T.
      • Shotelersuk V.
      Four novel mutations of FAM20A in amelogenesis imperfecta type IG and review of literature for its genotype and phenotype spectra.
      ). The patients exhibited nonlethal dental symptoms including hypoplastic enamel, gingival hyperplasia, and unerupted permanent teeth (
      • Nitayavardhana I.
      • Theerapanon T.
      • Srichomthong C.
      • Piwluang S.
      • Wichadakul D.
      • Porntaveetus T.
      • Shotelersuk V.
      Four novel mutations of FAM20A in amelogenesis imperfecta type IG and review of literature for its genotype and phenotype spectra.
      ). The majority of the alterations were frameshifts with increased chances of hypomorphism, truncation, deletion, complete loss of function, major structural effects with possible dissociation from the Fam20A–Fam20C complex. The alterations are listed in Table 2 and Figure 4, D and E.
      Table 2Fam20A genetic alterations in human disease
      Amino acidInheritanceDiseasePossible effectRef
      L12AfsHomo; CHAIDeletion/Hypomorphic(
      • Cho S.H.
      • Seymen F.
      • Lee K.E.
      • Lee S.K.
      • Kweon Y.S.
      • Kim K.J.
      • Jung S.E.
      • Song S.J.
      • Yildirim M.
      • Bayram M.
      • Tuna E.B.
      • Gencay K.
      • Kim J.W.
      Novel FAM20A mutations in hypoplastic amelogenesis imperfecta.
      ,
      • Cherkaoui Jaouad I.
      • El Alloussi M.
      • Chafai El Alaoui S.
      • Laarabi F.Z.
      • Lyahyai J.
      • Sefiani A.
      Further evidence for causal FAM20A mutations and first case of amelogenesis imperfecta and gingival hyperplasia syndrome in Morocco: A case report.
      ,
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      ,
      • Kantaputra P.N.
      • Bongkochwilawan C.
      • Kaewgahya M.
      • Ohazama A.
      • Kayserili H.
      • Erdem A.P.
      • Aktoren O.
      • Guven Y.
      Enamel-renal-gingival syndrome, hypodontia, and a novel FAM20A mutation.
      )
      C44AfsCHAI, NCDeletion/Hypomorphic(
      • Wang Y.P.
      • Lin H.Y.
      • Zhong W.L.
      • Simmer J.P.
      • Wang S.K.
      Transcriptome analysis of gingival tissues of enamel-renal syndrome.
      )
      A59PfsHomoAI29 bp duplication/Hypomorphic(
      • Cabral R.M.
      • Kurban M.
      • Rothman L.
      • Wajid M.
      • Shimomura Y.
      • Petukhova L.
      • Christiano A.M.
      Autosomal recessive gingival hyperplasia and dental anomalies caused by a 29-base pair duplication in the FAM20A gene.
      )
      R73XCHAI, NCDeletion/Hypomorphic, removes 87% of protein sequence(
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      )
      L117CfsHomoAI, EC, NCNonfunctional(
      • Kantaputra P.N.
      • Bongkochwilawan C.
      • Lubinsky M.
      • Pata S.
      • Kaewgahya M.
      • Tong H.J.
      • Ketudat Cairns J.R.
      • Guven Y.
      • Chaisrisookumporn N.
      Periodontal disease and FAM20A mutations.
      ,
      • Kantaputra P.N.
      • Kaewgahya M.
      • Khemaleelakul U.
      • Dejkhamron P.
      • Sutthimethakorn S.
      • Thongboonkerd V.
      • Iamaroon A.
      Enamel-renal-gingival syndrome and FAM20A mutations.
      )
      R136XHomoAI, NCInterfere with Fam20A–Fam20C dimer/tetramer formation, removes 75% of protein sequence(
      • O'Sullivan J.
      • Bitu C.C.
      • Daly S.B.
      • Urquhart J.E.
      • Barron M.J.
      • Bhaskar S.S.
      • Martelli-Junior H.
      • dos Santos Neto P.E.
      • Mansilla M.A.
      • Murray J.C.
      • Coletta R.D.
      • Black G.C.
      • Dixon M.J.
      Whole-exome sequencing identifies FAM20A mutations as a cause of amelogenesis imperfecta and gingival hyperplasia syndrome.
      ,
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      ,
      • Wang S.K.
      • Aref P.
      • Hu Y.
      • Milkovich R.N.
      • Simmer J.P.
      • El-Khateeb M.
      • Daggag H.
      • Baqain Z.H.
      • Hu J.C.
      FAM20A mutations can cause enamel-renal syndrome (ERS).
      ,
      • Pego S.P.B.
      • Coletta R.D.
      • Dumitriu S.
      • Iancu D.
      • Albanyan S.
      • Kleta R.
      • Auricchio M.T.
      • Santos L.A.
      • Rocha B.
