Structure and Function of a Prokaryotic Tyrosine Phosphoprotein

艾伦9318072 在真核生物中，酪氨酸蛋白激酶在调节细胞生长、发育和恶性转化的信号转导途径中占据关键位置。 虽然蛋白质磷酸化在原核生物中很重要，但酪氨酸残基的磷酸化很少。 研究表明，青枯假单胞菌（Pseudomonassolanacearum），一种重要的经济植物病原体，具有酪氨酸蛋白激酶活性，其磷酸化90kD的膜蛋白。 这种蛋白质底物已被部分纯化，动力学实验表明，它本身是一种酪氨酸激酶。 分子遗传学和生物化学的方法将被用来研究这种磷蛋白在植物发病机制中的作用。 青枯菌具有一系列导致细菌性枯萎病的毒力因子，这包括多聚半乳糖醛酸酶的产生和运动性。 最近的证据表明，酪氨酸磷酸化参与这些性状的调节。 过度产生多聚半乳糖醛酸酶生产的正调节剂的菌株没有可检测到的90 kD蛋白的磷酸化。 初步结果还表明，鞭毛蛋白，鞭毛丝蛋白，是磷酸化的酪氨酸。 因此，激酶活性也可能影响细菌运动性。 本研究的目的是确定激酶活性及其蛋白底物的功能，并对这些蛋白进行表征。 将使用分子遗传学和生物化学方法的组合。 衍生自纯化的酪氨酸磷蛋白的氨基酸序列的寡核苷酸探针将用于从青枯菌基因文库克隆同源tpp基因。 将tpp的DNA序列与真核酪氨酸激酶基因进行比较。 通过对tpp和多聚半乳糖醛酸酶调节突变体的表型分析，将检验tpp是植物诱导基因调节途径的一部分这一假设。 位点特异性诱变和生物化学方法将用于鉴定和表征90 kD磷蛋白的功能结构域。 %青枯假单胞菌在包括马铃薯、番茄、香蕉、辣椒和茄子在内的多种重要农作物上引起枯萎病。 这些疾病在美国东南部和许多发展中国家造成严重的经济损失。 有效控制这种病原体需要我们了解它引起疾病的机制。 该提案涉及对一种特定酶（一种蛋白激酶）活性的研究，这种酶调节这种生物体的生长，并且在癌症发展中也起着重要作用。 ***

9318072 Allen In eukaryotes, tyrosine protein kinases occupy key positions in signal transduction pathways that regulate cell growth, development, and malignant transformation. Although protein phosphorylation is important in prokaryotes, phosphorylation of tyrosine residues is rare. It was shown that Pseudomonas solanacearum, an economically important plant pathogen, has tyrosine protein kinase activity that phosphorylates a 90 kD membrane protein. This protein substrate has been partially purified and kinetic experiments suggest that it is itself a tyrosine kinase. Molecular genetic and biochemical approaches will be used to investigate the role of this phosphoprotein in plant pathogenesis. P. solanacearum has an array of virulence factors that contribute to bacterial wilt disease, this include polygalacturonase production and motility. Recent evidence suggests that tyrosine phosphorylation is involved in the regulation of these traits. Strains that overproduce a positive regulator of polygalacturonase production have no detectable phosphorylation of the 90 kD protein. Preliminary results also indicate that flagellin, the flagellar filament protein, is phosphorylated on tyrosine. Thus, the kinase activity may also affect bacterial motility. The objectives of this research are to determine the functions of the kinase activity and its protein substrates, and to characterize these proteins. A combination of molecular genetic and biochemical approaches will be used. Oligonucleotide probes derived from an amino acid sequence of the purified tyrosine phosphoprotein will be used to clone the cognate tpp gene from a P. solanacearum gene library. The DNA sequence of tpp will be compared to eukaryotic tyrosine kinase genes. The hypothesis that tpp is part of the regulatory pathway for plant-inducible genes will be tested by phenotypic analysis of tpp and polygalacturonase regulatory mutants. Site specific mutagenesis and biochemical methods will be used to identify and characterize functional domains with the 90 kD phosphoprotein. %%% Pseudomonas solanacearum causes wilt disease on a wide range of important crop plants, including potatoes, tomatoes, bananas, pepper and eggplant. These diseases cause severe economic losses in the Southeastern US and many developing countries. Effective control of this pathogen requires that we understand the mechanism by which it causes disease. The proposal addresses research on the activity of a specific enzyme, a protein kinase, that regulates the growth of this organism, and that also plays a major role in cancer development. ***