RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: Evidenced in vivo by an anti-farnesyl cysteine antibody (original) (raw)
BMC biochemistry, 2006
Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity. We describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) inc...
Expression Cloning of a Novel Farnesylated Protein, RDJ2, Encoding a DnaJ Protein Homologue
Archives of Biochemistry and Biophysics, 1997
isoprenoids, either 15-carbon farnesyl or 20-carbon ger-The CAAX farnesyltransferase is a heterodimeric en-anylgeranyl, can be found attached to proteins in thiozyme that attaches a farnesyl group to a single cysteine ether linkage to conserved cysteine residues at or near in cellular proteins which terminate in the sequence their carboxyl terminus. Most prenylated proteins are CAAX, where C is cysteine, A is an aliphatic amino acid, thought to serve as regulators of signal transduction and X is most often methionine or serine. Substrates and membrane trafficking, and prenylation has been include the p21 ras proteins, nuclear lamins, and a series shown to promote both the protein-protein and proof retinal proteins. To date, a limited number of subtein-membrane interactions of these molecules (2, 3). strates for the farnesyltransferase have been identified, Prenylation provides a mechanism for the membrane predominantly by demonstration of the attachment of localization of proteins which lack a transmembrane a farnesyl group to previously identified cDNA clones domain and appears to be a prerequisite for their in which encode proteins containing an appropriate carvivo activity.
The FASEB Journal
Isoprenylation is a posttranslational modification that involves the formation of thioether bonds between cysteine and isoprenyl groups derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. Numerous isoprenylated proteins have been detected in mammalian cells. Those identified include K-, N-, and H-p21, ras-related GTP-binding proteins such as G25K (G), nuclear lamin B and prelamin A, and the 'y subunits of heterotrimeric G proteins. The modified cysteine is located in the fourth position from the carboxyl terminus in every protein where this has been studied. For p2l, the last three amino I isopentlny-Pe Kin,,. Kiness D.csrboxyl.se isom.rso. Isop.nt.nyl-tRNA 4-DIm.thylallyl-PP DIm.thylallyl-PP Olmsthyisilyl Ad.nOsln. Trsnstsrss.
Monatshefte für Chemie - Chemical Monthly, 2006
Since 1979, when prenylation has been first discovered as chemical oddity of a yeast mating factor, the two forms of this posttranslational modification of proteins (farnesylation and geranylgeranylation) have been found as wide spread among proteins from Eukarya and their viruses. This review attempts to summarize as comprehensively as possible the enzymological processes of prenylation and the various aspects of their biological significance. The substrate proteins of prenyltransferases are known to carry a sequence signal composed of a cysteine-containing 4-5 residue stretch at the utmost C-terminal end that is N-terminally preceded by a flexible and polar linker region of ca. 10 residues. Postprenylation processing of substrate proteins can involve C-terminal proteolysis, C-terminal carboxyl methylation, and other steps of maturation. The prenyl anchor functions as module for membrane attachment or for protein-protein interaction. Prenyl anchor carrying proteins fulfill a large array of functions in signaling and regulation of cellular processes. Therefore, they are involved in the pathogenesis of a variety of human diseases, the most prominent one being cancer. Farnesyltransferase inhibitors show surprisingly high efficiency in controlling tumor growth in model systems but, so far, clinical trials with human patients have remained without the desired success. Interference into prenylation pathways appears also a promising treatment principle in a variety of parasitic diseases.
Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases
1991
Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an a/#? heterodimer, comprised of subunits having relative molecular masses of approximately 47 (a) and 46 kDa (#?). In the yeast Saccharomyces cerevisiae, two unlinked genes, RAMlIDPRl (R A M I) and RAM2, are required for FTase activity. To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses. cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase a-subunit were isolated. Comparison of the amino acid sequences deduced from the a-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase a-subunit. Antisera raised against the RAMl gene product reacted specifically with the #?-subunit of bovine FTase, suggesting that the RAMl gene product is analogous to the bovine FTase #?-subunit. Whereas a raml mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to R A M l , specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-11, respectively. In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-11. Antisera that specifically recognized the bovine FTase a-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-I1 activity. Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-I1 are comprised of different but homologous #?-subunits and that the a-subunits of FTase and GGTase-I share common features not shared by GGTase-11. Site-specific farnesylation or geranylgeranylation of cellular polypeptides a t a COOH-terminal cysteine residue is a functionally essential post-translational modification (see Ref. 1). Protein acceptor substrates in mammalian cells for farnesylation include the cell-transforming 20-kDa GTPase Ras, nuclear lamin B, and the y-subunit of retinal transducin. Substrates for geranylgeranylation include some 20-kDa
Journal of Biological Chemistry
~21'"" and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxylterminal sequence motif, Cuu(M/S/L), where u is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modifed preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rublB (GGCC), rub2 (GGCC), and rub5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Cuu(M/S/L) motif. We now show that
Nature Protocols, 2011
The importance of the post-translational lipid modifications farnesylation and geranylgeranylation in protein localization and function coupled with the critical role of prenylated proteins in malignant transformation has prompted interest in their biology and the development of farnesyl transferase and geranylgeranyl transferase inhibitors (FTIs and GGTIs) as chemical probes and anticancer agents. The ability to measure protein prenylation before and after FTI and GGTI treatment is important to understanding and interpreting the effects of these agents on signal transduction pathways and cellular phenotypes, as well as to the use of prenylation as a biomarker. Here we describe protocols to measure the degree of protein prenylation by farnesyl transferase or geranylgeranyl transferase in vitro, in cultured cells and in tumors from animals and humans. The assays use [ 3 H]farnesyl diphosphate and [ 3 H]geranylgeranyl diphosphate, electrophoretic mobility shift, membrane association using subcellular fractionation or immunofluorescence of intact cells, [ 3 H]mevalonic acid labeling, followed by immunoprecipitation and SDS-PAGE, and in vitro transcription, translation and prenylation in reticulocyte lysates. These protocols require from 1 day (enzyme assays) to up to 3 months (autoradiography of [ 3 H]-labeled proteins).
Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1
Nature, 2013
CAAX proteins play essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis 1 . The ~120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γsubunit of heterotrimeric GTPases, and several protein kinases and phosphatases 2 . Proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of their C-terminal CAAX motifs 3 (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve cysteine prenylation, -AAX tripeptide cleavage, and methylation of the carboxyl prenylated Cys residue. The major CAAX protease activity is mediated by the Ras and a-factor converting enzyme 1 (Rce1), an integral membrane protease of the endoplasmic reticulum 4,5 . Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here, we report the crystal structure of a Methanococcus maripaludis homolog of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site, are distinct from other intramembrane proteases (IMPs). Catalytic residues are located ~10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. The farnesyl lipid is proposed to bind to a site at the opening of two transmembrane α-helices, which then positions the scissile bond adjacent to a