Phosphorylation of Ser136 is critical for potent bone sialoprotein-mediated nucleation of hydroxyapatite crystals
Phosphorylation of Ser136 is critical for potent bone sialoprotein-mediated nucleation of hydroxyapatite crystals
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DOI：
10.1042/bj20091864
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发表时间：
2010-06-15
影响因子：
4.1
通讯作者：
Goldberg, Harvey A.
中科院分区：
文献类型：
--
作者：
Baht, Gurpreet S.;O'Young, Jason;Goldberg, Harvey A.
矿化组织如骨和牙本质的酸性磷蛋白被认为在羟基磷灰石成核和生长中起重要作用。BSP（骨唾液蛋白）是已知的血凝素最有效的成核体，其活性被认为依赖于该蛋白的磷酸化。本研究确定了磷酸基团在矿物形成中的作用。重组BSP和大鼠序列残基1-100和133-205对应的肽被CK2蛋白激酶磷酸化。磷酸化提高了BSP和BSP-(133-205)的成核活性，但对BSP-(1100)没有影响。质谱分析显示，BSP（133-205）中磷酸化的主要位点是Ser(136)，这是一个与先前参与HA成核的一系列连续谷氨酸残基相邻的位点。磷酸化的Ser（136）在血凝素成核中的关键作用已被定点诱变和功能分析证实。此外，在Ser上有或没有磷酸基团的情况下，合成了与大鼠BSP的133-148序列对应的肽（136）。不出所料，磷酸肽是一种更有效的成核剂。采用分子动力学模拟分析了BSP-(133 148)与HA{100}晶面的相互作用，探讨了其成核机理。磷酸化和非磷酸化的序列都以扩展构象的形式吸附在透明质酸上，与晶面接触或远离晶面的残基交替存在。然而，当Ser（136）被磷酸化时，这种交替残基模式更为明显。这些研究证明了Ser（136）磷酸化在bsp介导的HA成核中起关键作用，并确定了该蛋白成核位点与HA的{100}面之间的独特相互作用模式。
Acidic phosphoproteins of mineralized tissues such as bone and dentin are believed to play important roles in HA (hydroxyapatite) nucleation and growth. BSP (bone sialoprotein) is the most potent known nucleator of HA, an activity that is thought to be dependent on phosphorylation of the protein. The present study identifies the role phosphate groups play in mineral formation. Recombinant BSP and peptides corresponding to residues 1-100 and 133-205 of the rat sequence were phosphorylated with CK2 (protein kinase CK2). Phosphorylation increased the nucleating activity of BSP and BSP-(133-205), but not BSP-( l 100). MS analysis revealed that the major site phosphorylated within BSP(133-205) was Ser(136), a site adjacent to the series of contiguous glutamate residues previously implicated in HA nucleation. The critical role of phosphorylated Ser(136) in HA nucleation was confirmed by site-directed mutagenesis and functional analyses. Furthermore, peptides corresponding to the 133-148 sequence of rat BSP were synthesized with or without a phosphate group on Ser(136). As expected, the phosphopeptide was a more potent nucleator. The mechanism of nucleation was investigated using molecular-dynamics simulations analysing BSP-(133 148) interacting with the {100} crystal face of HA. Both phosphorylated and non-phosphorylated sequences adsorbed to HA in extended conformations with alternating residues in contact with and facing away from the crystal face. However, this alternating-residue pattern was more pronounced when Ser(136) was phosphorylated. These studies demonstrate a critical role for Ser(136) phosphorylation in BSP-mediated HA nucleation and identify a unique mode of interaction between the nucleating site of the protein and the {100} face of HA.