Evidence that Rap1 carboxylmethylation is involved in regulated insulin secretion

Margarita Leiser, Shimon Efrat, Norman Fleischer

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36 Scopus citations

Abstract

Protein carboxylmethylation is a reversible posttranslational modification that regulates protein function. We examined the carboxylmethylation of small GTP-binding proteins in a pancreatic β-cell line (β 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 (GTPγS). GTPγ 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) GTPγ, in the presence of cytosol, increased the amount of detectable membrane-associated Rap1. Studies in intact β 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.

Original languageEnglish (US)
Pages (from-to)2521-2530
Number of pages10
JournalEndocrinology
Volume136
Issue number6
DOIs
StatePublished - Jun 1995

ASJC Scopus subject areas

  • Endocrinology

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