RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: Evidenced in vivo by an anti-farnesyl cysteine antibody

Protein isoprenylation is a lipid posttranslational modification required f or the function of many proteins that share a carboxyl-terminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. W hen X is a methionine or serine. the farnesyl-transferase transfers a farne syl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I , a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the r ole of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequenc e expressed in baculovirus-infected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e.. RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclona l antibody against farnesyl cysteine methyl ester allowing prenylation anal ysis avoiding the metabolic labeling restrictions. We confirmed that the un ique modification of the RhoB CAAX box was sufficient to direct the RhoB di stinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chim era could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody sugges ted that wild-type RhoB is farnesylated in vivo. Taken together, our result s demonstrated that the three last carboxyl amino acids are the main determ inants for RhoB prenylation and described an anti-farnesyl cysteine antibod y as a useful tool for understanding the cellular control of protein farnes ylation.