©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Polylysine and CVIM Sequences of K-RasB Dictate Specificity of Prenylation and Confer Resistance to Benzodiazepine Peptidomimetic in Vitro(*)

(Received for publication, December 7, 1994; and in revised form, January 13, 1995)

Guy L. James (§) Joseph L. Goldstein Michael S. Brown

From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

BZA-5B, a benzodiazepine peptidomimetic, inhibits CAAX farnesyltransferase (FTase) and blocks attachment of farnesyl groups to oncogenic and wild-type H-Ras in animal cells. This compound slows the growth of cells transformed with oncogenic H-Ras at concentrations that do not affect the growth of nontransformed cells. This finding suggested that nontransformed cells may produce a form of Ras whose prenylation is resistant to BZA-5B. In the current studies, we found that FTase had a 50-fold higher affinity for K-RasB than for H-Ras in vitro. Farnesylation of K-RasB was inhibited by BZA-2B, the active form of BZA-5B, but only at concentrations that were 8-fold higher than those that inhibited farnesylation of H-Ras. K-RasB, but not H-Ras, was also a substrate for CAAX geranylgeranyltransferase-1 (GGTase-1), and its affinity for the enzyme was equal to that of Rap1B, an authentic leucine-terminated substrate for GGTase-1. Inhibition of the geranylgeranylation of K-RasB occurred only at high concentrations of BZA-2B. All of these properties of K-RasB were traced to the combined effects of its COOH-terminal CVIM sequence and the adjacent polylysine sequence, neither of which is present in H-Ras. These studies provide a potential explanation for the resistance of nontransformed cells to growth inhibition by BZA-5B. Inasmuch as the majority of Ras-related human cancers contain oncogenic versions of K-RasB rather than H-Ras, the current data suggest that in vitro studies of FTase inhibitors with potential anti-cancer activity should use authentic K-RasB as a substrate.


INTRODUCTION

Ras proteins are central to the process by which agents such as epidermal growth factor and platelet-derived growth factor stimulate the growth of animal cells (reviewed in refs. 1-3). Acting through tyrosine kinase receptors, these agents trigger a pathway that causes the Ras proteins to exchange GTP for GDP. In the GTP-bound form, Ras proteins initiate a protein kinase cascade that activates transcription of growth-controlling genes. Ras proteins return to the resting state by hydrolyzing the bound GTP to GDP. Mutant Ras proteins that lack GTPase activity transform cells to a malignant phenotype by providing an uninterrupted growth signal. Such mutations are found commonly in cancers of the pancreas and colon in humans.

The activity of normal and oncogenic Ras proteins requires their attachment to the inner leaflet of the plasma membrane. This process is initiated by the covalent attachment of a hydrophobic farnesyl group to a cysteine at the fourth position from the COOH terminus of the Ras protein. Mutation of this cysteine to a serine prevents farnesylation, abrogates membrane attachment, and abolishes the transforming ability of oncogenic Ras proteins(4, 5) .

Farnesylation of Ras proteins is catalyzed by a heterodimeric enzyme, CAAX farnesyltransferase (FTase), (^1)which was first purified and cloned from rat brain (6) and subsequently purified from bovine brain (7) . This Zn-containing enzyme catalyzes the Mg-dependent transfer of a farnesyl group from farnesyl pyrophosphate (FPP) to Ras proteins and several other proteins, including nuclear lamins(8) .

CAAX FTase is one of two enzymes that attach isoprenes to cysteines at the fourth position from the COOH terminus of proteins(9, 10) . The second enzyme, CAAX geranylgeranyl transferase, is also known as geranylgeranyl transferase-1 (GGTase-1). This enzyme transfers a 20-carbon geranylgeranyl group, which is even more hydrophobic than the 15-carbon farnesyl. Both prenyltransferases recognize COOH-terminal tetrapeptides that are designated as CAAX boxes in which C is cysteine, A stands for an aliphatic amino acid, and X is a variable amino acid that dictates the relative specificity of the protein for the two prenyltransferases. In most farnesylated proteins, including Ras proteins and nuclear lamins, X is methionine or serine. Geranylgeranylated proteins, including the GTP-binding protein Rap1B and the -subunits of heterotrimeric G proteins, usually terminate in leucine(9, 10, 11, 12) .

