Structure and Function of a Prokaryotic Tyrosine Phosphoprotein

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是罕见的。研究表明，茄酸假单胞菌具有酪氨酸蛋白激酶活性，可磷酸化一个90kd的膜蛋白。该蛋白底物已被部分纯化，动力学实验表明它本身就是酪氨酸激酶。分子遗传学和生物化学方法将用于研究该磷酸化蛋白在植物发病机制中的作用。solanacearum具有一系列致病因子，包括聚半乳糖醛酸酶的产生和活性。最近的证据表明酪氨酸磷酸化参与了这些性状的调节。过量产生聚半乳糖醛酸酶正调节因子的菌株没有检测到90 kD蛋白的磷酸化。初步结果还表明鞭毛蛋白（鞭毛丝蛋白）在酪氨酸上被磷酸化。因此，激酶的活性也可能影响细菌的运动。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。分子遗传学和生物化学方法的结合将被使用。从纯化的酪氨酸磷酸化蛋白的氨基酸序列中提取的寡核苷酸探针将被用于克隆茄青霉基因文库中的同源tpp基因。tpp的DNA序列将与真核酪氨酸激酶基因进行比较。tpp是植物诱导基因调控途径的一部分的假设将通过tpp和多半乳糖醛酸酶调控突变体的表型分析来验证。位点特异性诱变和生化方法将用于鉴定和表征90 kD磷酸化蛋白的功能域。solanacearum假单胞菌在许多重要作物上引起枯萎病，包括土豆、西红柿、香蕉、胡椒和茄子。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致病的机制。该提案涉及一种特定酶的活性研究，这种酶是一种蛋白激酶，它调节这种生物体的生长，并且在癌症的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. ***