MCB 9506929 Kevan Shokat Growth factors, transcription factors, hormones and cell cycle regulatory proteins utilize tyrosine kinases in their signaling cascades. The common molecular feature of these kinases is their activation of downstream proteins by phosphorylation of tyrosine residues. The main obstacle to dissecting the involvement of specific tyrosine kinases in signal transduction cascades has been their lack of substrate specificity in vitro and in vivo. The catalytic domains of tyrosine kinases possess little or no inherent substrate specificity. In fact catalytic domains from one tyrosine kinase can be substituted into a different tyrosine kinase with little change in the function of the latter. Other domains in the tyrosine kinases are responsible for assembling multiprotein complexes which guide the catalytic domain to the correct targets. The poor in vitro specificity makes it difficult to use purified enzymes and substrates to assess the involvement of an individual tyrosine kinase with phosphorylation of a particular substrate. The manifestation of this poor substrate specificity in vivo is that many genetic approaches, such as gene knock out experiments, give no phenotype due to compensation by other cellular tyrosine kinases. This proposal describes a method for tagging and identifying individual protein tyrosine kinases and their physiological substrates. The method is based on engineering the catalytic domain of a prototypical tyrosine kinase, v-src, to accept a non-natural ATP analog (A*TP). A yeast based selection syqtem will be used to screen v-src mutants which accept unnatural ATP analogs. Once the engineered v-src protein which accepts an unnatural ATP analog is identified it will be expressed in NIH 3T3 cells and gamma- 32P A*TP will be provided to the cell. Under these conditions, normal v-src induced signal transduction will cause all the physiological substrates of v-src to contain an easily identifiable radiolabel (32PO4-Y single letter amino acid c ode ). This methodology can also be used to label specific substrate tyrosine residues with the more stable phosphorothioate moiety resulting in a phosphotyrosine residue which is less sensitive to phosphatase catalyzed hydrolysis. This method will provide a novel approach for the study of specific phosphatase involvement in signaling pathways. The two types of experiments in concert will provide a powerful new tool for dissecting signaling cascades involving tyrosine kinases and phosphatases. The education plan of this project employs a multi-disciplinary approach to promote communication between biologists and chemists, and employs computer graphics to give the students a "hands-on" experience. This experience is provided for both undergraduates and graduate students. %%% Cells process information about their environment by using protein signaling cascades. Some types of cascades are so numerous and similar to one another that it has been difficult to study them using standard biochemical and genetic techniques. This proposal describes a chemical method for uniquely "tagging" one signaling protein at a time inside a cell which will allow us to decipher the molecular logic of signaling cascades. The education plan is designed to improve the quality of both undergraduate and graduate education, and to promote communication between disciplines in science. ***
