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The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcatIKM 1 x 106 M-s-at 24°C) as GAP purified from bovine brain or full-length GAP expressed in E.coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E.coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.

Biologically active forms of Ras complexed to GTP can bind to the GTPase-activating protein (GAP), which has been implicated as possible target of Ras in mammalian cells. In order to study the structural features of Ras required for this interaction, we have evaluated a series of mutant ras proteins for the ability to bind GAP and a series of Ras peptides for the ability to interfere with this interaction. Point mutations in the putative effector region of Ras (residues 32-40) that inhibit biological activity also impair Ras binding to GAP. An apparent exception is the Thr to Ser substitution at residue 35; [Ser-35]Ras binds to GAP as effectively as wild-type Ras even though this mutant is biologically weak in both mammalian and S. cerevisiae cells. In vitro, [Ser-35]Ras can also efficiently stimulate the S. cerevisiae target of Ras, adenylyl cyclase, indicating that other factors may influence Ras/protein interactions in vivo. Peptides having Ras residues 17-44 and 17-32 competed with the binding of Ras to E. coli-expressed GAP with IC50 values of 2.4 and 0.9 microM, respectively, whereas Ras peptide 17-26 was without effect up to 400 microM. A related peptide from the yeast GTP-binding protein YPT1 analogous to Ras peptide 17-32 competed with an IC50 value of 19 microM even though the YPT1 protein itself is unable to bind to GAP. These results suggest that determinants within Ras peptide 17-32 may be important for Ras binding to GAP.