Four Ras proteins, designated H-Ras, N-Ras, K-RasA, and K-RasB, are expressed in animal cells(1) . These proteins resemble each other closely with the exception of their COOH-terminal domains. All of them are believed to function similarly in activating cell growth. All four terminate in CAAX boxes with the following sequences: CVIM (K-RasB), CIIM (K-RasA), CVVM (N-Ras), and CVLS (H-Ras). K-RasA and K-RasB are alternatively spliced products of a single gene(1) . K-RasB differs from the other three forms of Ras because it contains a prominent string of lysine residues (8 lysines among the 10 residues immediately adjacent to the farnesylated cysteine). These lysines assist in the membrane attachment of farnesylated K-RasB, presumably by binding to negatively charged phospholipids on the inner surface of the plasma membrane(13) . H-Ras is the form most often studied experimentally in cell culture. However, mutations in K-RasB are by far the most frequent in human tumors(1) .

When CAAX tetrapeptides were studied as competitive inhibitors of rat brain FTase, the enzyme showed a strong preference for methionine, serine, glutamine, or cysteine at the X position(11) . The affinity for CVIM, which corresponds to the COOH terminus of K-RasB, was 20-fold higher than the affinity for CVLS, which corresponds to the COOH terminus of H-Ras. Leucine-terminated tetrapeptides were recognized with 70-fold lower affinity(11) .

CAAX FTase and GGTase-1 are both alpha/beta heterodimers(9, 10, 14, 15) . The alpha-subunits of the two enzymes are identical(16, 17) , and the two rat beta-subunits show 28% identity(17) . Genetic studies in yeast confirm that the alpha-subunits of the two enzymes are the product of the same gene(5) . So far it has not been possible to separate the alpha- and beta-subunits without denaturation, nor is it possible to produce high levels of one without the other by overexpression in animal (18) or Sf9 cells(19, 20) . The beta-subunit of rat brain FTase binds the CAAX substrate, as determined from cross-linking studies(21) . The role of the alpha-subunit has not yet been delineated(22) .

The existence of a shared alpha-subunit suggests that the two CAAX prenyltransferases may have some overlapping substrate specificity. Consistent with this notion, studies with partially purified GGTase-1 showed that its substrate specificity overlaps that of FTase. Yokoyama et al.(23) showed that GGTase-1 will transfer [^3H]geranylgeranyl to a peptide corresponding to the COOH-terminal 10 residues of lamin B, which terminates in serine, albeit at much lower efficiency than was observed for leucine-terminated peptides. Moreover, the enzyme was able to attach [^3H]farnesyl as well as [^3H]geranylgeranyl to peptides that terminate in leucine. In yeast, Trueblood et al.(24) showed that the consequences of a deletion mutant of the beta-subunit of CAAX FTase could be overcome partially by overexpression of the beta subunit of the GGTase-1 and vice versa.

In a search for agents that block the growth of Ras-dependent tumors, several laboratories have isolated compounds that inhibit CAAX FTase by competing with the FPP or the Ras protein substrate(20, 25, 26) . Among the most potent of these agents are the benzodiazepine peptidomimetics, which were designed to mimic the postulated structure of a CAAX tetrapeptide bound to the Zn at the active site of FTase(20) . The compound designated BZA-5B has an N-methylated cysteine attached to the C-3 position of a benzodiazepine nucleus and a methyl-esterified methionine attached to position N-1. Inside cells, the methyl ester is believed to be cleaved, and the compound is thereby converted to BZA-2B, a potent inhibitor of CAAX FTase(20) . In studies with rat fibroblasts, BZA-5B abolished the farnesylation of overexpressed H-Ras and inhibited the growth of cells transformed by the oncogenic form of this protein(20, 27) . Surprisingly, this compound did not inhibit the growth of untransformed cells, even though it blocked farnesylation of their endogenous H-Ras. This finding was unexpected because Ras proteins are thought to be essential for normal cell growth. Indeed, mutant yeast that lack Ras proteins are nonviable (5) . Moreover, normal cells and oncogenic H-Ras-transformed cells treated with BZA-5B retained the ability to respond to epidermal growth factor with an increase in mitogen-activated protein kinase activity, an event that is thought to require farnesylated Ras proteins(27) .

