All organisms possess a family of enzymes known collectively as aminoacyl-tRNA synthetases that covalently attach amino acids to cognate tRNAs capable of inserting them in the correct positions during mRNA-directed protein synthesis. The biochemical properties of theses enzymes are sufficiently different between prokaryotes and eukaryotes that they are attractive targets for antibiotic development. The tryptophan specific enzyme (WRS) from mammals is unique in that it is also an autophosphorylating protein kinase. Further is the only aminoacyl-tRNA synthetase gene sensitive to regulation by gamma-interferon. These properties strongly suggest a regulatory role for this enzyme in addition to it's housekeeping function of aminoacylation. The research to be accomplished is intended to determine the function(s) of the unique protein kinase/autophosphorylation activities of the human WRS.
The specific aims are to 1) determine whether phosphorylation affects catalytic properties by assaying the kinetics of aminoacylation and protein phosphation with the phospho and dephospho enzyme as well as mutants with the site of phosphorylation, S467, mutated to either Asp or Ala. 2) Characterize the affects of tRNA on the autophosphorylation and exogenous protein kinase activities of human WRS. 3) Identify protein-protein interactions of HsWRS with potential kinase substrates using the yeast two-hybrid system, co-immunoprecipitation from human cells in culture transfected with normal and mutant forms of WRS, and in vitro phosphorylation of peptide libraries. 4) determine the effect(s) of overexpression of normal HsWRS and mutants with altered kinase and phosphorylation properties on global and gene-specific transcription and translation in transfected mammalian cells 5) identify site-specific mutations in HsWRS that selectively inactive the kinase activity. These mutants will be used to enhance the resolution of experiments done in specific aims 1-4. Results from these experiments will determine how the kinase activities are regulated, what other proteins are modified, and what structural features of the enzyme are responsible for these properties.
