(Received for publication, December 7, 1994; and in revised form, January 13, 1995)
From the
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.
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), ()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 /
heterodimers(9, 10, 14, 15) . The
-subunits of the two enzymes are
identical(16, 17) , and the two rat
-subunits
show 28% identity(17) . Genetic studies in yeast confirm that
the
-subunits of the two enzymes are the product of the same
gene(5) . So far it has not been possible to separate the
- and
-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
-subunit of rat brain FTase binds the CAAX substrate, as
determined from cross-linking studies(21) . The role of the
-subunit has not yet been delineated(22) .
The
existence of a shared -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 [
H]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 [
H]farnesyl as well as
[
H]geranylgeranyl to peptides that terminate in
leucine. In yeast, Trueblood et al.(24) showed that
the consequences of a deletion mutant of the
-subunit of CAAX FTase could be overcome partially by overexpression of the
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.
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.
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, 3 mM MgCl
, 20 mM KCl, 1 mM DTT, 0.2% (w/v)
octyl-
-D-glucoside, 0.5 or 0.6 µM [
H]FPP (14,093-16,876 dpm/pmol), 20 ng
of recombinant FTase, and various concentrations of the desired
His
-tagged protein substrate. After incubation for 30 min
at 37 °C, the amount of [
H]farnesyl
transferred to each substrate was measured by ethanol/HCl precipitation
as described above.
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 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
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
was 0.2 µM,
which was 50-fold lower than the S
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
value by
approximately 6-fold (1.5 µM). Insertion of the polylysine
sequence of K-RasB into H-Ras also lowered the S
by
5-fold (panel C). Insertion of both the polylysine sequence
and CVIM into H-Ras lowered the S
by a further 10-fold,
which now approximated the S
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 [H]FPP (14,093 dpm/pmol), and the indicated
concentration of H-Ras (
), K-RasB (
), or the indicated
chimeric H-Ras (
,
,
; Table I). After incubation for
30 min at 37 °C, the amount of [
H]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 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
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 [H]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 [
H]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
(
), 4.1 (
), 2.9 (
), 11.8 (
), and 4.5 (
)
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 [H]geranylgeranyl from
[
H]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
values for the FTase and GGTase-1 were
0.3
µM and 1.5 µM, respectively. The affinity of
GGTase-1 for [
H]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
values were 0.17
and 0.13 µM, respectively.
Figure 4:
Prenylation of K-RasB (), H-Ras
(
), and Rap1B (
) by recombinant GGTase-1 (A) and
CAAX FTase (B). Panel A, each reaction
contained 100 ng of recombinant GGTase-1, 0.5 µM [
H]GGPP (33,000 dpm/pmol), and the indicated
concentration of K-RasB (
), H-Ras (
), or Rap1B (
). Panel B, each reaction contained 20 ng of recombinant FTase,
0.5 µM [
H]FPP (16, 244 dpm/pmol),
and the indicated concentration of K-RasB (
), H-Ras (
), or
Rap1B (
). After incubation for 30 min at 37 °C, the amount of
[
H]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
- and
-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 () or GGTase-1 (
). 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 [
H]FPP (15,234 dpm/pmol) (
) or 100 ng
recombinant GGTase-1 and 0.5 µM [
H]GGPP (33,000 dpm/pmol) (
) 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
[H]GGPP in a GGTase-1 assay with Rap1B (
)
or K-RasB (
) 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 (
) or 3 µM Rap1B
(
), and the indicated concentration of
[
H]GGPP (33,000 dpm/pmol). Blank values ranging
from 0.005 to 0.025 pmol were determined at each
[
H]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
(
] 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
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 [H]GGPP (33,000 dpm/pmol) and the
indicated concentration of either wild-type H-Ras (
) or
wild-type K-RasB (
). 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 and
subunits. The resulting heterodimeric
enzyme was purified by virtue of a His
tag placed at the
NH
terminus of the
-subunit. The ability of this
enzyme to geranylgeranylate K-RasB with high affinity was not dependent
on the NH
-terminal His
tag. When the behavior
of the His
-tagged enzyme was compared directly with
untagged recombinant GGTase-1, we observed no difference in the K
or V
for the two enzymes
with K-RasB as the peptide substrate (data not shown).
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 [H]farnesyl derived
from [
H]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 -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
-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.