The above findings raised the possibility that animal cells may continue to grow in the presence of BZA-5B because they continue to prenylate an endogenous form of Ras other than H-Ras, most likely K-RasB(27) . In the current studies we demonstrate that K-RasB is prenylated in vitro with high affinity by the two CAAX prenyltransferases, FTase, and GGTase-1. Prenylation of K-RasB by both enzymes is relatively resistant to inhibition by BZA-2B, the active nonmethylated form of BZA-5B. These properties of K-RasB are attributable to the combined effects of the polylysine sequence and the CVIM sequence at the COOH terminus. The data are consistent with the notion that normal cells resist the growth inhibitory effects of BZA-5B because they continue to produce prenylated K-RasB.


EXPERIMENTAL PROCEDURES

Materials

We purchased all-trans-[^3H]farnesyl pyrophosphate and all-trans-[^3H]geranylgeranyl pyrophosphate from DuPont NEN and American Radiolabeled Chemicals, respectively; pRSET and pTrcHis bacterial expression vectors from Invitrogen; and BL21(DE3) bacteria from Novagen. Various plasmids were obtained from the following: pTrcHis-Rap1B, Doug Andres (University of Kentucky Medical School); rat CAAX GGTase-1 beta-subunit(17) , Pat Casey (Duke University Medical School); and pZIP-KRas4B(28) , Channing Der (University of North Carolina School of Medicine). BZA-2B (20) was provided by James C. Marsters, Jr. and Mark Reynolds of Genentech, Inc.

Bacterial Expression Constructs

All Ras proteins and Rap1B were expressed as fusion proteins in which six histidine residues were placed at the NH(2) terminus. The coding region of human H-Ras was amplified from pRcCMV-H-Ras (27) by PCR and subcloned into the XhoI site of pRSETA (which contains the coding sequence for His(6)) to yield pRSET-H-Ras. The human K-RasB coding region was amplified from pZIP-KRas4B by PCR and subcloned into the HindIII and NcoI sites of pRSETB, yielding pRSET-K-Ras4B. Plasmids encoding chimeric H-Ras proteins were constructed by amplifying a 357-base pair fragment of pRcCMV-H-Ras between codon 71 and the stop codon of H-Ras. The 3` PCR primers encoded the desired COOH-terminal amino acids (Fig. 1) followed by a stop codon and a NotI restriction site. Each PCR product was digested with BstXI plus NotI, and the resulting 247-base pair fragments were ligated into the corresponding region of pRcCMV-H-Ras. These fragments were introduced into pRSET-H-Ras using a similar domain swap strategy to yield pRSET-H-RasCVIM, pRSET-H-Ras(K(n))CVLS, and pRSET-H-Ras(K(n))CVIM. The coding region of Rap1B (29) was amplified by PCR from human lymphocyte B cell cDNA and subcloned into the BamHI site of pTrcHis. The structures of all plasmids were confirmed by restriction mapping and DNA sequencing of the PCR-amplified fragments.


Figure 1: COOH-terminal sequences of wild-type and chimeric Ras proteins used in this study. All wild-type proteins and wild-type amino acid sequences are given in regular type. Bold type denotes K-RasB derived sequences substituted into H-Ras proteins. All sequences are those of human proteins.



Purification of Prenyltransferase Substrates

Plasmids encoding the desired His(6)-tagged proteins were transformed into BL21(DE3) bacteria. Cultures were grown and the proteins were purified by Ni-Sepharose affinity chromatography as described previously(30) . Peak fractions were pooled and dialyzed for 16 h in 6 liters of buffer containing 20 mM Tris-HCl (pH 7.5), 3 mM MgCl(2), 1 mM sodium EDTA, 0.1 M NaCl, 5 mM DTT, and 0.1 mM GDP. The dialyzed proteins were concentrated to 5-20 mg/ml with a Centriprep 10 concentrator (Amicon) and stored in multiple aliquots at -80 °C.

