Crystal structure of the Golgi casein kinase

Abstract

The family with sequence similarity 20 (Fam20) kinases phosphorylate extracellular substrates and play important roles in biomineralization. Fam20C is the Golgi casein kinase that phosphorylates secretory pathway proteins within Ser-x-Glu/pSer motifs. Mutations in Fam20C cause Raine syndrome, an osteosclerotic bone dysplasia. Here we report the crystal structure of the Fam20C ortholog from Caenorhabditis elegans. The nucleotide-free and Mn/ ADP-bound structures unveil an atypical protein kinase-like fold and highlight residues critical for activity. The position of the regulatory αC helix and the lack of an activation loop indicate an architecture primed for efficient catalysis. Furthermore, several distinct elements, including the presence of disulfide bonds, suggest that the Fam20 family diverged early in the evolution of the protein kinase superfamily. Our results reinforce the structural diversity of protein kinases and have important implications for patients with disorders of biomineralization.P rotein phosphorylation is a fundamental mechanism that regulates numerous physiological processes (1, 2). Protein kinases have evolved to function as dynamic switches relaying signals in response to various stimuli by transferring a phosphate from ATP to target proteins (3-5). Most phosphoproteins are found within the cell, residing in the nuclei and cytosol; however, secreted proteins, including casein and other members of the secretory calciumbinding phosphoprotein family, are phosphorylated as well (6). We recently identified a family of kinases that reside in the secretory pathway and function to phosphorylate extracellular substrates (7,8). One member of this family, Fam20C (family with sequence similarity 20, member C), is the physiological casein kinase that phosphorylates multiple secreted proteins within a Ser-x-Glu/pSer motif (7, 9, 10). The importance of this discovery is underscored by the fact that some 75% of the phosphoproteins identified in human serum and cerebrospinal fluid contain phosphate within this motif (11-13). Furthermore, mutations in FAM20C cause Raine syndrome, a deadly osteosclerotic bone dysplasia characterized by generalized osteosclerosis, ectopic calcifications, and characteristic facial features (14-16). Most individuals with Raine syndrome die within a few weeks after birth; however, nonlethal cases with dental abnormalities and clinical features of hypophosphatemia have been reported (17,18). Loss of Fam20C in mice also results in severe bone and tooth anomalies, as well as hypophosphatemia (19)(20)(21).Two other closely related Fam20C paralogs, Fam20A and Fam20B, are present in humans (22). Fam20B is ubiquitously expressed and phosphorylates xylose within the tetrasaccharide linkage region of proteoglycans (23). This phosphorylation event may influence glycosaminoglycan biosynthesis (24). Genetic deletion of Fam20B in mice results in embryonic lethality at E13.5, and mutations in Danio rerio result in reduced cartilage matrix production and skeletal defects (19,25). The substra...

Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis

“…The Fam20B insert from the pCCF vector was also subcloned into the pQCXIP retroviral vector for generating stable cell lines. GalT-II and Fam20B ORFs were subcloned into a modified pSMBP baculovirus vector for protein production (19).…”

“…Fam20B belongs to a recently identified family of secretory pathway kinases that includes Fam20C, which phosphorylates secretory proteins within a highly conserved Ser-X-Glu/pSer motif (7,18). Although Fam20B shares nearly 60% sequence similarity with Fam20C, it does not phosphorylate proteins; rather, Fam20B has been reported to phosphorylate the xylose residue within the tetrasaccharide linkage of α-thrombomodulin (6,19). To test whether authentic PGs could serve as substrates for Fam20B, we took advantage of the fact that xylose phosphorylation on mature PGs has been reported to be substoichiometric (20).…”

Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa

“…The presence of these various VWA and chitin-binding domains suggests that these proteins might participate in structuring the chitin/protein nacre scaffold. In addition, we identified other ECM-related proteins, such as DMP-like, MSP130-like, mytilin3-like, ANF-receptor containing or Ca-binding proteins, for which homologues have previously been described from other proteins associated with biomineralization [30][31][32]. The involvement of these proteins in biomineralization processes is discussed below.…”

Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis

“…Notably, Rafaelsen et al (12) identified a novel missense mutation in Fam20C that substituted Thr 268 with a Met in two compound heterozygous brothers who had elevated serum intact FGF23 and hypophosphatemia. Threonine 268 (Thr 175 in Caenorhabditis elegans) is highly conserved within the Fam20C subfamily, and the C. elegans Fam20 crystal structure revealed that this Thr is located in the glycine-rich loop (β1-β2), a region important for nucleotide binding (35) (Fig. 5 A and B).…”