      • Martelli-Junior H.
      Enamel-renal syndrome in 2 patients with a mutation in FAM20 A and atypical hypertrichosis and hearing loss phenotypes.
      )
      L173RHomoAI, NCImpaired folding, L173 participates in hydrophobic interactions(
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      )
      D197_I214delinsVCHAIFam20C interface, disulfide disruption, reduced secretion and activity(
      • Cho S.H.
      • Seymen F.
      • Lee K.E.
      • Lee S.K.
      • Kweon Y.S.
      • Kim K.J.
      • Jung S.E.
      • Song S.J.
      • Yildirim M.
      • Bayram M.
      • Tuna E.B.
      • Gencay K.
      • Kim J.W.
      Novel FAM20A mutations in hypoplastic amelogenesis imperfecta.
      )
      L205CfsCHAIHypomorphic, alters 62% of protein sequence(
      • Nitayavardhana I.
      • Theerapanon T.
      • Srichomthong C.
      • Piwluang S.
      • Wichadakul D.
      • Porntaveetus T.
      • Shotelersuk V.
      Four novel mutations of FAM20A in amelogenesis imperfecta type IG and review of literature for its genotype and phenotype spectra.
      ,
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      )
      I214NfsCHAI, NCDestabilization, interferes with Fam20A–Fam20C dimer/tetramer formation, alters 60% of protein sequence(
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      )
      Q241-R271delHomoAI, NCInterferes with Fam20A–Fam20C dimer/tetramer formation, destabilization(
      • Wang S.K.
      • Aref P.
      • Hu Y.
      • Milkovich R.N.
      • Simmer J.P.
      • El-Khateeb M.
      • Daggag H.
      • Baqain Z.H.
      • Hu J.C.
      FAM20A mutations can cause enamel-renal syndrome (ERS).
      )
      R243XCHAI, NCDestabilization, R243 participates in polar contacts, removes 45% of protein sequence(
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      )
      E245GfsCHAI, NCInterferes with Fam20A–Fam20C dimer/tetramer formation, alters 55% of protein sequence(
      • Wang Y.P.
      • Lin H.Y.
      • Zhong W.L.
      • Simmer J.P.
      • Wang S.K.
      Transcriptome analysis of gingival tissues of enamel-renal syndrome.
      )
      F252delCHAI, NCInterferes with Fam20A–Fam20C dimer/tetramer formation(
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      )
      Y253CCHAIDestabilization, Y253 participates in polar contacts, interferes with Fam20A–Fam20C dimer/tetramer formation(
      • Nitayavardhana I.
      • Theerapanon T.
      • Srichomthong C.
      • Piwluang S.
      • Wichadakul D.
      • Porntaveetus T.
      • Shotelersuk V.
      Four novel mutations of FAM20A in amelogenesis imperfecta type IG and review of literature for its genotype and phenotype spectra.
      )
      R271SfsHomoAIDestabilization, alters 50% of protein sequence(
      • Cho S.H.
      • Seymen F.
      • Lee K.E.
      • Lee S.K.
      • Kweon Y.S.
      • Kim K.J.
      • Jung S.E.
      • Song S.J.
      • Yildirim M.
      • Bayram M.
      • Tuna E.B.
      • Gencay K.
      • Kim J.W.
      Novel FAM20A mutations in hypoplastic amelogenesis imperfecta.
      )
      R276XCHAILoss of “kinase” domain, removes 49% of protein sequence(
      • Cho S.H.
      • Seymen F.
      • Lee K.E.
      • Lee S.K.
      • Kweon Y.S.
      • Kim K.J.
      • Jung S.E.
      • Song S.J.
      • Yildirim M.
      • Bayram M.
      • Tuna E.B.
      • Gencay K.
      • Kim J.W.
      Novel FAM20A mutations in hypoplastic amelogenesis imperfecta.
      )
      S303CfsHomoAI, NCDestabilization, alters 44% of protein sequence(
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      )
      F305LfsCH; HomoAI, NCInterferes with Fam20A–Fam20C dimer/tetramer formation(
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      ,
      • Kantaputra P.N.
      • Bongkochwilawan C.
      • Lubinsky M.
      • Pata S.
      • Kaewgahya M.
      • Tong H.J.
      • Ketudat Cairns J.R.
      • Guven Y.
      • Chaisrisookumporn N.
      Periodontal disease and FAM20A mutations.
      ,
      • Kantaputra P.N.
      • Kaewgahya M.
      • Khemaleelakul U.
      • Dejkhamron P.
      • Sutthimethakorn S.
      • Thongboonkerd V.
      • Iamaroon A.
      Enamel-renal-gingival syndrome and FAM20A mutations.
      ,
      • Hassib N.F.
      • Shoeib M.A.
      • ElSadek H.A.
      • Wali M.E.
      • Mostafa M.I.