Production of Recombinant Baculoviruses

A recombinant baculovirus that expresses the beta-subunit of rat CAAX GGTase-1 (17) was constructed by Scott Armstrong. (^2)The alpha-subunit was obtained by subcloning the alpha-subunit of rat CAAX FTase (31) into the EcoRI and XbaI sites of the baculoviral transfer plasmid pVL1392 (Invitrogen) to yield pVL1392-FTalpha(20) . A 197-base pair fragment from this plasmid was amplified by PCR with a 3` primer located beyond the EagI restriction site at codon 40 and a 5` primer designed so that the product would encode a BglII restriction site followed by the amino acid sequence Met-Ala-(His)(6) fused to codon 2 of the FTase alpha-subunit. This PCR product was digested with BglII and EagI and subcloned into the corresponding region of pVL1392-FTalpha to yield pVL1392-(His)(6)-FTalpha. The segment amplified by PCR was sequenced on both strands, and this plasmid was then used to construct a recombinant baculovirus according to standard procedures(32) .

CAAX GGTase-1 Purification

A 500-ml culture of Spodoptera frugiperda (Sf9) cells was coinfected with recombinant baculoviruses that express the GGTase-1 beta-subunit and the His(6)-FTase alpha-subunit (described above). The cells were harvested 48 h after infection by centrifugation and washed once in 50 mM Tris-HCl (pH 7.5) and 0.1 M NaCl. The cell pellet was resuspended in 30 ml of buffer containing 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 2 mM MgCl(2), 20 µM ZnCl(2), 5 mM imidazole, 1 mM beta-mercaptoethanol, 3 units/ml DNase I, 0.5 mM phenylmethylsulfonyl fluoride, and 5 µg/ml each of aprotinin, leupeptin, and pepstatin, and the cells were lysed by two passes through a French press. The lysate was centrifuged at 4 °C for 10 min at 10,000 times g, and the resulting supernatant was centrifuged for 30 min at 10^5 times g. His(6)-tagged GGTase-1 was purified from the 10^5 times g supernatant by Ni-Sepharose affinity chromatography as described for Rab proteins(30) . Peak fractions were pooled and dialyzed for 16 h in 6 liters of buffer containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1 mM DTT, and 20 µM ZnCl(2). The dialyzed enzyme was concentrated to 1 mg/ml with a Centriprep 10 concentrator and stored in multiple aliquots at -80 °C.

Prenyltransferase Assays

CAAX GGTase-1 assays were performed in 12 times 75-mm glass tubes in a final volume of 50 µl. Each reaction contained 50 mM sodium Hepes (pH 7.2), 5 mM MgCl(2), 1 mM DTT, 0.3 mM Nonidet P-40, 0.5 µM [^3H]GGPP (33,000 dpm/pmol), 100 ng of purified recombinant His(6)-tagged GGTase-1, and various concentrations of the desired recombinant His(6)-tagged protein substrate. Each protein substrate was diluted to five times the desired final concentration with 50 mM sodium Hepes (pH 7.2) and added to the reaction in a volume of 10 µl. Following a 30-min incubation at 37 °C, the amount of [^3H]geranylgeranyl transferred to each substrate was measured by precipitation with ethanol/HCl (33) with minor modification as described previously(34) .

CAAX FTase assays were performed with purified recombinant rat FTase as described previously(20) . Each reaction, in a final volume of 50 µl, contained 50 mM Tris-Cl (pH 7.2), 10 µM ZnCl(2), 3 mM MgCl(2), 20 mM KCl, 1 mM DTT, 0.2% (w/v) octyl-beta-D-glucoside, 0.5 or 0.6 µM [^3H]FPP (14,093-16,876 dpm/pmol), 20 ng of recombinant FTase, and various concentrations of the desired His(6)-tagged protein substrate. After incubation for 30 min at 37 °C, the amount of [^3H]farnesyl transferred to each substrate was measured by ethanol/HCl precipitation as described above.


RESULTS

Fig. 1shows the COOH-terminal sequences of H-Ras, K-RasB, and the three chimeric versions that were used in the present study. The chimeras were designed to contain the sequence of H-Ras into which we substituted the polylysine sequence of K-RasB, the COOH-terminal CVIM, or both. The figure also shows the COOH-terminal sequence of Rap1B, which terminates in leucine, and is a classic substrate for GGTase-1 ((35) ).

