Although tyrosine phosphorylation is an important controlling element in cell signaling, no tRNA suppressor for phosphotyrosine (pTyr) incorporation has yet been made. We propose to use directed molecular evolution of several aminoacyl tRNA synthetases (aaRSs) to identify mutations that enable binding of pTyr to the aaRS. Specifically, ATP molecules will be attached to beads, which will then be incubated with free pTyr and a phage-display library of a mutated aaRS. If a mutant aaRS can catalyze the formation of pTyr-AMP, which is the intermediate for the generation of charged tRNA, it will bind to the beads and can be enriched. The mutated aaRSs will be used in in vitro translation to incorporate the pTyr into the protein structure of an assayable gene (for example ? galactosidase). Mass Spectrometry and a set of already existing anti-pTyr specific antibodies will be used to validate incorporation of the pTyr in the assayable protein. Phase I is focused on in vitro incorporation.
The ability to generate tyrosyl-phosphorylated proteins will have significant utility in studying the role of phosphoproteins that are involved in cell signalin, inflammation, cancer and other diseases. Conventional approaches toward making phosphorylated proteins require kinases, which are promiscuous and often lead to phosphorylation at multiple undesirable residues. Successful completion of this proposal will be the first method to allow site-specific incorporation of phosphotyrosine in a protein.
