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

Abstract

Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxylterminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, 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 role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirusinfected 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 polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation. P rotein isoprenylation is a posttranslational modification by lipid recently discovered (1-3) that affects about 0.5% of cellular proteins and is essential for protein biological activity (4). Two prenyltransferases catalyze, through thioether bonds, the covalent attachment of prenyl groups from prenylpyrophosphates to the carboxyl-terminal cysteine of the protein included in either a CA 1 A 2 X motif (C is a cysteine, A 1 and A 2 usually aliphatic amino acids) or a CC, CCXX, or CXC motif (5). The CAAX protein family, which contains numerous proteins such as members of the Ras small G protein family (6-8), the nuclear lamins (9), or the ␥ subunit of trimeric G proteins (10-13), are prenylated by the farnesyltransferase (FTase) or geranylgeranyltransferase I (GGTase I), which transfer a 15-carbon farnesyl or a 20-carbon geranylgeranyl from the corresponding prenyl-pyrophosphate to the sulfhydryl group of the carboxyl-terminal cysteine, respectively (5). Mammalian FTase and GGTase I, which both are zinc metalloenzymes, initially were identified and purified from rat brain cytosol as ␣͞␤ heterodimers (14-16). The 48-kDa ␣ subunit is shared by both enzymes, whereas the 46-kDa ␤ subunit of FTase is 30% identical with the 43-kDa of the GGTase I (17, 18).It was concluded from earlier studies that the nature of the carbo...

Farnesylated RhoB inhibits radiation-induced mitotic cell death and controls radiation-induced centrosome overduplication

“…Cells transfected with RhoB-F were treated with R115777 at 1 nM and the inhibition of RhoB-F farnesylation was checked by immunoprecipitation of the farnesylated proteins with the selective antifarnesylated cysteine antibody we described previously. 17 No signal was detected after treatment with either 20 mM lovastatin or with 1 nM R115777 treatment (Figure 3a upper gel), although at the same time no significant difference in RhoB expression was observed (Figure 3a lower gel). This finding indicated that an inhibition of RhoB-F farnesylation occurred after treatment with 1 nM R115777.…”

“…To investigate this aspect, we used NIH3T3 cells encoding for RhoB mutants that had been mutated to produce specific prenylation, either CAIM for farnesylation (RhoB-F cells) or CLLL for geranylgeranylation (RhoB-GG cells), or a CAAX deletion mutant that did not permit RhoB prenylation (RhoB-D cells) as well as wild-type CAAX (RhoB cells), as described previously 17,24 ( Figure 2a). We first determined the SF2 value of different clones for each construction (Figure 2b).…”

“…The rabbit F-Cys Ab was produced in this laboratory. 17 Nitrocellulose and nonfat-dried milk protein were obtained from Biorad (Ivry/Seine, France). The ECL system and Hyperfilm MP were purchased from Amersham (les Ulis, France).…”

“…15 In contrast to other Rho proteins which are solely geranylgeranylated, RhoB is present in both a farnesylated and a geranylgeranylated form in vivo. 16,17 Previous data have suggested that this protein might be a potential target of the antitumoral effects of FTIs. 13,18 Moreover, the role of RhoB in cell death mechanisms, and more precisely in apoptosis regulation, has been widely reported in fibroblasts and epithelial cells.…”

Farnesyltransferase and geranylgeranyltransferase I inhibitors upregulate RhoB expression by HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter

“…Whether inhibition of RhoB farnesylation is involved in the mechanism of action of FTIs is unclear at this time (Lebowitz and Prendergast, 1998;Prendergast, 2001;Sebti and Der, 2003). Whereas most Rho proteins (e.g., RhoA, Rac1 and Cdc42) are modified by geranylgeranylation only, RhoB exists normally in vivo in either a farnesylated form (RhoB-F) or geranylgeranylated form (RhoB-GG) (Lebowitz et al, 1997;Baron et al, 2000). We have shown that RhoB prenylation mutants with mutations in the CAAX sequence that force RhoB to be either farnesylated (RhoB-F) or geranylgeranylated (RhoB-GG) are both equally efficient in inhibiting human cancer cell growth, transformation, oncogenic signaling and tumor growth in nude mice .…”