Enzymatic modification of proteins with a geranylgeranyl isoprenoid.

Casey, Patrick J.; Thissen, Julia A.; Moomaw, John F.

doi:10.1073/pnas.88.19.8631

Cited by 166 publications

(133 citation statements)

References 26 publications

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“…This is consistent with recent reports that low-molecular-mass GTP-binding proteins which have a COOH-terminal leucine, such as racl, are geranylgeranyl modified, while those with serine or methionine in this position (e.g. ras proteins) are farnesyl modified (Kinsella et al, 1991;Casey et al, 1991;Maltese, 1990). The biological role of the addition of different prenyl groups to these proteins remains to be ascertained.…”

supporting

confidence: 78%

“…The two protein prenyltransferases, farnesyltransferase and geranylgeranyltransferase, were resolved from bovine brain, as described by Casey et al (1991). The proteins to be tested as substrates were incubated at 4 pM in 50 mM Tris/ HC1, pH 7.7, 5 mM MgC12, 50 pM ZnC12, 2 mM dithiothreitol, 2 pM of either [3H]farnesyl diphosphate or [3H]-geranylgeranyl diphosphate and either 5 pg farnesyltransferase or 12 pg of geranylgeranyltransferase.…”

Section: Prenylation Of Proteinsmentioning

confidence: 99%

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Rac1, a low‐molecular‐mass GTP‐binding‐protein with high intrinsic GTPase activity and distinct biochemical properties

Ménard

Tomhave

Casey

et al. 1992

European Journal of Biochemistry

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Racl, a member of the family of low-molecular-mass GTP-binding proteins, functions in phagocytic leukocytes as a component necessary for activation of the respiratory burst. To characterize the biochemical properties of racl, the protein was expressed as a fusion protein in Escherichia coli and purified to greater than 99% homogeneity by affinity chromatography. Racl protein bound maximally bound and hydrolyzed GTP under low free-Mg2+ concentrations. Under those conditions, (45 nm free Mg"), purified racl exhibited a steady-state GTPase activity of 18 nmol . min-' . mg protein-' (turnover number M 0.39 min-' at 37"C), which is 40-fold higher than H-ras. The high intrinsic GTPase activity of racl under low free Mg2+ was mainly due to an increased k,,,, the rate constant for hydrolysis of bound GTP, which was 0.29 min-' for racl vs 0.007 min-l for H-ras (at 20°C). Racl also released bound GDP faster than H-ras (k,,, . G D P = 1.02 min-l for racl vs 0.33 min-' for H-ras at 20 "C). In contrast, racl released bound guanosine 5'-[y-thioltriphosphate (GTPIS]) at a slower rate than H-ras (k,ff.GTp[S] M 0.04 min-l for racl vs 0.31 min-' for H-ras at 20°C). Racl was a very good substrate for in vitro geranylgeranylation (C,,) but not for farnesylation (C15), whereas the converse is true for H-ras. Surprisingly, racl was a very poor substrate for in vitro ADPribosylation by the C3 component of Clostridium botulinum toxin compared to rhoA. As a further characterization of racl, a mutant was made in which the Thrll5 was replaced by asparagine. This protein (referred to as [ThrllS -+ Asnlracl) contains the consensus amino acids of all four GTPbinding domains of H-ras. The koff.GDP of [ThrllS --f Asnlracl was reduced to that of H-ras, but Asnlracl also suggests that the region responsible for the increased k,,, of racl is not within the consensus amino acids of the four guanine-nucleotidebinding domains.

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supporting

confidence: 78%

Section: Prenylation Of Proteinsmentioning

confidence: 99%

Rac1, a low‐molecular‐mass GTP‐binding‐protein with high intrinsic GTPase activity and distinct biochemical properties

Ménard

Tomhave

Casey

et al. 1992

European Journal of Biochemistry

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“…identity between DPR1 and the f-subunit of the mammalian Another type of protein prenylation involves the addition of a FTase as well as between RAM2 and the a-subunit of the geranylgeranyl group, and protein geranylgeranyltransferases mammalian FTase [19,20]. In addition, FTase activity was (GGTases) are responsible for this type of modification [6][7][8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

Gomez

Goodman

Tripathy

et al. 1993

Biochemical Journal

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Protein farnesyltransferase (FTase) catalyses the addition of a farnesyl group to a cysteine within the so-called ‘CAAX box’ at the C-terminus of various proteins. In the present paper we report purification of Saccharomyces cerevisiae FTase to near-homogeneity. This was accomplished by constructing a yeast strain overproducing FTase approx. 100-fold. The purified enzyme was a heterodimer of approx. 90 kDa and consisted of 43 kDa and 34 kDa subunits. The 43 kDa subunit was shown to be the product of the DPR1 gene by using antibody raised against baculovirus-produced DPR1 polypeptide. The purified enzyme required Mg2+, showed a pH optimum of 7.8 and was most active at 50 degrees C. The Km values for farnesyl pyrophosphate and GST-CIIS (glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein), KmFpp and KmGST CIIS, were 8.1 and 5.1 microM respectively. The enzyme was capable of farnesylating GST-CIIL (the same as GST-CIIS, except that the C-terminal serine is changed to leucine), a substrate protein for the enzyme geranylgeranyltransferase, although with a higher apparent Km than for GST-CIIS. Like its mammalian counterpart, yeast FTase activity was inhibited by peptides containing the C-terminal CAAX sequence (that is, one where C = cysteine, A = aliphatic amino acid and X = any amino acid). These results provide direct evidence for the idea that the yeast and mammalian FTases are structurally and functionally very similar.

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“…The enzymes FTase 1 and GGTase I recognize a tetra peptide sequence at the C terminus of a protein (5,6). This sequence has been designated the CAAX box, and it determines whether a protein will be prenylated (7).…”

mentioning

confidence: 99%

Direct Demonstration of Geranylgeranylation and Farnesylation of Ki-Ras in Vivo

Rowell

Kowalczyk

Lewis

et al. 1997

Journal of Biological Chemistry

342

227

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It has recently been reported that Ki-Ras protein can be modified in vitro by farnesylation or geranylgeranylation. However, a previous analysis of Ki-Ras prenylation in vivo found only farnesylated Ki-Ras. In this report it is shown that under normal conditions, Ki-Ras is farnesylated in vivo and when cells are treated with the farnesyl transferase inhibitors B956 or B957, farnesylation is inhibited and Ki-Ras becomes geranylgeranylated in a dose dependent manner. These results have strong implications in the design of anticancer drugs based on inhibition of prenylation.Post-translational modifications of small molecular weight GTP-binding proteins have been the subject of intense studies since the first reports of prenylation appeared (1-3). Many of these proteins, and specifically the Ras proteins, are involved in tumorigenesis. Since prenylation is necessary for the transformation potential of Ras proteins, a number of groups have been dedicated to the design of inhibitors of prenylation as a strategy to inhibit tumor formation (4).Two types of prenyl modifications have been described: farnesylation and geranylgeranylation. The enzymes FTase 1 and GGTase I recognize a tetra peptide sequence at the C terminus of a protein (5, 6). This sequence has been designated the CAAX box, and it determines whether a protein will be prenylated (7). In this sequence, C stands for cysteine and A for any amino acid. The amino acid X specifies whether the protein will be farnesylated (methionine or serine) or geranylgeranylated (leucine or phenylalanine) by the appropriate enzyme. This rule is followed in the majority of cases. However, recent reports have shown that Ki-Ras, whose C terminus CVIM predicts farnesylation, can also be geranylgeranylated in vitro, (8) and that its prenylation in cells is inhibited by a GGTase I inhibitor (9). Since activated Ki-Ras is the type of Ras most frequently found in human cancers, it is very important to determine how it is modified in vivo and whether this modification can be inhibited. Cell labeling with [ 3 H]mevalonate in the presence of inhibitors of hydroxymethylglutaryl-CoA reductase such as lovastatin allows the radioactive labeling of cholesterol and its intermediate isoprenoids. However, a major drawback of this approach is the low level of [ 3 H]mevalonate incorporation into cells. The fortuitous identification of the gene for a mevalonate transport membrane protein (pMev) has provided a tool that makes the identification of isoprenoid and non-steroidal isoprenoid intermediates easier (10, 11). Transfection of cells expressing either Ha-Ras or Ki-Ras oncogenes with the mevalonate transporter and growth of the cells under conditions in which the endogenous pools of farnesyl diphosphate and geranylgeranyl diphosphate are radioactively labeled enabled us to demonstrate that both Ha-Ras and Ki-Ras are farnesylated under normal conditions. In the presence of the farnesyl transferase inhibitor B956, farnesylation of both Ha-Ras and Ki-Ras was inhibited in a dose-dependent manner. Ho...

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Enzymatic modification of proteins with a geranylgeranyl isoprenoid.

Cited by 166 publications

References 26 publications

Rac1, a low‐molecular‐mass GTP‐binding‐protein with high intrinsic GTPase activity and distinct biochemical properties

Rac1, a low‐molecular‐mass GTP‐binding‐protein with high intrinsic GTPase activity and distinct biochemical properties

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

Direct Demonstration of Geranylgeranylation and Farnesylation of Ki-Ras in Vivo

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