PROTEIN-KINASES OF CULTURED CHICKEN OSTEOBLASTS THAT PHOSPHORYLATE EXTRACELLULAR BONE PROTEINS

Cytosolic and microsomal protein kinase preparations from cultured chi cken osteoblasts were found to phosphorylate up to six major proteins with M(r)s 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract, Use of heparin led to the conclusion that these proteins were predominant ly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations, It was confirmed that m icrosomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the b one proteins, Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions, The identify of the p rotein kinases was clearly established by using a series of synthetic peptide substrates, Quantitative analysis utilizing pure protein kinas es and purified chicken bone OPN, recombinant mouse OPN, and bovine bo ne OPN and BSP led to introduction of similar to 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 kinas e C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, where as cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP, Consistent with the above results, sites of phosphoryl ation identified for OPN (metabolically labeled) and BSP (labeled by c asein kinase II) revealed that predominant phosphorylated sites have r ecognition sequences for FIPK.