Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity

We have introduced a variety of amino acid substitutions into carboxyl-terminal CA₁A₂X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic [Val¹²]Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells. While all substitutions were tolerated at the A₁ position, substitutions at A₂ and X reduced transforming activity. The A₂ residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only. Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system. Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (≈50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity. Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAX-signaled modifications may have different contributions to the function of different CAAX-containing proteins.