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首页> 外文期刊>Journal of Molecular Biology >Mass Spectrometry and Site-directed Mutagenesis Identify Several Autophosphorylated Residues Required for the Activity of PrkC, a Ser/Thr Kinase from Bacillus subtilis.
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Mass Spectrometry and Site-directed Mutagenesis Identify Several Autophosphorylated Residues Required for the Activity of PrkC, a Ser/Thr Kinase from Bacillus subtilis.

机译：质谱和定点诱变确定了PrkC（一种来自枯草芽孢杆菌的Ser / Thr激酶）的活性所需的几个自磷酸化残基。

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We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may beessential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.
机译：最近我们发现参与枯草芽孢杆菌的发育过程的PrkC是具有真核Hanks激酶受体激酶家族特征的Ser / Thr激酶。在这项研究中，我们从大肠杆菌表达并纯化了PrkC的胞质结构域，该结构域包含该激酶和一个短近膜区。我们将该片段称为PrkCc，并在大肠杆菌中进行自身磷酸化。 PrkCc显然在体外通过转激酶分子间反应进一步自磷酸化。 PrkC还显示髓磷脂碱性蛋白的激酶活性。使用高质量的电喷雾串联质谱（LC-MS / MS）和纳米电喷雾串联质谱，我们在PrkCc中鉴定了7个磷酸化苏氨酸和1个丝氨酸残基。通过系统的定点诱变替换所有相应的残基，并测试纯化的突变蛋白的体外激酶活性。单个和多个置换的四个苏氨酸残基聚集在一个假定的激活环中的残基162和167之间，显着降低了激酶活性，并且效果明显加和。聚簇在残基290和320之间的其他三个苏氨酸残基对活性的影响相对较小。相反，在密切相关的受体激酶样细菌蛋白中保守的Ser214取代独立地影响活性，可能代表了一种新的调节机制。当投影到以已知Hanks激酶的结构为模型的PrkC的3D结构上时，磷酸苏氨酸残基的第一簇恰好落在激活环中，控制底物和ATP进入许多真核受体激酶的催化位点，而第二个簇位于近膜区。这些结果表明，PrkC激酶活性的调节（可能是自身磷酸化）包括保守的激活环机制。近膜磷酸苏氨酸残基可能是必需的，例如对于募集PrkC信号级联反应或与其他信号通路偶联所必需的其他蛋白质。这是汉克斯家族细菌受体样激酶的首次结构功能分析。

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