Salih E, Ashkar S, Gerstenfeld L C, Glimcher M J
Harvard Medical School, Department of Orthopaedic Surgery, Boston, Massachusetts, USA.
J Bone Miner Res. 1996 Oct;11(10):1461-73. doi: 10.1002/jbmr.5650111013.
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 proteins of the bone ECM were phosphorylated by the kinase(s) found in both osteoblast cellular fractions. The purification and N-terminal sequence analysis of three of the above proteins, M(r)'s 66 and 58 kD (+50 kD), identified them as chicken bone sialoprotein (BSP) and osteopontin (OPN), respectively. Heparin, a specific inhibitor of factor-independent protein kinase (FIPK) activity, blocked the phosphorylation of all six ECM proteins by the microsomal kinase(s) but only inhibited the phosphorylation of the 66, 50, and 36 kD by the cytosolic enzyme(s). Casein kinase II (a known FIPK) showed a similar phosphorylation pattern of the same bone ECM proteins as the FIPK(s) found in osteoblast cell extracts, while purified cyclic adenosine monophosphate (cAMP)-dependent protein kinase did not phosphorylate any of the ECM proteins. Use of dephosphorylated casein showed that in comparison with casein kinase II, casein was a poor substrate for the FIPK found in the osteoblast cellular extracts. Further studies, using FIPK(s) of osteoblasts and purified chicken OPN or bacterially produced recombinant murine OPN as a substrate, showed that both species of OPN were excellent substrates for the FIPK(s) found in osteoblasts. The phosphorylation of the purified chicken and recombinant mouse OPNs were evaluated by quantitative analysis using commercially available protein kinases. cAMP-dependent kinase showed no phosphorylation of either protein, and cyclic guanodine monophosphate (cGMP)-dependent kinase and protein kinase C incorporated 1.2 and 0.5 mol phosphate/mol OPN, respectively. However, both chicken and mouse OPNs were significantly phosphorylated by casein kinase II (9.3 and 9.0 mol of phosphate/mol of OPN, respectively). These results demonstrate that the noncollagenous proteins of the bone ECM, and in particular OPN, are predominantly phosphorylated by FIPK(s), and this class of kinase is the major enzyme found within the microsomal fraction of osteoblasts.
对鸡胚颅骨成骨细胞胞质和微粒体组分中主要激酶的酶活性进行了测定，以检测它们对骨的各种非胶原蛋白细胞外基质（ECM）蛋白的特异性。在骨ECM的30多种非胶原蛋白中，至少有6种分子量分别为66、58、50、36、30和22 kD的蛋白被成骨细胞两个组分中的激酶磷酸化。对上述三种蛋白（分子量分别为66和58 kD（+50 kD））进行纯化和N端序列分析，结果表明它们分别为鸡骨唾液蛋白（BSP）和骨桥蛋白（OPN）。肝素是一种非依赖性蛋白激酶（FIPK）活性的特异性抑制剂，它可阻断微粒体激酶对所有六种ECM蛋白的磷酸化，但仅抑制胞质酶对66、50和36 kD蛋白的磷酸化。酪蛋白激酶II（一种已知的FIPK）对相同骨ECM蛋白的磷酸化模式与在成骨细胞提取物中发现的FIPK相似，而纯化的环磷酸腺苷（cAMP）依赖性蛋白激酶未使任何一种ECM蛋白磷酸化。使用去磷酸化酪蛋白的结果表明，与酪蛋白激酶II相比，酪蛋白是成骨细胞提取物中FIPK的较差底物。进一步的研究以成骨细胞的FIPK以及纯化的鸡OPN或细菌产生的重组小鼠OPN作为底物，结果表明两种OPN都是成骨细胞中FIPK的优良底物。使用市售蛋白激酶通过定量分析评估纯化的鸡OPN和重组小鼠OPN的磷酸化情况。cAMP依赖性激酶未使任何一种蛋白磷酸化，环磷酸鸟苷（cGMP）依赖性激酶和蛋白激酶C分别将1.2和0.5摩尔磷酸盐/摩尔OPN掺入。然而，鸡和小鼠的OPN均被酪蛋白激酶II显著磷酸化（分别为9.3和9.0摩尔磷酸盐/摩尔OPN）。这些结果表明，骨ECM的非胶原蛋白，尤其是OPN，主要被FIPK磷酸化，并且这类激酶是成骨细胞微粒体组分中发现的主要酶。
