Developing and optimizing a method to analyze substrate specificity of tyrosine kinases

Protein Tyrosine Kinases (PTKs) are crucial enzymes which aid in cellular signal transduction pathways as well as cell cycle regulation. This is accomplished by phosphorylating downstream targets specifically on the hydroxyl group of tyrosine residues. Equally important, extensive research has established a connection between overexpression of PTKs and the development of certain cancers. One example of this is seen in Chronic Myeloid Leukemia(CML) where a chromosomal translocation occurs in patients which results in a hybrid, overexpressed BCR/ABL fusion PTK ultimately leading to tumor formation. Thus, understanding PTKs along with what substrates they act on is of utmost importance. Along with understanding the structure and function of PTKs, identifying which substrates they phosphorylate is crucial as well. There are several methods both in vitro and in vivo which allow the study of PTK activity on their specific substrates. Each method has some setbacks. Some methods can be time-consuming to set up and use while other methods such as studying PTKs in mammalian cells can lack consistency due to other factors in the cell that can act on these enzymes and alter experimental data. A rapid and efficient system to allow characterization of PTK/substrate relationships would be highly valuable. In the following research, a bacterial system has been developed, optimized and tested which allows a researcher to screen both wild type and mutant protein tyrosine kinases for their phosphorylation activity on chosen substrates. In this screening model, PTKs Abl, Csk and Src and their respective substrates (crk l, kdSrc) were used to demonstrate the effectiveness of this method. Once demonstrated, chosen mutants of the above PTKs and substrates were then tested to further validate this screening method. In both wild type and mutant testing using this applied in vivo screening system, the expected phosphorylation activity was demonstrated. This system can be used to identify substrates for a known kinase, or kinase mutants with certain substrate specificity. It is a useful platform for understanding the mechanisms of PTK substrate specificity. Once this method is developed and proven, the protocol was optimized to allow rapid colony screening of PTKs for substrate specificity. This method can be used to screen mutant PTKs generated for the substrates they phosphorylate in a rapid, effective manner in an effort to further understanding PTKs