A critical early event in signal transduction by the epidermal growth factor (EGF) receptor is the activation of the intrinsic protein tyrosine kinase activity of the receptor. Activation of tyrosine kinase activity is a common element in the mitogenic pathways of various polypeptide growth factors and oncogenes. The first goal of the proposed research is to better understand the mechanisms of regulation of the EGF receptor/tyrosine kinase at the enzyme level. Biochemical, biophysical, and molecular biological approaches will be combined to study the structural basis of tyrosine kinase activation and the autophosphorylation reactions. The subjects of these studies will be recombinant receptor tyrosine kinase domain (TKD) forms, which will be expressed in a baculovirus/insect cell system. The TKD is the enzyme component of the receptor and is free of the membrane-spanning and growth factor binding domains of the holoreceptor polypeptide. The TKD is subject to regulation by divalent metal ions, certain basic proteins, and a specific antibody reagent. A second goal of the plan is to understand the physical basis of the protein-protein interactions involved in the formation of signal transduction complexes between growth factor receptors and proteins incorporating src homology domain 2 (SH2). The hypothesis that the acidic domains of the EGF receptor, the related erbB3 tyrosine kinase, and the transforming middle T antigen (mT) interact specifically with the SH2 domain will be tested by expressing the individual domains as bacterial fusion proteins. Convenient assays will be developed for monitoring the interactions between the fusion proteins, so that the factors which modulate these protein-protein interactions can be studied. In particular, the role of protein phosphorylation will be addressed. Finally, as the c-src tyrosine kinase has been recently implicated in signal transduction by the EGF receptor and is the prototypical SH2 domain protein, attempts will be made to reconstitute functional interactions between the EGF receptor and c-src in vitro. A permeabilized cell system, in which the regulatory phosphorylations of the c-src kinase have been observed, would appear to be ideal for these investigations. It is hoped that the understanding of tyrosine kinase mechanism and the methods developed in the course of these three studies will have broad applications in the study of tyrosine kinase signal transduction in both normal growth pathways and transformed cells.
