Abstract
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.
Highlights
Tals have received increasing attention (8 –11)
In a separate study in this laboratory using cultured chicken osteoblast cytosolic and microsomal enzyme preparations, we have shown that the cytosolic preparations contained several protein kinases (FIPK, cGMP- and cAMP-dependent kinases, protein kinase C, and Ca2ϩ/calmodulin-dependent kinase), whereas the microsomal preparation contained predominantly factor-independent protein kinase (FIPK)
Phosphorylation of Bone Sialoproteins by Protein Kinase preparations in phosphorylation of purified chicken OPN and recombinant mouse OPN in combination with specific inhibitors and “qualitative analysis” led to the conclusion that these proteins were predominantly phosphorylated by FIPK [31, 32]
Summary
Isolation and Purification of Bovine BSP and OPN—Cortical bone of the mid-portion of the femora of 5– 6-week-old calves was prepared and extracted in dilute HCl, and initially fractionated by DE52 ion exchange chromatography as described previously [3, 4], with the exception that 7 M urea was included in the buffers. Quantification of Number of Moles of Phosphate/Mol of OPN or BSP Introduced by FIPK—32P-Labeled samples of OPN and BSP (free of excess [32P]ATP, and phosphorylation reaction buffers from “Phosphorylation of Bovine OPN and BSP by Factor-independent Protein Kinase” and “Phosphorylation of Bovine OPN and BSP by cGMP-dependent Protein Kinase and Protein Kinase C” as described above) were each resuspended separately in 0.8 ml of H2O and the following aliquots were removed from each for analysis: (a) 5 l for 32P count, (b) 0.15 ml for Lowry’s protein assay, (c) 0.15 ml for total amino acid analysis and phosphoamino acid analysis, (d) 0.15 ml for thrombin digestion followed by SDS-PAGE and autoradiography (Fig. 5). Specific activity of the stock working [32P]ATP was 11.05 Ci/mol (or 11.05 mCi/mmol)
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