FAM20C phosphorylation of the RGDSVVYGLR motif in osteopontin inhibits interaction with the αvß3 integrin.

Osteopontin (OPN) is a ubiquitously expressed, multifunctional, and highly phosphorylated protein. OPN contains two neighboring integrin-binding motifs, RGD and SVVYGLR, which mediate interaction with cells. Phosphorylation and proteolytic processing affect the integrin-binding activities of OPN. Here we report that the kinase, FAM20C, phosphorylates Ser146 in the 143 RGDSVVYGLR152 motif of OPN and that Ser146 is phosphorylated in vivo in human and bovine milk. Ser146 is located right next to the RGD motif and close by the regulatory thrombin and plasmin cleavage sites in the OPN sequence. Phosphorylation of Ser146 could potentially affect the proteolytic processing and the integrin-binding activities of OPN. We show that phosphorylation of Ser146 does not affect the susceptibility of OPN for thrombin or plasmin cleavage. However, phosphorylation of Ser146 significantly reduces the RGD-mediated interaction with the αv ß3 integrin in MDA-MB-435 and Moαv cells. This suggests a new mechanism by which specific phosphorylation of OPN can regulate interaction with the αv ß3 integrin and thereby affect OPN-cell interaction.

Ras-transformation reduce FAM20C expression and osteopontin phosphorylation.

Family with sequence similarity 20, member C (FAM20C) is the main kinase of secreted phosphoproteins, including the multifunctional protein and cytokine, osteopontin (OPN). The phosphorylation of OPN varies greatly among cell types, tissues and species, and the different phospho-isoforms contribute to the multifunctionality of the protein. Expression of OPN is increased in human malignancies, and less phosphorylated isoforms of the protein have been associated with this phenotype. Here, we compared OPN from ras-transformed fibroblasts with that from their non-transformed parental cells, and found that OPN was less phosphorylated after ras-transformation. Furthermore, we demonstrated that expression of FAM20C mRNA was reduced five-fold in ras-transformed fibroblasts compared with non-transformed fibroblasts. Transfection with FAM20C of the ras-transformed fibroblasts restored the FAM20C mRNA expression but the phosphorylation of OPN was not increased proportionally. Likewise, the mRNA level of FAM20C was reduced in the malignant ras-transformed mammary cell line MCF10ACA1a compared with its non-transformed parental cell line MCF10A. These results suggest that expression of the FAM20C kinase is reduced after oncogenic ras-transformation, which potentially affects the phosphorylation of secreted phosphoproteins.

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

Matrix extracellular phosphoglycoprotein (MEPE) is expressed in bone and teeth where it has multiple functions. The C-terminus of MEPE contains a mineral-binding, acidic serine- and aspartate-rich motif (ASARM) that is also present in other noncollagenous proteins of mineralized tissues. MEPE-derived ASARM peptides function in phosphate homeostasis and direct inhibition of bone mineralization in a phosphorylation-dependent manner. MEPE is phosphorylated by family with sequence similarity 20, member C (FAM20C), which is the main kinase phosphorylating secreted phosphoprotein. Although the functional importance of protein phosphorylation status in mineralization processes has now been well-established for secreted bone and tooth proteins (particularly for osteopontin), the phosphorylation pattern of MEPE has not been previously determined. Here we provide evidence for a very high phosphorylation level of this protein, reporting on the localization of 31 phosphoresidues in human MEPE after coexpression with FAM20C in HEK293T cells. This includes the finding that all serine residues located in the canonical target sequence of FAM20C (Ser-x-Glu) were phosphorylated, thus establishing the major target sites for this kinase. We also show that MEPE has numerous other phosphorylation sites, these not being positioned in the canonical phosphorylation sequence. Of note, and underscoring a possible important function in mineralization biology, all nine serine residues in the ASARM were phosphorylated, even though only two of these were positioned in the Ser-x-Glu sequence. The presence of many phosphorylated amino acids in MEPE, and particularly their high density in the ASARM motif, provides an important basis for the understanding of structural and functional interdependencies in mineralization and phosphate homeostasis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.