Family with sequence similarity member 20C is the primary but not the only kinase for the small‐integrin‐binding ligand N‐linked glycoproteins in bone

Abstract

Recent studies have identified family with sequence similarity member 20C (FAM20C) as a kinase that phosphorylates the Ser in Ser-X-Glu/phospho-Ser (pSer) motifs in the small-integrin-binding ligand N-linked glycoproteins (SIBLINGs). There is no in vivo evidence that validates this finding, and it is unclear whether FAM20C is the only kinase for SIBLINGs. We extracted bone noncollagenous proteins (NCPs) from Fam20C-knockout (KO) mice and analyzed the phosphorylation levels. The total NCPs were separated into osteopontin-, bone sialoprotein-, and dentin matrix protein-1-enriched fractions by anion-exchange chromatography and analyzed by SDS-PAGE, native PAGE, and Western immunoblot analysis. The NCP phosphorylation level in the KO mice was lower than that in the wild-type (WT). On the native gel, the SIBLINGs from KO mice showed a lower migration rate (M r ) than those from the WT. Calf intestine phosphatase treatment shifted SIBLINGs from the WT mice to the level adjacent to the KO, but failed to shift the latter, suggesting a phosphorylation loss of SIBLINGs in the KO mice. Mass spectrometry identified less pSers in the SIBLINGs from the KO mice [including the region of the acidic Ser-and aspartate-rich motif (ASARM) peptides]. In an intriguing finding, several pSers in the Ser-X-Glu motifs in the KO mice maintained their phosphorylation, whereas several others in non-Ser-X-Glu motifs did not. Phospho-Tyrs and phospho-Thrs in the SIBLINGs did not appear to be associated with FAM20C. Our results indicate that FAM20C is the primary, but not the only, kinase for the SIBLINGs.-Yang, X., Yan, W., Tian, Y., Ma, P., Opperman, L. A., Wang, X. Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone. FASEB J. 30, 121-128 (2016 The organic phase of the extracellular matrix (ECM) in the bone is mainly composed of type I collagen and noncollagenous proteins (NCPs) synthesized and secreted by osteoblasts. A prominent category of NCPs in bone ECM is termed the small-integrin-binding ligand N-linked glycoprotein (SIBLING) family, which includes osteopontin (OPN), bone sialoprotein (BSP), and dentin matrix protein (DMP)-1. These polyanionic proteins play pivotal roles in osteogenesis and biomineralization, and their biologic functions are closely related to posttranslational modifications, such as phosphorylation (1-7).OPN is commonly recognized as an effective inhibitor of hydroxyapatite formation and growth (8). Rat bone OPN contains 12 phospho-Sers (pSers) and 1 phospho-Thr, and has considerable heterogeneity in degree of phosphorylation (9). In contrast, bovine milk OPN has 27 pSers and 1 phospho-Thr and appears to be maximally phosphorylated (10). The extent of phosphorylation of OPN, as well as of the specific sites, may play an important role in its physiologic function. The activity of OPN in regulating biomineralization is lost after the removal of the phosphates (2, 11). BSP in bone has been reported to have 11 phosp...

Specific ablation of mouse Fam20C in cells expressing type I collagen leads to skeletal defects and hypophosphatemia

“…These Sox2-Cre;Fam20C flox/flox mice also showed enamel and dentin defects 14 . The phosphorylation levels of non-collagenous proteins including DMP1 and OPN in the bone and dentin of Sox2-Cre ; Fam20C flox/flox mice were lower than in the normal mice 6 .…”

“…DMP1 is a substrate of FAM20C 2, 6 . Without FAM20C, DMP1 cannot be properly phosphorylated, which may lead to the loss of its functions.…”

Osteopontin -- a promising biomarker for cancer therapy

“…The five proteins do, however, share some structural similarities, such as the integrin-binding tripeptide Arg-Gly-Asp (RGD), several post-translational phosphorylation and N-glycosylation sites 8 . Traditionally, the SIBLING families are classified by exon-intron similarities and their biochemical properties in osteogenesis and biomineralization 9 . However, previous research suggested that SIBLING proteins expression is not only observed in mineralized tissues, but it is also observed in different tumor stages which are coupled with Matrix Metalloproteinases (MMPs).…”

FAM20C‐Mediated Phosphorylation of MEPE and Its Acidic Serine‐ and Aspartate‐Rich Motif

“…In FAM20C KO mice, OPN has been shown to be still phosphorylated at FAM20C recognition motifs. (31) This indicates that other kinases apart from FAM20C can phosphorylate the SIBLING proteins. In the present study, 22 phosphorylated residues were identified by LC-MS/MS in MEPE (not including the ASARM sequence) after coexpression with FAM20C, whereas only a single phosphoserine residue was identified when MEPE was expressed alone.…”