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.