Lam 9728399 1. Technical This study focuses on protein tyrosine kinases (PTKs), which play an important role in signal transduction for a broad spectrum of cellular activities, including T-cell and B-cell activation, responses to extracellular stimuli, mitogenesis, differentiation, and oncogenesis. The objective is to further our understanding of the range and substrate specificity of these enzymes, particularly peptide substrates and inhibitors for p60c-src PTK. The structure-activity relationship, kinetic parameters, physico-chemical interaction, and biological and biochemical effects on v-src transfected cells or cells that have constitutively elevated c-src activity are basic issues that are studied. Experiments include: evaluation "one-bead one-compound" combinatorial peptide libraries with p60c-src PTK for identification of additional substrate motifs; design of potent pseudosubstrate-based peptide inhibitors for p60c-src PTK; detailed kinetic studies on the identified substrates and inhibitors; probing the biochemical effects of the peptide inhibitors on the phosphorylation of cellular proteins; examining the biological and biochemical effects of the peptide inhibitors on v-src transfected 3T3 and other cell lines; computer modelling ( DOCK program) the interaction between the peptide substrates or inhibitors with the known crystal structure of p60c-src PTK; and determining the X-ray crystal structure of the co-crystal (in collaboration with Professor Stephen Harrison and Michael Eck of Harvard University). 2. Non-technical Protein tyrosine kinases represent a class of enzymes that play extremely important roles in various cellular activities such as cell division, cell death, and cancer formation. Despite their great importance, very little is known about the mechanism of action of these enzymes. The objective of this study is to build on what has already been discovered in this laboratory in the last two years (NSF Grant MCB 9506217) on the p60c-src protein tyrosine ki nase project. The peptide substrates and inhibitors that have already been identified will be used to further study the mechanism of action of this enzyme. Kinetic studies will also be performed with these peptides. Additionally, Dr. Lam has established a collaboration with Drs. Eck and Harrison of Harvard University to determine the X-ray structure of the peptide-enzyme complex. Also planned is the use of computer modelling techniques to understand how these peptides interact with the enzyme. Finally, different approaches will be followed to deliver the peptide inhibitors into cells and study their fundamental biological effect on cell lines which have an elevated level of protein for p60c-src tyrosine kinase.
