Antibodies to synthetic peptide from the residue 33 to 42 domain of c-Ha-ras p21 block reconstitution of the protein with different effectors.

Abstract

Residues 32 to 40, which are conserved among ras proteins from different species, are likely to participate in interactions with the p21 effector system. With the goal of understanding the structural basis of the regulatory functions of c-Ha-ras p21, we produced rabbit antisera against a synthetic peptide corresponding to amino acids 33 to 42 of the protein. The affinity-purified antibodies interacted specifically with p21 and with the antigenic peptide. The epitope recognized by the antibodies appeared to be centered on threonine 35. The antibodies inhibited both in vitro p21-induced production of cyclic AMP in detergent extracts of RAS-defective yeast membranes and GAP-stimulated GTPase activity. However, monoclonal anti-ras antibodies Y13-259 and Y13-238 were not capable of specifically inhibiting interactions of p21 with these two putative effector proteins. The apparent inhibitory effect of Y13-259 on stimulation of p21 by GAP was due to a greatly reduced rate of exchange of nucleotides in the binding pocket of the protein. These findings provide additional support for the essential role of the residue 32 to 40 domain as the true effector site and further evidence of the involvement of GAP as a cellular effector of ras proteins.Because ras oncogenes activated by single nucleotide substitutions have been associated with a significant percentage of human tumors of diverse histological origins (2), there is intense interest in elucidating the biological functions of the ras product. One question of current interest is the identity of the target for the action of ras proteins, and there have been several recent advances in this area: first, it was demonstrated that single substitutions in residues 32 to 40 of oncogenic p21 (the 32-40 region) can eliminate transforming activity (32, 38). The mammalian ras proteins effectively substitute for the RAS proteins in yeast cells (5,6,21), and the residues of p21 that are important for this substitution are the same as those that determine the oncogenic properties of a mutated ras protein (32). Then McCormick and colleagues demonstrated that a cytosolic factor called GAP (GTPase-activating protein) stimulated, in mammalian cells, the GTPase activity of normal ras p21 by 100-fold, with no effect on the oncogenic form of the protein (36). The same 32-40 region was found to be critical for p21-GAP interaction (1, 3). However, some questions remained unanswered about the identity of the binding site for GAP (or other ras targets). The possibility has not been ruled out that GAP interacts at a different site that is conformationally affected by single substitutions in the 32-40 region. Moreover, it has recently been shown that the monoclonal antibodies Y13-238 and Y13-259, originally prepared by Furth et al. (12), which recognized different epitopes on p21, could prevent GAPinduced increase of GTP hydrolysis (1, 20). To more clearly define which portion of the protein is involved in transduction, we produced rabbit antisera against the p21 putative effector domain and compar...

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