Chen Q, Engelberg-Kulka H, Amster-Choder O
Department of Molecular Biology, The Hebrew University-Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel.
J Biol Chem. 1997 Jul 11;272(28):17263-8. doi: 10.1074/jbc.272.28.17263.
BglG, the response regulator of the bgl sensory system, was recently shown to be phosphorylated on a histidine residue. We report here the localization of the phosphorylation site to histidine 208. Localization of the phosphorylated histidine was carried out in two steps. We first engineered BglG derivatives with a specific protease (factor Xa) cleavage site that allowed asymmetric splitting of each prephosphorylated protein to well defined peptides, of which only one was labeled by radioactive phosphate. This allowed the localization of the phosphorylation site to the last 111 residues. Subsequently, we identified the phosphorylated histidine by mutating each of the three histidines located in this region to an arginine and following the ability of the resulting mutants to be in vivo regulated and in vitro phosphorylated by BglF, the bgl system sensor. Histidine 208 was the only histidine which failed both tests. The use of simple techniques to map the phosphorylation site should make this protocol applicable for the localization of phosphorylation sites in other proteins. The bgl system represents a new family of sensory systems. Thus, the mapping reported here is an important step toward the definition of the functional domains involved in the transduction of a signal by the components that constitute systems of this novel family.
BglG是bgl传感系统的应答调节蛋白，最近研究表明它在一个组氨酸残基上发生了磷酸化。我们在此报告磷酸化位点位于组氨酸208。磷酸化组氨酸的定位分两步进行。我们首先构建了带有特定蛋白酶（因子Xa）切割位点的BglG衍生物，该位点可将每个预磷酸化蛋白不对称切割成明确的肽段，其中只有一个被放射性磷酸盐标记。这使得磷酸化位点定位到最后111个残基。随后，我们通过将位于该区域的三个组氨酸中的每一个突变为精氨酸，并观察所得突变体在体内受调节以及在体外被bgl系统传感器BglF磷酸化的能力，从而鉴定出磷酸化的组氨酸。组氨酸208是唯一在这两项测试中均未通过的组氨酸。使用简单技术来定位磷酸化位点应使该方案适用于其他蛋白质磷酸化位点的定位。bgl系统代表了一个新的传感系统家族。因此，此处报道的定位是朝着定义由构成这个新家族系统的组件转导信号所涉及的功能域迈出的重要一步。
