Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five O-glycosylation sites and their biological implications

Christensen, Brian; Nielsen, Mette S.; Haselmann, Kim F.; Petersen, Torben E.; Sørensen, Esben S.

doi:10.1042/bj20050341

Cited by 207 publications

(220 citation statements)

References 47 publications

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“…Other studies have identified several phospho-Thrs in the SIBLINGs, with phosphorylation that is not governed by FAM20C (3,28,39). We confirmed the existence of some of these phospho-Thrs in the Fam20C-KO mice.…”

Section: Discussionsupporting

confidence: 77%

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

et al. 2015

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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...

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Section: Discussionsupporting

confidence: 77%

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

et al. 2015

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“…For example, both bovine and human milk OPN have a large number of phosphorylated serine residues (28 and 32 respectively, mostly in motifs implicating casein kinase 2). These are located in clusters of 3-5 sites and these sites are absent from the RGD region as well as from the glycosylated region (Christensen et al, 2005;. In contrast, rat bone OPN (at least as isolated) contains only 10-11 phosphorylated residues (Keykhosravani et al, 2005).…”

Section: Post-translational Modifications (Ptm)mentioning

confidence: 99%

“…This OPN is phosphorylated at a few randomly located serine/threnine sites (out of some 30 potential sites) and presumably behaves as un-phosphorylated OPN (Christensen et al, 2005). OPN -/-mice were divided into 3 groups: (A) control group, (B) CRS group injected with PBS (C) CRS group injected with OPN in PBS.…”

Section: Administration Of Purified Opnmentioning

confidence: 99%

Osteopontin: Role in immune regulation and stress responses

Wang¹,

Denhardt²

2008

Cytokine & Growth Factor Reviews

609

558

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“…In OPN, tyrosine sulfation has been linked to tissue mineralization (Nagata et al 1989), and has been found in OPN isolated from rat bone (Keykhosravani et al 2005), mouse osteoblasts (Ecarot-Charrier et al 1989) and also from human urine (Christensen et al 2008), but not in human milk OPN (Christensen et al 2005). To our knowledge, there is at present no literature regarding the role of OPN sulfation on malignant properties of cancer cells.…”

Section: Post-translational Modifications Of Opnmentioning

confidence: 99%

Pre- and post-translational regulation of osteopontin in cancer

Anborgh¹,

Mutrie

Tuck

et al. 2011

J. Cell Commun. Signal.

105

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Osteopontin (OPN) is a matricellular protein that binds to a number of cell surface receptors including integrins and CD44. It is expressed in many tissues and secreted into body fluids including blood, milk and urine. OPN plays important physiological roles in bone remodeling, immune response and inflammation. It is also a tumour-associated protein, and elevated OPN levels are associated with tumour formation, progression and metastasis. Research has revealed a promising role for OPN as a cancer biomarker. OPN is subject to alternative splicing, as well as post-translational modifications such as phosphorylation, glycosylation and proteolytic cleavage. Functional differences have been revealed for different isoforms and post-translational modifications. The pattern of isoform expression and post-translational modification is cell-type specific and may influence the potential role of OPN in malignancy and as a cancer biomarker.

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Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five O-glycosylation sites and their biological implications

Cited by 207 publications

References 47 publications

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

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

Osteopontin: Role in immune regulation and stress responses

Pre- and post-translational regulation of osteopontin in cancer

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