Evidence that Rap1 carboxylmethylation is involved in regulated insulin secretion.
Protein carboxylmethylation is a reversible posttranslational modification that regulates protein function. We examined the carboxylmethylation of small GTP-binding proteins in a pancreatic beta-cell line (beta TC cells). In vitro assays showed that carboxylmethylation of a membrane 23-kDa protein was induced by guanine nucleotides, best demonstrated by the nonhydrolyzable GTP analog, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). GTP gamma S also induced translocation of this 23-kilodalton (kDa) protein from cytosol to particulate fractions. Immunoblotting with antiserum sc-65 raised against Rap1 identified the carboxyl-methylated 23-kDa protein as Rap1. 1) The 23-kDa carboxyl-methylated protein separated by two-dimensional electrophoresis overlapped with the 23-kDa protein detected by immunoblotting. 2) GTP gamma S, in the presence of cytosol, increased the amount of detectable membrane-associated Rap1. Studies in intact beta TC cells demonstrated the carboxylmethylation of the 23-kDa protein in response to glucose and depolarizing concentrations of potassium, an effect that was abolished by the calcium channel inhibitor, D600. Similarly, N-acetyl-S-trans,trans-farnesyl-L-cysteine, an inhibitor of in vivo carboxylmethylation at COOH-terminal S-farnesylcysteine by methyltransferase, inhibited carboxylmethylation of the 23-kDa protein in intact cells and reduced insulin secretion in response to glucose and potassium. These data establish a correlation between insulin secretion and carboxylmethylation of a 23-kDa protein that comigrates with Rap1.
Leiser
M
M
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Efrat
S
S
Fleischer
N
N
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008745:Methylation; D008970:Molecular Weight; D013347:Subcellular Fractions / Q000201:enzymology; D020744:rac GTP-Binding Proteins; D020668:rap GTP-Binding Proteins
D007328:Insulin; D008565:Membrane Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; C068268:N-acetyl-S-farnesylcysteine; D000111:Acetylcysteine