Salih E, Zhou H Y, Glimcher M J
Department of Orthopaedic Surgery, Harvard Medical School and The Children's Hospital, Boston, Massachusetts 02115, USA.
J Biol Chem. 1996 Jul 12;271(28):16897-905. doi: 10.1074/jbc.271.28.16897.
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.
细胞外磷蛋白骨桥蛋白（OPN）和骨唾液酸蛋白（BSP）上大量共价结合的磷酸盐与诸如矿物质沉积和破骨细胞结合等生物学功能有关。在本研究中，通过使用一系列蛋白激酶进行体外³²P标记和定量来评估BSP和OPN的磷酸化状态。纯化的牛BSP和OPN均被[³²P]ATP和不依赖因子的蛋白激酶进行放射性标记。对去磷酸化的BSP和OPN上掺入的³²P放射性进行定量，分别得到每摩尔掺入6.6和8.9摩尔磷酸盐。天然OPN通过不依赖因子的蛋白激酶每摩尔掺入1.07摩尔磷酸盐，BSP每摩尔掺入2.46摩尔磷酸盐。这些数据得出计算结果，即OPN和BSP在天然状态下分别每摩尔含有7.83和4.14摩尔磷酸盐。³²P标记的BSP经凝血酶消化后，放射性与大约分子量为30 kDa的片段（N端半段）相关，而与40 kDa片段（C端半段）未观察到放射性相关。对³²P标记的OPN进行的类似实验提供了两个放射性标记的凝血酶片段，分子量分别为30 kDa（N端半段）和20 kDa（C端半段），两者均有放射性。主要的磷酸化与含有7.0摩尔磷酸盐的N端半段相关，1.9摩尔磷酸盐与C端半段相关。还进行了其他几种已知蛋白激酶对OPN和BSP进行体外磷酸化的额外实验。cAMP依赖性蛋白激酶未显示对OPN或BSP的磷酸化，而蛋白激酶C和cGMP依赖性蛋白激酶导致少量磷酸化，后者每种激酶每摩尔OPN和BSP分子引入约1摩尔磷酸盐。