Fig. 2shows saturation curves for the CAAX FTase reaction as a function of Ras concentration using recombinant rat FTase purified from Sf9 insect cells. As previously noted(6, 7) , these saturation curves were not simple rectangular hyperbolae. To compare the relative affinities for the different Ras proteins, we calculated the S(0.5) value, which is defined as the substrate concentration that gives approximately 50% of the maximum velocity obtained in the experiment. The saturation curve for H-Ras was sigmoidal, and the S(0.5) was 10 µM (panel A). The maximum velocity for K-RasB was lower than that for H-Ras (panel B), and there was evidence of substrate inhibition at higher concentrations. The calculated S(0.5) was 0.2 µM, which was 50-fold lower than the S(0.5) for H-Ras. Replacement of the COOH-terminal sequence of H-Ras (CVLS) with the COOH-terminal sequence of K-RasB (CVIM) lowered the S(0.5) value by approximately 6-fold (1.5 µM). Insertion of the polylysine sequence of K-RasB into H-Ras also lowered the S(0.5) by 5-fold (panel C). Insertion of both the polylysine sequence and CVIM into H-Ras lowered the S(0.5) by a further 10-fold, which now approximated the S(0.5) value for K-RasB (0.2 µM) (panel B). These data indicate that the apparent high affinity of FTase for K-RasB is attributable both to the polylysine sequence and to the COOH-terminal CVIM.


Figure 2: Saturation curves for various Ras proteins farnesylated by recombinant CAAX FTase. Panels A-C, each reaction contained 20 ng of recombinant FTase, 0.6 µM [^3H]FPP (14,093 dpm/pmol), and the indicated concentration of H-Ras (), K-RasB (), or the indicated chimeric H-Ras (box, circle, up triangle; Table I). After incubation for 30 min at 37 °C, the amount of [^3H]farnesyl transferred to each protein was determined as described under ``Experimental Procedures.'' A blank value, determined in parallel reactions containing no protein substrate, was subtracted from each value (0.04 pmol). Each value is the average of duplicate determinations.



We next compared the ability of BZA-2B to inhibit farnesylation of the various Ras proteins (Fig. 3). We used a concentration of each Ras protein that was approximately 2-fold above the S(0.5) value. Under these conditions, an 8-fold higher concentration of BZA-2B was required for 50% inhibition of farnesylation of K-RasB as opposed to H-Ras (13 nMversus 1.6 nM). Resistance to inhibition was not conferred when we inserted either the COOH-terminal CVIM or the polylysine sequence separately into H-Ras. However, when the two sequences were inserted together, the I(0.5) value increased to that seen with K-RasB. These data indicate that the farnesylation of K-RasB is relatively resistant to inhibition by BZA-2B and that this resistance is attributable to a combination of the effects of the polylysine sequence and the CVIM. This resistance is not restricted to the benzodiazepine class of inhibitors. When compared to farnesylation of H-Ras, K-RasB showed a 10-20-fold higher resistance to inhibition by CVFM, a tetrapeptide inhibitor of FTase (20) (data not shown).


Figure 3: BZA-2B mediated inhibition of farnesylation of various Ras proteins. Each reaction contained 20 ng of recombinant FTase, 0.6 µM [^3H]FPP (16,876 dpm/pmol), the indicated concentration of BZA-2B, and one of the following native or chimeric Ras proteins: 20 µM H-Ras, 0.5 µM K-RasB, 2 µM H-RasCVIM, 3 µM H-Ras (K)CVLS, or 0.5 µM H-Ras(K)CVIM. BZA-2B was dissolved in dimethyl sulfoxide and added to the reactions as described previously(20) . After a 30-min incubation at 37 °C, the amount of [^3H]farnesyl transferred to each protein was determined as described under ``Experimental Procedures.'' Each value represents a single incubation, except for the values taken as 100%, which are the average of triplicate determinations. The ``100% of control'' values were 9.9 (up triangle), 4.1 (), 2.9 (circle), 11.8 (up triangle), and 4.5 (box) pmol/tube. A blank value, determined in parallel reactions containing no protein substrate, was subtracted from each value (0.03 pmol). Inset, the approximate concentration of BZA-2B required to inhibit 50% of the farnesylation of each substrate was determined from the appropriate curve.



