Structure and Function of a Prokaryotic Tyrosine Phosphoprotein

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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. ***