STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RAS P21 PROTEINS

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A series of ras p21 proteins carrying activating point mutations at position 12, 59 or 61, as well as the normal protein, have been expressed in E. coli. The purified proteins were analyzed for in vitro and in vivo activities. We have found that there are at least two different potential mechanisms of activation of transforming properties of ras p21 proteins. One is by means of a reduction of the GTPase activity of the protein, as found in some of the activating point mutations at position 12 or 61. Another mechanism involves acceleration of the interchange of bound nucleotides, as found in mutants with substitutions of Ala to Thr at position 59. Both mechanisms are consistent with the hypothesis that ras p21s are G-binding proteins which act through their association with GTP. After hydrolysis of GTP to GDP + Pi, the p21 molecule loses its ability to interact with other component(s) in the pathway which regulates cell growth. In parallel studies, we have expressed in E. coli a series of deletion mutants of the Harvey-ras p21 protein and utilized them to map epitopes recognized by a series of newly generated or previously reported monoclonal antibodies. By means of this strategy, we have been able to map different regions of the ras p21 molecule related to its biochemical activities. Two regions, from 5-69 and 107-164 are related to GTP-binding activity. A region from 70 to 106 was found to be related to the biological activity of the protein. Finally, microinjection experiments have been performed in which the mitogenic activity of ras p21 proteins has been linked to very early pH alterations induced in microinjected cells. Attempts are being made to correlate pH alterations induced by ras p21 proteins and other protein kinase activities.