PROTEIN-KINASES OF CULTURED OSTEOBLASTS - SELECTIVITY FOR THE EXTRACELLULAR-MATRIX PROTEINS OF BONE AND THEIR CATALYTIC COMPETENCE FOR OSTEOPONTIN

The enzyme activities of the major kinases found within the cytosolic and microsomal fractions of embryonic avian calvaria osteoblasts were assayed for their specificity for various noncollagenous extracellular matrix (ECM) proteins of bone. At least 6 proteins with M(r)'s of 66, 58, 50, 36, 30, and 22 kD out of more than 30 of the noncollagenous p roteins of the bone ECM were phosphorylated by the kinase(s) found in both osteoblast cellular fractions. The purification and N-terminal se quence analysis of three of the above proteins, M(r)'s 66 and 58 kD (50 kD), identified them as chicken bone sialoprotein (BSP) and osteopo ntin (OPN), respectively. Heparin, a specific inhibitor of factor-inde pendent protein kinase (FIPK) activity, blocked the phosphorylation of all six ECM proteins by the microsomal kinase(s) but only inhibited t he phosphorylation of the 66, 50, and 36 kD by the cytosolic enzyme(s) . Casein kinase II (a known FIPK) showed a similar phosphorylation pat tern of the same bone FCM proteins as the FIPK(s) found in osteoblast cell extracts, while purified cyclic adenosine monophosphate (cAMP)-de pendent protein kinase did not phosphorylate any of the ECM proteins. Use of dephosphorylated casein showed that in comparison with casein k inase II, casein was a poor substrate for the FIPK found in the osteob last cellular extracts. Further studies, using FIPK(s) of osteoblasts and purified chicken OPN or bacterially produced recombinant murine OP N as a substrate, showed that both species of OPN were excellent subst rates for the FIPK(s) found in osteoblasts. The phosphorylation of the purified chicken and recombinant mouse OPNs were evaluated by quantit ative analysis using commercially available protein kinases. cAMP-depe ndent kinase showed no phosphorylation of either protein, and cyclic g uanodine monophosphate (cGMP)-dependent kinase and protein kinase C in corporated 1.2 and 0.5 mol phosphate/mol OPN, respectively. However, b oth chicken and mouse OPNs were significantly phosphorylated by casein kinase II (9.3 and 9.0 mol of phosphate/mol of OPN, respectively). Th ese results demonstrate that the noncollagenous proteins of the bone E CM, and in particular OPN, are predominantly phosphorylated by FIPK(s) , and this class of kinase is the major enzyme found within the micros omal fraction of osteoblasts.