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 for the function of many proteins that share a carboxyl-terminal 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 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 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.
Baron
R
R
Endocrinologie et Communications Cellulaires Institut National de la Santé et de la Recherche Médicale Unité 397, Toulouse, France.
Fourcade
E
E
Lajoie-Mazenc
I
I
Allal
C
C
Couderc
B
B
Barbaras
R
R
Favre
G
G
Faye
J C
JC
Pradines
A
A
D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D020816:Amino Acid Motifs; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003545:Cysteine / Q000276:immunology* / Q000378:metabolism; D017931:DNA Primers; D051231:Farnesyltranstransferase; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D020743:rhoB GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D017931:DNA Primers; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D020743:rhoB GTP-Binding Protein; D003545:Cysteine