EXTL2, a Member of the EXT Family of Tumor Suppressors, Controls Glycosaminoglycan Biosynthesis in a Xylose Kinase-dependent Manner

Nadanaka, Satomi; Zhou, Shuping; Kagiyama, Shoji; Shoji, Naoko; Sugahara, Kazuyuki; Sugihara, Kazushi; Asano, Masahide; Kitagawa, Hiroshi

doi:10.1074/jbc.m112.416909

Cited by 84 publications

(92 citation statements)

References 35 publications

(45 reference statements)

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“…4C). The phosphoxylose may also affect the in vitro activities of other downstream glycosyltransferases in GAG synthesis, but our results indicate its dominant effect is modulating GalT-II activity (31,32).…”

Section: Fam20b-dependent Xylose Phosphorylation Stimulates Synthesis Ofcontrasting

confidence: 38%

“…We next depleted Fam20B in MRC-5 lung fibroblast cells using lentiviral-based short hairpin RNAs and metabolically labeled the cells with [ 32 P]orthophosphate. Endogenous DCN immunoprecipitated from Fam20B knockdown cells contained a significantly decreased amount of 32 P compared with DCN imunoprecipitated from control cells (Fig. 1D).…”

Section: Significancementioning

confidence: 99%

“…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). We therefore immunopurified FLAG-tagged decorin from conditioned medium of HEK293T cells and performed an in vitro kinase reaction with recombinant Fam20B in the presence of [γ- 32 P]ATP (21). Fam20B indeed efficiently phosphorylated DCN in a time-dependent manner, whereas mutation of the conserved metal binding Asp to Ala (D309A, Fam20B-D/A) abolished its kinase activity ( Fig.…”

Section: Significancementioning

confidence: 99%

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Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis

Wen¹,

Xiao²,

Rahdar³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Most eukaryotic cells elaborate several proteoglycans critical for transmitting biochemical signals into and between cells. However, the regulation of proteoglycan biosynthesis is not completely understood. We show that the atypical secretory kinase family with sequence similarity 20, member B (Fam20B) phosphorylates the initiating xylose residue in the proteoglycan tetrasaccharide linkage region, and that this event functions as a molecular switch to regulate subsequent glycosaminoglycan assembly. Proteoglycans from FAM20B knockout cells contain a truncated tetrasaccharide linkage region consisting of a disaccharide capped with sialic acid (Siaα2-3Galβ1-4Xylβ1) that cannot be further elongated. We also show that the activity of galactosyl transferase II (GalT-II, B3GalT6), a key enzyme in the biosynthesis of the tetrasaccharide linkage region, is dramatically increased by Fam20B-dependent xylose phosphorylation. Inactivating mutations in the GALT-II gene (B3GALT6) associated with Ehlers-Danlos syndrome cause proteoglycan maturation defects similar to FAM20B deletion. Collectively, our findings suggest that GalT-II function is impaired by loss of Fam20B-dependent xylose phosphorylation and reveal a previously unappreciated mechanism for regulation of proteoglycan biosynthesis.secretory kinase | proteoglycan | xylose phosphorylation | Fam20B | GalT-II

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Section: Fam20b-dependent Xylose Phosphorylation Stimulates Synthesis Ofcontrasting

confidence: 38%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis

Wen¹,

Xiao²,

Rahdar³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…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).…”

mentioning

confidence: 99%

Crystal structure of the Golgi casein kinase

Xiao

Tagliabracci

Wen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…26 In a recent report, Nadanaka et al hypothesized that the phosphorylation of xylose by FAM20B facilitates the formation of the linkage region and thereby leads to enhanced GAG biosynthesis. 27 Our observation of xylose phosphorylation at the GAG core and intermediates suggests that recombinant CHO expression system may contain residual xylose kinase activity. Wakabayashi et al reported the attachment of sulfate at the terminal GlcA of GAG core in human urine α-thrombomodulin (αTM) that contains a typical sequence motif for syntheses of proteoglycans.…”

Section: Discussionmentioning

confidence: 99%