The long-term objectives of this grant proposal are to identify the function of the 36 kD protein, the best known and perhaps major substrate of pp60v-src tyrosine kinase and to characterize a new post-translational modification of the 36 kD protein described by us. The latter acyl modification will be localized in terms of the previously described post-translational phosphorylation. Therefore, there are two specific goals. 1) We have recently reported (Science, in press) the myristylation of the 36 kD protein in a transformation sensitive manner. In this grant proposal, we describe a series of experiments whereby the 36 kD protein will be isolated/purified following metabolic labeling of intact cells with 3H-myristate, 35S-methionine, or 32P-orthophosphate. Utilizing the new purification technique outlined in this grant proposal, the localization of the metabolic labels on similar or different 36K molecules will be identified and substantiated by two dimensional gel electrophoresis. The extent of labeling by myristate will be accomplished by means of Edman degradation of the end terminal amino acid, tryptic peptide maps, and the superimposition of the two dimensional gel patterns (autoradiography) of the 36K isoforms. Blockage of fatty acid acylation, by means of an inhibitor in intact cells, will also be carried out as will a series of similar experiments utilizing chicken embryo fibroblasts transformed by a ts mutant. In a minor vein, the role of myristylation and/or phosphorylation on the assembly, disassembly and reassembly of the 36K multimer will be studies utilizing column chromatography, subunit fractionation, and density gradient ultracentrifugation. 2) Our recently developed simple chromatography technique for the purification of the 36K protein has provided us with an additional clue to function. These studies will include gel overlay techniques using labeled 36 kD protein as a molecular probe for possible 36 kD-binding proteins. The affinity of the 36 kD protein for metal ions and functional dependence will be performed, which should confirm the ability of the 36K molecule to interact with other proteins and ligands.
