Phosphorylation of purified bovine bone sialoprotein and osteopontin by protein kinases.
The large number of covalently bound phosphates on the extracellular phosphoproteins osteopontin (OPN) and bone sialoprotein (BSP) have been implicated in biological functions such as mineral deposition and osteoclast binding. In the present study the state of phosphorylation of BSP and OPN was evaluated by in vitro 32P labeling using a series of protein kinases and quantification. Both the purified bovine BSP and OPN were radiolabeled by [32P]ATP and factor-independent protein kinase. Quantification of 32P radioactivity incorporated on dephosphorylated BSP and OPN provided 6.6 and 8.9 mol of phosphate incorporated/mol, respectively. Native OPN incorporated 1.07 and BSP 2.46 mol of phosphate/mol by factor-independent protein kinase. These data led to calculations that OPN and BSP, respectively, contain 7.83 and 4.14 mol of phosphate/mol in their natural state. Thrombin digests of 32P-labeled BSP showed radioactivity to be associated with fragment of approximately molecular mass values 30 kDa (N-terminal half), with no observable radioactivity associated with the 40-kDa fragment (C-terminal half). Similar experiments with 32P-labeled OPN provided two radiolabeled thrombin fragments, with molecular mass 30 kDa (N-terminal half) and 20 kDa (C-terminal half), both were radioactive. The major phosphorylation was associated with the N-terminal half containing 7.0 mol of phosphate, and 1.9 mol of phosphate were associated with the C-terminal half. Additional experiments of in vitro phosphorylation of OPN and BSP by several other known protein kinases were carried out. cAMP-dependent protein kinase showed no phosphorylation of OPN or BSP, while protein kinase C and cGMP-dependent protein kinase led to minor phosphorylation, each of the latter introduced about 1 mol of phosphate/mol of OPN and BSP molecule.
Salih
E
E
Department of Orthopaedic Surgery, Harvard Medical School and The Children's Hospital, Boston, Massachusetts 02115, USA.
Zhou
H Y
HY
Glimcher
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001842:Bone and Bones / Q000378:metabolism*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D058574:Integrin-Binding Sialoprotein; D008969:Molecular Sequence Data; D053495:Osteopontin; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
D058574:Integrin-Binding Sialoprotein; D010761:Phosphorus Radioisotopes; D012795:Sialoglycoproteins; D053495:Osteopontin; D011494:Protein Kinases; D013917:Thrombin