This proposal is intended to advance knowledge concerning the role(s) of the src family of tyrosine kinases in transformation and differentiation.
Specific aims for the next project period have been chosen from the many possible experiments based on two criteria. They are designed (1) to provide information on fundamental gaps in current understanding of the src family and (2) to make use of the considerable expertise and supply of reagents which has been built in the previous grant period. Understanding of the mechanism of action of a kinase necessarily requires a knowledge of its physiological substrates. Thus, the excellent anti-phosphotyrosine monoclonal antibody which was developed will be utilized to find previously uncharacterized src substrates. Of the hundreds of possible substrates attention will be focused on substrates shared with growth factor receptors (an area which we pioneered in our work on the PI3 kinase) and those unique to the src mediated differentiation of M1 cells. Intense effort will also be trained on structure function studies of the src family. Work will be concentrated on the region of the molecule which has been heretofore neglected, the kinase domain itself, once again using special reagents which have been developed in the previous grant period. Key questions to be answered are: (1) how important conserved residues in the kinase domain really are, (2) how the kinase picks out its substrates. The latter point seems particularly important now that the role of the N-terminus in substrate selection is being questioned. An attempt will also be made to create a very useful class of mutants, those which are resistant to kinase inhibitors. This type of mutant is essential to understanding inhibitor based experiments. A foray will also be made into the more contested turf of genetic studies of the SH2 region. Finally the large variety of baculovirus overexpression vectors available in the lab will be used to explore in detail the mechanism by which v-src activates the Raf-1 kinase.
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