Protein kinases of cultured chicken osteoblasts that phosphorylate extracellular bone proteins.
Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.
Salih
E
E
Department of Orthopaedic Surgery, Harvard Medical School, Boston, MA, USA.
Ashkar
S
S
Zhou
H Y
HY
Gerstenfeld
L
L
Glimcher
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002642:Chick Embryo; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D058574:Integrin-Binding Sialoprotein; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology / Q000201:enzymology*; D053495:Osteopontin; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000378:metabolism*
D016326:Extracellular Matrix Proteins; C546872:Ibsp protein, mouse; D058574:Integrin-Binding Sialoprotein; D010455:Peptides; D010750:Phosphoproteins; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D011494:Protein Kinases