We next sought to determine whether K-RasB is a substrate for recombinant rat GGTase-1 purified from Sf9 insect cells. We compared the activity of this enzyme with the activity of recombinant rat FTase (Fig. 4). Surprisingly, GGTase-1 was able to transfer [^3H]geranylgeranyl from [^3H]GGPP to K-RasB (panel A). The affinity for K-RasB was about the same as the affinity for Rap1B, an authentic leucine-terminated substrate for GGTase-1, and the maximum velocity appeared to be higher for K-RasB than for Rap1B. GGTase-1 did not transfer geranylgeranyl groups to H-Ras (panel A). For comparative purposes, panel B in Fig. 4shows the farnesylation of the Ras proteins by FTase in the same experiment as that shown in panel A. As shown in Fig. 2, the enzyme farnesylated K-RasB with high affinity, and it also farnesylated H-Ras (panel B). The enzyme did not transfer farnesyl to Rap1B (panel B). Fig. 5compares the farnesylation and geranylgeranylation of K-RasB by FTase and GGTase-1, respectively. The S(0.5) values for the FTase and GGTase-1 were 0.3 µM and 1.5 µM, respectively. The affinity of GGTase-1 for [^3H]GGPP, calculated by Lineweaver-Burk plots, was similar with K-RasB or Rap1B as substrates (Fig. 6). In both cases a hyperbolic saturation curve was obtained, and the calculated K(m) values were 0.17 and 0.13 µM, respectively.


Figure 4: Prenylation of K-RasB (), H-Ras (bullet), and Rap1B (circle) by recombinant GGTase-1 (A) and CAAX FTase (B). Panel A, each reaction contained 100 ng of recombinant GGTase-1, 0.5 µM [^3H]GGPP (33,000 dpm/pmol), and the indicated concentration of K-RasB (), H-Ras (bullet), or Rap1B (circle). Panel B, each reaction contained 20 ng of recombinant FTase, 0.5 µM [^3H]FPP (16, 244 dpm/pmol), and the indicated concentration of K-RasB (), H-Ras (bullet), or Rap1B (circle). After incubation for 30 min at 37 °C, the amount of [^3H]prenyl group transferred to each protein was determined as described under ``Experimental Procedures.'' A blank value, determined for each panel in parallel reactions containing no protein substrate, was subtracted from each value (0.04 pmol). Each value is the average of duplicate determinations. Insets, 5 µg of either recombinant GGTase-1 (A) or FTase (B) were subjected to electrophoresis on an 8% SDS-polyacrylamide mini-gel and visualized by staining with Coomassie Blue. The alpha- and beta-subunits of each enzyme are denoted on the right. The position of the 45-kDa molecular mass standard is indicated on the left.




Figure 5: Saturation curves for K-RasB prenylated by recombinant CAAX FTase (bullet) or GGTase-1 (circle). Assays were performed at 37 °C for 30 min as described under ``Experimental Procedures'' in the presence of either 20 ng of recombinant FTase and 0.5 µM [^3H]FPP (15,234 dpm/pmol) (bullet) or 100 ng recombinant GGTase-1 and 0.5 µM [^3H]GGPP (33,000 dpm/pmol) (circle) and the indicated concentrations of K-RasB. Blank values determined in parallel reactions in the absence of K-RasB (0.03 pmol for each enzyme) were subtracted from each value. Each value is the average of duplicate determinations.




Figure 6: Saturation curves for [^3H]GGPP in a GGTase-1 assay with Rap1B (bullet) or K-RasB (circle) as protein substrate. Assays were performed at 37 °C for 30 min as described under ``Experimental Procedures'' in the presence of 100 ng of recombinant GGTase-1, either 1 µM K-RasB (circle) or 3 µM Rap1B (bullet), and the indicated concentration of [^3H]GGPP (33,000 dpm/pmol). Blank values ranging from 0.005 to 0.025 pmol were determined at each [^3H]GGPP concentration in the absence of protein substrate and subtracted from the appropriate value. Each value is the average of duplicate determinations.



To determine the structural features of K-RasB that are responsible for its ability to act as a substrate for GGTase-1, we studied the various chimeric proteins (Fig. 7). As shown above, GGTase-1 attached geranylgeranyl groups to K-RasB, but not to H-Ras (panel A). Insertion of the polylysine sequence of K-RasB into H-Ras increased its ability to act as a substrate, but the apparent affinity remained relatively low ( in panel B). A similar low affinity was obtained when we substituted the COOH-terminal sequence of K-RasB (CVIM) into H-Ras (bullet] in panel B). However, when both substitutions were made, the affinity of chimeric H-Ras became similar to that of authentic K-RasB ( in panel B). In three experiments not shown, we found that BZA-2B inhibited the geranylgeranylation of K-RasB by GGTase-1 (I(0.5) 42-120 nM), whereas it did not inhibit the geranylgeranylation of Rap1B at concentrations up to 3 µM.


