The ABCs of the atypical Fam20 secretory pathway kinases

Abstract

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: Golgi; biomineralization; endoplasmic reticulum (ER); enzyme mutation; enzyme structure; extracellular matrix; phosphorylation; secretion; signal transduction.

Figure 1

Fam20 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 2

Structure 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.

Figure 3

Structure 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.

Figure 4

Structure 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.

Figure 5

Roles of Fam20 secretory pathway kinases. The overarching roles of the Fam20 kinases identified to date are in nutrition, biomineralization, blood, cardiac function, proteoglycan biosynthesis, allosteric kinase activation, and endoplasmic reticulum proteostasis. Fam20 paralogs are localized in the secretory pathway and phosphorylate multiple substrates playing essential roles in animal physiology.