Protein tyrosine kinases are thought to be involved in the processes of cell division, migration and differentiation occurring during embryonic development, but their physiological functions, regulatory properties and mechanism of action are largely unknown. Proteins phosphorylated on tyrosine have been detected in embryonic tissues using antibodies specific for phosphotyrosine residues. However, a large fraction of the protein tyrosine kinases involved in developmental processes may not be known at this time. Using anti-phosphotyrosine antibodies to screen chicken embryo cDNA libraries will allow the isolation of the tyrosine kinase genes that are expressed during development. The cloned protein tyrosine kinases will then be identified by sequencing the appropriate cDNAs and characterized using specific polyclonal antibodies to detect them in embryonic tissues or cultured cells. Antibodies will be prepared by injecting the cloned kinases expressed in bacteria as fusion proteins and used in immunoprecipitation, immunoblotting and immunofluorescence microscopy experiments. Finally, the effects that one of the newly identified tyrosine kinases has on cellular growth and differentiation will be investigated by producing eukaryotic cell lines that express the chosen tyrosine kinase cDNA, in its wild type and mutated forms, and by characterizing their phenotypes. The DNA and antibody probes generated in the course of the proposed project will be useful for further studies leading to a detailed characterization of protein tyrosine kinases and their role in embryonic development. The cloning and characterization of novel protein tyrosine kinases may also contribute to the recognition of new oncogenes encoding tyrosine kinases and of their roles in the events leading to oncogenic transformation.
