title: Phosphorylation of Ser136 is critical for potent bone sialoprotein-mediated nucleation of hydroxyapatite crystals.
abstract: Acidic phosphoproteins of mineralized tissues such as bone and dentin are believed to play important roles in HA (hydroxyapatite) nucleation and growth. BSP (bone sialoprotein) is the most potent known nucleator of HA, an activity that is thought to be dependent on phosphorylation of the protein. The present study identifies the role phosphate groups play in mineral formation. Recombinant BSP and peptides corresponding to residues 1-100 and 133-205 of the rat sequence were phosphorylated with CK2 (protein kinase CK2). Phosphorylation increased the nucleating activity of BSP and BSP-(133-205), but not BSP-(1-100). MS analysis revealed that the major site phosphorylated within BSP-(133-205) was Ser136, a site adjacent to the series of contiguous glutamate residues previously implicated in HA nucleation. The critical role of phosphorylated Ser136 in HA nucleation was confirmed by site-directed mutagenesis and functional analyses. Furthermore, peptides corresponding to the 133-148 sequence of rat BSP were synthesized with or without a phosphate group on Ser136. As expected, the phosphopeptide was a more potent nucleator. The mechanism of nucleation was investigated using molecular-dynamics simulations analysing BSP-(133-148) interacting with the {100} crystal face of HA. Both phosphorylated and non-phosphorylated sequences adsorbed to HA in extended conformations with alternating residues in contact with and facing away from the crystal face. However, this alternating-residue pattern was more pronounced when Ser136 was phosphorylated. These studies demonstrate a critical role for Ser136 phosphorylation in BSP-mediated HA nucleation and identify a unique mode of interaction between the nucleating site of the protein and the {100} face of HA.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, ON N6A5C1, Canada.
authors.forename: Gurpreet S
authors.initials: GS
authors.lastname: Baht
authors.forename: Jason
authors.initials: J
authors.lastname: O'Young
authors.forename: Antonia
authors.initials: A
authors.lastname: Borovina
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Coralee E
authors.initials: CE
authors.lastname: Tye
authors.forename: Mikko
authors.initials: M
authors.lastname: Karttunen
authors.forename: Gilles A
authors.initials: GA
authors.lastname: Lajoie
authors.forename: Graeme K
authors.initials: GK
authors.lastname: Hunter
authors.forename: Harvey A
authors.initials: HA
authors.lastname: Goldberg
mesh_terms: D000818:Animals; D001665:Binding Sites; D017886:Durapatite / Q000737:chemistry* / Q000378:metabolism; D058574:Integrin-Binding Sialoprotein; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D012795:Sialoglycoproteins; D012694:Serine; D017886:Durapatite