Figure 7: Saturation curves for various Ras proteins prenylated by recombinant GGTase-1. Assays were performed at 37 °C for 30 min as described under ``Experimental Procedures''. Panel A, recombinant GGTase-1 (100 ng) was incubated with 0.5 µM [^3H]GGPP (33,000 dpm/pmol) and the indicated concentration of either wild-type H-Ras () or wild-type K-RasB (bullet). Panel B, same as Panel A except that the indicated chimeric H-Ras protein (Table I) was used as a substrate. A blank value determined in a parallel reaction containing no Ras substrate (0.04 pmol) was subtracted from each value. Each value is the average of duplicate determinations.



The GGTase-1 used in the above experiments was produced in Sf9 cells by coinfection with recombinant baculoviruses that encoded the alpha and beta subunits. The resulting heterodimeric enzyme was purified by virtue of a His(6) tag placed at the NH(2) terminus of the alpha-subunit. The ability of this enzyme to geranylgeranylate K-RasB with high affinity was not dependent on the NH(2)-terminal His(6) tag. When the behavior of the His(6)-tagged enzyme was compared directly with untagged recombinant GGTase-1, we observed no difference in the K(m) or V(max) for the two enzymes with K-RasB as the peptide substrate (data not shown).


DISCUSSION

The current data demonstrate that the prenylation of K-RasB in vitro differs profoundly from the previously described process for prenylation of H-Ras(6, 7, 8, 9, 10, 11, 12) . In comparison with H-Ras, K-RasB exhibited: 1) a 50-fold higher affinity for CAAX FTase; 2) an 8-fold decrease in sensitivity to the FTase inhibitor BZA-2B; and 3) a susceptibility to high affinity geranylgeranylation by GGTase-1. All of these properties of K-RasB were attributed to the combined effects of the CAAX box (CVIM) and the polylysine sequence immediately preceding the CAAX box.

Previous studies of the substrate specificity of CAAX FTase have used short peptides corresponding to the various CAAX sequences (11, 12) or variants of yeast Ras that were mutagenized so as to change the sequence of the CAAX box(7, 36) . These studies concluded that the kinetics of the farnesyl transfer reaction were complex and that the nature of the prenyl pyrophosphate substrate determined in part the relative affinities of the enzyme for different CAAX boxes(7) . None of these studies, including those in our laboratory, used authentic mammalian K-RasB with its polylysine sequence upstream of the CVIM.

We were led to study the farnesylation of authentic K-RasB because of the paradoxical finding that BZA-5B, a benzodiazepine peptidomimetic, inhibited the farnesylation of H-Ras in wild-type animal cells at concentrations that neither inhibited cell growth nor blocked the acute response to epidermal growth factor(27) . Both of the latter processes are thought to require prenylated Ras. These findings could be explained most simply if the cells contained a form of Ras whose prenylation was not inhibited by BZA-5B. The most logical candidate was K-RasB, since its CAAX box (CVIM) was known to have a higher affinity than the H-Ras CAAX box (CVLS) for the CAAX FTase(11) .

If the prenylation reaction proceeds in vivo as it does in vitro, the present data raise the possibility that the prenylation of K-RasB is not inhibited efficiently by BZA-5B or by the BZA-2B that is produced from BZA-5B within the cell. Prenylation might persist for either of two reasons: 1) farnesylation of K-RasB is not inhibited by the same concentrations of BZA-5B that inhibit the farnesylation of H-Ras and 2) K-RasB, but not H-Ras, can be geranylgeranylated by GGTase-1 in a reaction that is relatively resistant to inhibition by BZA-5B. Previous studies have shown that geranylgeranylated Ras proteins support transformation of animal cells (37) and growth of yeast cells(24) . Testing of this hypothesis will require antibodies that uniquely recognize K-RasB at the low concentrations that are present in normal animal cells. These antibodies are not yet available.

