Carboxyl methylation of platelet rap1 proteins is stimulated by guanosine 5'-(3-O-thio)triphosphate.
Carboxyl methylation of platelet ras-related proteins, known as rap proteins, was investigated in this study. Platelet membrane proteins of Mr 23,000 incorporated radioactivity in the presence of S-[methyl-3H]adenosylmethionine and platelet cytosol. About 97% of the radioactivity present in the Mr 23,000 proteins was liberated as volatile methanol under basic (1 M sodium hydroxide) conditions. Cycloheximide, an inhibitor of protein synthesis, inhibited incorporation of S-[methyl-3H]adenosylmethionine by 25%. These results suggest that at least 75% of the radioactivity present in the Mr 23,000 proteins is due to carboxyl methylation and not due to the incorporation of S-[methyl-3H]adenosylmethionine into proteins or due to the incorporation of base-stable methyl groups into side chains of arginine, histidine, or lysine residues. Protein methylation did not occur if membranes or cytosol alone was incubated with S-[methyl-3H]adenosylmethionine. Guanosine 5'(3-O-thio)triphosphate increased methylation of the Mr 23,000 proteins in a time- and concentration-dependent manner. Acetyl-farnesylcysteine, a synthetic substrate for carboxyl methyltransferases, completely blocked methylation of the Mr 23,000 membrane proteins. On the basis of one- and two-dimensional Western blots using rap-specific antisera, the Mr 23,000 methylated proteins were identified as rap1 proteins. The existence of the carboxyl-terminal CAAX motif in rap1 proteins, similar to the CAAX motif present in p21ras as well as in the yeast mating factors, leads us to suggest that methylation of rap1 proteins possibly occurs at the alpha-carboxyl-terminal cysteine.
Huzoor-Akbar
Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Winegar
D A
DA
Lapetina
E G
EG
D000255:Adenosine Triphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008745:Methylation; D008970:Molecular Weight; D013873:Thionucleotides / Q000494:pharmacology*; D020668:rap GTP-Binding Proteins
D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins