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A Mechanism for Posttranslational Modifications of Proteins by Yeast Protein Farnesyltransferase (Ras/Prenyl Transfer/Farnesyl Diphosphate) JULIA M

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A Mechanism for Posttranslational Modifications of Proteins by Yeast Protein Farnesyltransferase (Ras/Prenyl Transfer/Farnesyl Diphosphate) JULIA M Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5008-5011, May 1995 Biochemistry A mechanism for posttranslational modifications of proteins by yeast protein farnesyltransferase (Ras/prenyl transfer/farnesyl diphosphate) JULIA M. DOLENCE AND C. DALE POULTER* Department of Chemistry, University of Utah, Salt Lake City, UT 84112 Communicated by Thomas C. Bruice, University of California, Santa Barbara, CA, February 13, 1995 (received for review November 29, 1994) ABSTRACT Protein farnesyltransferase catalyzes the al- modification may be necessary for the cycling of proteins kylation of cysteine in C-terminal CaaX sequences ofa variety between membranes and the cytosol (6). of proteins, including Ras, nuclear lamins, large G proteins, Protein farnesyltransferase (PFTase) and protein gera- and phosphodiesterases, by farnesyl diphosphate (FPP). nylgeranyltransferase I prenylate proteins containing C- These modifications enhance the ability of the proteins to terminal CaaX consensus sequences, where the a is an ali- associate with membranes and are essential for their respec- phatic amino acid and X is one of several amino acids, as tive functions. The enzyme-catalyzed reaction was studied by illustrated in Fig. 1. The enzymes are heterodimers that share using a series of substrate analogs for FPP to distinguish a common a subunit but differ in their 13 subunits (7). Whether between electrophilic and nucleophilic mechanisms for prenyl the protein substrate is modified by a farnesyl or geranylgera- transfer. FPP analogs containing hydrogen, fluoromethyl, nyl group is determined by the X residue. If X is Ser, Ala, or and trifluoromethyl substituents in place of the methyl at Met, the protein is farnesylated; if X is Leu, the protein is carbon 3 were evaluated as alternative substrates for alkyla- geranylgeranylated (8, 9). Two other prenylation-recognition tion ofthe sulfhydryl moiety in the peptide dansyl-GCVIA. The motifs have been identified. In the Rab GTP-binding proteins, analogs were alternative substrates for the prenylation reac- both cysteines of C-terminal -CC or -CXC sequences are tion and were competitive inhibitors against FPP. A compar- geranylgeranylated by protein geranylgeranyltransferase II (10). ison of k,.t for FPP and the analogs with ksolv, the rate PFTase was purified from bovine brains (11), rat brains (12), constants for solvolysis of related p-methoxybenzenesulfonate and yeast (13). Recotnbinant human (14) and yeast (15) derivatives, indicated that protein prenylation occurred by an PFTases with C-terminal Glu-Glu-Phe (EEF) epitopes in the electrophilic mechanism. 1 subunit were overproduced in Escherichia coli and purified by immunoaffinity chromatography. Short peptides conform- Approximately 30% of human cancers are associated with ing to the CaaX rule were competitive inhibitors of PFTase, mutations in Ras proteins. These include pancreatic adeno- acting either as alternative substrates or true dead-end inhib- carcinomas (90%), colon adenocarcinomas and adenomas itors (usually when the second aliphatic amino acid is Phe, Tyr, (50%), thyroid carcinomas and adenomas (50%), lung adeno- or Trp) (16). Detailed kinetic analyses of the PFTase reaction carcinomas (30%), myeloid leukemias (30%), and melanomas were performed for the purified bovine and human enzymes. (20%) (1, 2). Ras proteins are major components in the signal Bovine PFTase appears to add substrates by a random se- transduction pathway leading to cell division. They bind GIDP quential mechanism (17), and the binding mechanism for the when inactive and, upon stimulation, exchange GDP for GTP. human enzyme is apparently an ordered sequential process An activating protein (GAP) stimulates Ras to hydrolyze GTP (18) with farnesyl diphosphate (FPP) binding first. Preliminary to GDP, which terminates the signal. Mutant Ras proteins bind experiments showed that the yeast PFTase also prefers to bind GTP but do not hydrolyze it efficiently. The resulting stimu- FPP first by an ordered sequential mechanism (13). Although lation of downstream protein kinases leads to uncontrolled cell inhibition studies with substrate analogs and alternative sub- growth. Both normal and mutant forms of Ras must associate strates were performed for PFTase in an intense search for with the inner potent inhibitors of Ras farnesylation, to our knowledge, there surface of the outer membrane to participate in are no reports that bear on the chemical mechanism offarnesyl signal transduction (3). The discovery that farnesylation is transfer to the cysteine residue. The design of potent PFTase required for mutant forms of Ras to manifest their transform- inhibitors, especially transition-state analogs, ing activity has promoted widespread interest in protein should be facili- prenylation (4, 5). tated by understanding the mechanism of the reaction. A large group of proteins has been identified that are posttranslationally modified by the attachment of a prenyl MATERIALS AND METHODS group to a cysteine residue near the C terminus through a Materials. Diphosphates 1-4 (see Fig. 2) were synthesized thioether bond. Although many are "true" Ras proteins or from their corresponding alcohols by the method of Davisson other members of the Ras superfamily (Rab, Ral, Rac, and et at (19). The syntheses of alcohols 2-4 are available from the Rap), some non-Ras proteins such as some nuclear lamins, authors upon request. Dansyl-GCVIA was synthesized by large G proteins, and phosphodiesterases are also prenylated solid-phase peptide methods. (4). Addition of a hydrophobic prenyl group increases the Prenyl Transfer Assays. Recombinant yeast PFTase was protein's affinity for a membrane and assists in membrane purified by immunoaffinity chromatography as described (15). recognition (4). Although prenylated proteins selectively as- Catalytic rate constants (kcat) were measured by using a sociate with different membranes in a cell, there is currently no fluorescence assay that continuously monitored farnesylation conclusive evidence for a highly selective receptor-mediated of dansylated pentapeptide dansyl-GCVIA (20, 21) with a affiliation. For some proteins such as rhodopsin kinase, prenyl Spex FluoroMax model spectrofluorimeter with Aex = 340 nm The publication costs of this article were defrayed in part by page charge Abbreviations: FPP, farnesyl diphosphate; PFTase, protein farnesyl- payment. This article must therefore be hereby marked "advertisement" in transferase; TFA, trifluoroacetic acid; FPPSase, FPP synthase. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5008 Downloaded by guest on September 30, 2021 Biochemistry: Dolence and Poulter Proc. NatL Acad Sci. USA 92 (1995) 5009 PPO SH Mg2+ / 7n2+ Protein- N CO-a-a-X Protein - NCO - a-a-X H H FIG. 1. Reaction catalyzed by PFTase. (slit width = 5.1 or 8.5 nm) and Acm = 486 nm (slit width = 30°C for 5 hr. Coinjections consisted of the reaction mixture 5.1 or 8.5 nm) and 3-mm square cuvettes. Assays (250 pl) were plus 5 nmol of the corresponding synthetic standard. conducted at 30°C in 50 mM Tris HCl/10 mM MgCl2/10 ,uM ZnC12/5 mM dithiothreitol/0.04% n-dodecyl f-D-maltoside, RESULTS pH 7.0. Diphosphate compounds (1-4) were present at satu- rating concentrations (10-20 ,uM). Due to substrate inhibition Kinetic Measurements. Kinetic constants for alkylation of (21), a saturating concentration of the peptide substrate was the cysteine in dansyl-GCVIA by diphosphates 1-4 (Fig. 2) not used, and the concentration that gave a maximal rate (2.4 were determined from initial velocity measurements by using the fluorescence assay of et at and a ,uM) was chosen instead. PFTase (1-47 nM) was used to Pompliano (20) buffer specifically optimized for yeast PFTase (21). The results are initiate the reactions. Initial rates were measured from the given in Table 1. Substitution of the methyl group at carbon 3 linear region of each experiment, and all measurements were of FPP by CH2F (2), H (3), and CF3 (4) led to a progressive made in duplicate. Rates were measured in cps/s and con- decrease in kcat, with the CF3 analog (4) being 770-fold less verted to units of s-I by using a conversion factor calculated reactive than FPP. Km values for the allylic substrate also from the slope of a line generated in a plot of concentration decreased progressively from 1.3 ,uM for FPP to 36 nM for of synthetic dansyl-G(S-farnesyl)CV1A (dansyl-GCFVIA) compound 3. We were unable to measure the Km for com- (where F is farnesyl) vs. fluorescence intensity. For each pound 4. Yeast PFTase was saturated by the trifluoromethyl alternative substrate, compounds 2-4, the S-farnesylated dan- diphosphate down to a concentration of 20 nM. The fluores- sylated product was synthesized from the corresponding flu- cence assay was not sufficiently sensitive to accurately measure orinated or desmethyl alcohol and dansyl-GCVIA and used to initial rates at lower substrate concentrations. generate calibration curves. Values for each conversion factor Analogs 2-4 were competitive inhibitors of FPP and non- did not appreciably differ. competitive inhibitors of dansyl-GCVIA (Table 2). These Synthesis ofFarnesylated Products. The farnesylated products patterns are consistent with a mechanism where the analogs were synthesized essentially as described by Pompliano et at (20). exert their influence by binding selectively to the FPP site (22). Alcohols 2 and 3 were converted to their corresponding bromides Trifluoromethyl derivative 4 (Ki = 11 nM) is
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