Interactions between Bacterial Tyrosine Kinases and Phosphatases

描述（申请人提供）：酪氨酸磷酸化，在真核生物中长期研究，开始被认为在细菌生理中起着关键的调节作用。这一过程是由一类新的蛋白质酪氨酸激酶催化的，即by -激酶，它在革兰氏阴性和革兰氏阳性细菌中都是保守的，但与真核细菌不同，具有独特的结构和调节特征。by激酶介导的信号转导在多糖的合成中起着核心作用，多糖负责胶囊和生物膜的形成，前者作为许多致病物种的抗吞噬毒力因子，后者保护细胞免受各种环境胁迫。by激酶的调控模块是位于胞质催化结构域c末端末端的一簇酪氨酸残基（YC）。YC以分子间方式自磷酸化，并通过低分子量蛋白酪氨酸磷酸酶（LMW-PTP）去磷酸化。真核蛋白激酶依赖于一个或多个调节残基的定量磷酸化状态，充当活性/非活性或“开-关”开关，与之不同的是，在by激酶中，磷酸化和去磷酸化的物种都是最佳多糖合成所必需的。因此，由于激酶和磷酸酶活性之间的平衡，磷酸化和去磷酸化形式之间的动态平衡似乎是正常细胞功能的关键。因此，确定by激酶和LMW-PTPs之间相互作用的性质对于充分理解细菌中酪氨酸磷酸化介导的信号传导的理化方面具有重要意义。虽然一些by激酶催化结构域和细菌LMW-PTPs的结构是可用的，但这些并没有提供任何关于它们相互作用的性质或破坏它们的生理后果的见解。利用大肠杆菌（K12） by -激酶Wzc的催化结构域和相应的lw - ptp Wzb作为模型激酶-磷酸酶系统，并利用最先进的溶液核磁共振方法结合位点定向诱变，我们将确定定义激酶-磷酸酶界面的关键残基。这些核磁共振确定的相互作用在完整宿主生物生理背景下的相关性将通过测定表达多糖合成和干燥下存活的突变蛋白的细胞来确定。我们希望我们目前的研究仅限于大肠杆菌Wzc/Wzb系统，将有助于开展更详细的研究项目，涉及其他细菌by -激酶/ lw - ptp对，包括来自致病物种的研究，并建立by -激酶介导的细菌酪氨酸磷酸化的综合机制模型。

DESCRIPTION (provided by applicant): Tyrosine phosphorylation, long studied in eukaryotes, is beginning to be recognized as playing a key regulatory role in bacterial physiology. This process is catalyzed by a novel class of protein tyrosine kinases, the BY- kinases, which are conserved in both Gram-negative and Gram-positive bacteria, but are distinct from their eukaryotic counterparts, with unique structural and regulatory features. BY-kinase mediated signal transduction plays a central role in the synthesis of polysaccharides responsible for capsule and biofilm formation, the former acting as antiphagocytic virulence factors for many pathogenic species, and the latter protecting the cell from a variety of environmental stresses. The regulatory module for BY-kinases is a cluster of tyrosine residues (YC) in the C-terminal tail of its cytosolic catalytic domain. The YC is autophosphorylated in an intermolecular fashion and dephosphorylated by low molecular weight protein tyrosine phosphatases (LMW-PTP). Unlike eukaryotic protein kinases which act as active/inactive or "on-off" switches dependent on the quantitative phosphorylation states of one or more regulatory residues, in BY-kinases both phosphorylated and dephosphorylated species are required for optimal polysaccharide synthesis. Therefore it appears that the dynamic equilibrium between phosphorylated and dephosphorylated forms, resulting from a balance between the kinase and phosphatase activities, is key to proper cellular function. Thus, determining the nature of the interactions between BY-kinases and LMW-PTPs is of fundamental importance in fully understanding the physiochemical aspects of tyrosine phosphorylation mediated signaling in bacteria. Although the structures of several BY-kinase catalytic domains and bacterial LMW-PTPs are available, these do not provide any insight into the nature of their mutual interactions or the physiological consequences of disrupting them. Using the catalytic domain of Wzc, an E. coli (K12) BY-kinase and Wzb, the corresponding LMW-PTP, as a model kinase-phosphatase system, and utilizing state-of-the-art solution NMR methods combined with site-directed mutagenesis, we will ascertain the key residues that define the kinase- phosphatase interface. The relevance of these NMR-determined interactions in the context of the physiology of the intact host organism will be ascertained by assaying cells expressing mutant proteins for polysaccharide synthesis and survival under desiccation. We expect that our present studies, limited to the E. coli Wzc/Wzb system, will facilitate the development of a more elaborate research project involving other bacterial BY-kinase/LMW-PTP pairs including those from pathogenic species, and the construction of a comprehensive mechanistic model of BY-kinase mediated tyrosine phosphorylation in bacteria.