Interactions between Bacterial Tyrosine Kinases and Phosphatases

细菌酪氨酸激酶和磷酸酶之间的相互作用

• 批准号: 8541689
• 负责人: RANAJEET GHOSE
• 金额: $ 18.39万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-07 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8541689
• 关键词: Affect; Anti-Bacterial Agents; Antibiotic Resistance; Bacteria; Bacterial Physiology; Binding; Biochemical; Biological Assay; C-terminal; Catalytic Domain; Cell physiology; Cells; Characteristics; Desiccation; Development; Enzymes; Equilibrium; Escherichia coli; Escherichia coli K12; Eukaryota; Face; Family; Goals; Gram-Positive Bacteria; Human; In Vitro; Individual; Knowledge; Laboratories; Lead; Length; Measurement; Measures; Mediating; Methods; Microbial Biofilms; Modeling; Molecular Weight; Mutation; NMR Spectroscopy; Nature; Organism; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Physiological Processes; Physiology; Play; Polysaccharides; Process; Production; Protein Dephosphorylation; Protein Kinase; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Regulation; Relaxation; Research; Research Project Grants; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Solutions; Spin Labels; Staphylococcus aureus; Streptococcus pneumoniae; Stress; Structure; Surface; System; Tail; Testing; Tyrosine; Tyrosine Phosphorylation; Virulence; Virulence Factors; Walkers; base; capsule; colanic acid; design; enteropathogenic Escherichia coli; genetic manipulation; in vivo; insight; mutant; novel; pathogen; protein folding; public health relevance; response; therapeutic target

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.

描述（由申请人提供）：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学中起关键调节作用。这一过程由一类新的蛋白酪氨酸激酶BY-激酶催化，BY-激酶在革兰氏阴性和革兰氏阳性细菌中都是保守的，但与它们的真核对应物不同，具有独特的结构和调控特征。BY-kinase介导的信号转导在多糖的合成中起着核心作用，多糖负责荚膜和生物膜的形成，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-激酶的调节模块是其胞质催化结构域的C-末端尾部中的一簇酪氨酸残基（YC）。YC以分子间方式自磷酸化，并被低分子量蛋白酪氨酸磷酸酶（LMW-PTP）去磷酸化。与依赖于一个或多个调节残基的定量磷酸化状态而充当活性/非活性或“开-关”开关的真核蛋白激酶不同，在BY-激酶中，磷酸化和去磷酸化物质都是最佳多糖合成所需的。因此，似乎磷酸化和去磷酸化形式之间的动态平衡（由激酶和磷酸酶活性之间的平衡引起）是适当细胞功能的关键。因此，确定BY-激酶和LMW-PTP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生理化学方面具有根本重要性。虽然几个BY-激酶催化结构域和细菌LMW-PTP的结构是可用的，这些并没有提供任何洞察其相互作用的性质或破坏它们的生理后果。利用Wzc的催化结构域，构建了一个E. coli（K12）BY-激酶和相应的LMW-PTP Wzb作为模型激酶-磷酸酶系统，并利用最先进的溶液NMR方法结合定点突变，我们将确定定义激酶-磷酸酶界面的关键残基。这些NMR测定的相互作用在完整宿主生物体的生理学背景下的相关性将通过测定表达突变蛋白的细胞在干燥条件下的多糖合成和存活来确定。我们希望，我们目前的研究，仅限于E。coliWzc/Wzb系统的构建，将有助于进一步开展包括致病菌在内的其他细菌BY-kinase/LMW-PTP对的研究，以及构建细菌BY-kinase介导酪氨酸磷酸化的综合机制模型。