STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RAS P21 PROTEINS

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The structural and functional properties of bacterially expressed ras p21 proteins were investigated by means of in vitro and in vivo analysis. Aseries of ras proteins, including BALB-MSV, Harvey-MSV and Kirsten-MSV, were expressed in E. coli and the products purified to greater than 95% purity by extraction of bacterial pellets with 7 M urea followed by a sephadex G-100 chromatography. The same procedure was utilized to obtain deleted mutants of Harvey-MSV protein and to generate BALB-, Harvey- and Kirsten-MSV chimeric proteins carrying the normal 12th codon. Small deletions were generated at both amino and carboxy termini. Furthermore, larger deletions spanning almost the whole coding sequence generated a series of p21 derivatives lacking from 30 to 115 amino acid residues from the carboxy terminus. In vitro analysis of GTP binding, autophosphorylation and GTPase activities of all the expressed proteins shown that at least two regions are required to generate all the activities. Amino acid sequences between positions 6-23 and 153-165 are necessary but not sufficient. In addition, monoclonal antibodies were generated against native p21 ras-H and the epitopes localized by means of deleted mutants. Mononclonals directed against positions 1-69 and 130-152 showed a complete blockage of GTP binding and related activities. Both sets of experiments indicate that at least these two regions are required for the in vitro activities of the p21 ras proteins. In addition, microinjection of NIH/3T3 cells by deletion mutants that showed lack of GTP binding activity showed a clear correlation between GTP binding, GTPase and transforming activity of the protein. The deleted derivatives of p21 were utilized as well to characterize a new functional domain by means of the localization of the epitope which is recognized by the monoclonal antibody Y13-259. This antibody has been proved to be able to block the normal activity of p21 proteins and revert the transformed phenotype of ras-, fms-, fes- and raf-transformed cells.