      • Abdel-Hamid M.S.
      Two new families with enamel renal syndrome: A novel FAM20A gene mutation and review of literature.
      )
      E326GfsCHAIDestabilization, alters 40% of protein sequence(
      • Kantaputra P.N.
      • Bongkochwilawan C.
      • Lubinsky M.
      • Pata S.
      • Kaewgahya M.
      • Tong H.J.
      • Ketudat Cairns J.R.
      • Guven Y.
      • Chaisrisookumporn N.
      Periodontal disease and FAM20A mutations.
      )
      C330RCHAIDisruption of disulfide(
      • Poulter J.A.
      • Smith C.E.
      • Murrillo G.
      • Silva S.
      • Feather S.
      • Howell M.
      • Crinnion L.
      • Bonthron D.T.
      • Carr I.M.
      • Watson C.M.
      • Inglehearn C.F.
      • Mighell A.J.
      A distinctive oral phenotype points to FAM20A mutations not identified by Sanger sequencing.
      )
      G331DHomoAIDestabilization, introduces steric clash(
      • Wang S.K.
      • Aref P.
      • Hu Y.
      • Milkovich R.N.
      • Simmer J.P.
      • El-Khateeb M.
      • Daggag H.
      • Baqain Z.H.
      • Hu J.C.
      FAM20A mutations can cause enamel-renal syndrome (ERS).
      )
      R361CCHAIDestabilization, R361 participates in polar contacts, interferes with Fam20A–Fam20C dimer/tetramer formation(
      • Nitayavardhana I.
      • Theerapanon T.
      • Srichomthong C.
      • Piwluang S.
      • Wichadakul D.
      • Porntaveetus T.
      • Shotelersuk V.
      Four novel mutations of FAM20A in amelogenesis imperfecta type IG and review of literature for its genotype and phenotype spectra.
      )
      R392PfsHomoAI, NCDestabilizing, alters 28% of protein sequence(
      • Cho S.H.
      • Seymen F.
      • Lee K.E.
      • Lee S.K.
      • Kweon Y.S.
      • Kim K.J.
      • Jung S.E.
      • Song S.J.
      • Yildirim M.
      • Bayram M.
      • Tuna E.B.
      • Gencay K.
      • Kim J.W.
      Novel FAM20A mutations in hypoplastic amelogenesis imperfecta.
      ,
      • Koruyucu M.
      • Seymen F.
      • Gencay G.
      • Gencay K.
      • Tuna E.B.
      • Shin T.J.
      • Hyun H.K.
      • Kim Y.J.
      • Kim J.W.
      Nephrocalcinosis in amelogenesis imperfecta caused by the FAM20A mutation.
      )
      D403NCHAIImpaired folding, disrupts multiple polar contacts(
      • Wang S.K.
      • Reid B.M.
      • Dugan S.L.
      • Roggenbuck J.A.
      • Read L.
      • Aref P.
      • Taheri A.P.
      • Yeganeh M.Z.
      • Simmer J.P.
      • Hu J.C.
      FAM20A mutations associated with enamel renal syndrome.
      )
      D410PfsCHAIDestabilization, D410 participates in polar contacts, alters 24% of protein sequence(
      • Jaureguiberry G.
      • De la Dure-Molla M.
      • Parry D.
      • Quentric M.
      • Himmerkus N.
      • Koike T.
      • Poulter J.
      • Klootwijk E.
      • Robinette S.L.
      • Howie A.J.
      • Patel V.
      • Figueres M.L.
      • Stanescu H.C.
      • Issler N.
      • Nicholson J.K.
      • et al.
      Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations.
      )
      F417VfsCHAIDestabilization, alters 23% of protein sequence(
      • Nitayavardhana I.
      • Theerapanon T.
      • Srichomthong C.
      • Piwluang S.
      • Wichadakul D.
      • Porntaveetus T.
      • Shotelersuk V.
      Four novel mutations of FAM20A in amelogenesis imperfecta type IG and review of literature for its genotype and phenotype spectra.
      )
      A432TCHAIDestabilization, larger side chain introduces steric clashes(
      • Poulter J.A.
      • Smith C.E.
      • Murrillo G.
      • Silva S.
      • Feather S.
      • Howell M.
      • Crinnion L.
      • Bonthron D.T.
      • Carr I.M.
      • Watson C.M.
      • Inglehearn C.F.
      • Mighell A.J.
      A distinctive oral phenotype points to FAM20A mutations not identified by Sanger sequencing.
      )
      G436EHomoAIInterferes with salt bridges(
      • Kantaputra P.N.
      • Bongkochwilawan C.
      • Lubinsky M.
      • Pata S.
      • Kaewgahya M.
      • Tong H.J.
      • Ketudat Cairns J.R.
      • Guven Y.
      • Chaisrisookumporn N.
      Periodontal disease and FAM20A mutations.