The data of Fig. 5suggest that most K-RasB in cells should be farnesylated rather than geranylgeranylated based on the relative affinities of the two enzymes for K-RasB. In vitro, GGTase-1 had the same affinity for K-RasB as it did for Rap1B, an authentic leucine-terminated substrate. However, we cannot extrapolate from the in vitro data to the in vivo situation. The intracellular concentrations of GGPP and FPP are unknown, as are the concentrations of the nonfarnesylated Ras substrates and the relative activities of the GGTase and the FTase. In addition, it is possible that the GGTase-1 uses GGPP that is metabolically channeled from the GGPP synthetase, and this may alter the affinity of the enzyme for different protein substrates.

As far as we can determine, only one previous study determined the nature of the prenyl group attached to Ras proteins in animal cells (38) . This study was performed before the existence of geranylgeranyl modifications was widely recognized. The authors showed that Ras proteins, including K-Ras4B, contained covalently bound [^3H]farnesyl derived from [^3H]mevalonic acid. These studies were not designed to search for relatively small amounts of another isoprenoid such as geranylgeranyl. We do not therefore know whether K-RasB contains any geranylgeranyl groups in vivo. Although cells may preferentially attach farnesyl residues to K-RasB under normal conditions, it is possible that they switch to geranylgeranyl when the FTase is inhibited by a compound such as a benzodiazepine peptidomimetic.

Kohl et al.(39) reported that a non-benzodiazepine CAAX peptidomimetic, L-739,749, inhibited the anchorage-independent growth of Rat-1 cells transformed with oncogenic K-Ras4B, as well as with other forms of oncogenic Ras, but not Raf-transformed cells. It is not clear that the growth inhibition is attributable solely to inhibition of Ras farnesylation since this compound also inhibits the anchorage-dependent growth of nontransformed Rat-1 cells(40) . It also exerts effects on the cytoskeleton that are believed to be independent of Ras farnesylation(40) . Data on the ability of L-739,749 to inhibit farnesylation or geranylgeranylation of native or oncogenic K-RasB in vitro have not been reported.

The studies reported here, together with previous data(7, 8, 16, 21, 23, 24, 33) indicate that the CAAX prenyltransferases are highly unusual two-substrate enzymes with substrate specificities that are plastic and can be overlapping. The two enzymes share a common alpha-subunit, whose catalytic role has not yet been defined(8, 14, 15, 16, 22) . The FTase forms a stable noncovalent complex with FPP(8, 21) . This binding is inhibited competitively by GGPP. Whereas the bound FPP is transferred to H-Ras, the bound GGPP is not(8) . GGTase-1 also forms a stable complex with GGPP and to a lesser extent with FPP(15) . This enzyme appears able to transfer both prenyl groups(15) . In general, the COOH-terminal amino acid of the CAAX box determines specificity for the two enzymes(9, 11, 12) . Methionine-terminated CAAX boxes such as CVIM have only low affinity for GGTase-1 (12, 41) . The current data show that the affinity of the GGTase-1 for the methionine-terminated K-RasB is enhanced markedly by the polylysine stretch immediately upstream of the CAAX box (Fig. 7). The polylysine sequence also increases the affinity of FTase for K-RasB (Fig. 2). Considered together, these findings raise the possibility that both CAAX prenyltransferases contain a binding site for the polylysine sequence, most likely on the alpha-subunit that is shared by the two enzymes.

The studies described in this paper call attention to the need to use authentic K-RasB in any future studies of FTase inhibitors and to measure the ability of these agents to inhibit geranylgeranylation as well as farnesylation of K-RasB.


FOOTNOTES

*
This research was supported by Grant HL 20948 from the National Institutes of Health and by the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a postdoctoral fellowship from the Helen Hay Whitney Foundation.

(^1)
The abbreviations used are: FTase, farnesyltransferase; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; GGTase-1, geranylgeranyl transferase-1; PCR, polymerase chain reaction; DTT, dithiothreitol.

(^2)
S. A. Armstrong, V. C. Hannah, M. S. Brown, and J. L. Goldstein, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Debra D. Morgan and Kara Robinson for excellent technical assistance; Jeff Cormier and Michelle Laremore for DNA sequencing; James Marsters, Mark Reynolds, and colleagues (Genentech, Inc.) for providing BZA-2B; and Channing Der, Pat Casey, and Doug Andres for providing plasmids.


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