(Received for publication, December 23, 1994)
From the
RhoB, a small GTP-binding protein, was shown previously to contain farnesyl (C-15) as well as geranylgeranyl (C-20) groups (Adamson, P., Marshall, C. J., Hall, A., and Tilbrook, P. A. (1992) J. Biol. Chem. 267, 20033-20038). The COOH-terminal sequence of the protein is CCKVL. According to current rules of prenylation, the COOH-terminal leucine should render the protein a substrate for CAAX geranylgeranyl transferase (GGTase-1), but not for CAAX farnesyltransferase (FTase). To determine the mechanism of farnesylation, we prepared recombinant RhoB and incubated it with recombinant preparations of either FTase or GGTase-1. RhoB was neither farnesylated nor geranylgeranylated efficiently by FTase, but it was farnesylated as well as geranylgeranylated by GGTase-1. The enzyme attached farnesyl more efficiently than geranylgeranyl to RhoB. Neither farnesylation nor geranylgeranylation required the cysteine at the fifth position from the COOH terminus. However, replacement of the cysteine at the fourth position abolished attachment of both prenyl groups. We conclude that the previously observed farnesylation of RhoB is attributable to the FTase activity of GGTase-1. These data, and other accumulating data, indicate that GGTase-1 is a highly unusual enzyme that efficiently transfers both farnesyl and geranylgeranyl groups and that the choice of prenyl group is dictated by the nature of the protein acceptor.
Two enzymes transfer prenyl groups from prenyl pyrophosphates to
cysteine residues at the fourth position from the COOH terminus of
various proteins (reviewed in (1, 2, 3) ).
These prenyltransferases recognize cysteine in the context of the
CAAX consensus, where C is cysteine, A is an
aliphatic amino acid, and X is a COOH-terminal amino acid that
specifies which transferase will act. Early studies suggested simple
rules for CAAX specificity(4, 5, 6, 7) .
CAAX farnesyltransferase (FTase) ()prefers
methionine or serine at the X position and transfers a
15-carbon farnesyl group. CAAX geranylgeranyl transferase
(also known as GGTase-1) prefers leucine at the X position and
transfers a 20-carbon geranylgeranyl (GG) group. These conclusions were
based on studies in which CAAX-terminated peptides were used
as substrates (4, 5, 6) or in which the
COOH-terminal CAAX sequence of a known substrate was mutated
at the X position so as to change enzyme
specificity(7) . More recent studies suggest that the
determinants of enzyme recognition may be much more complex,
particularly for GGTase-1. (
)
The earliest indication of
this complexity was revealed by the finding that both CAAX prenyl transferases are /
heterodimers that share a
single
-subunit(8, 9, 10) . Both
prenyltransferases have the unusual ability to form stable noncovalent
complexes with their respective prenyl
pyrophosphates(5, 11, 12) . These complexes
can be isolated by size exclusion chromatography, after which the
prenyl pyrophosphate can be transferred to a CAAX-terminated
protein. CAAX FTase binds geranylgeranyl pyrophosphate (GGPP)
as well as farnesyl pyrophosphate (FPP), but only the FPP is
transferred to CAAX boxes(11, 12) . The
situation with GGTase-1 is even more complex. In the initial studies
with a partially purified enzyme preparation, Yokoyama et al.(6) showed that this enzyme can transfer either farnesyl
or GG to leucine-terminated CAAX boxes, although farnesyl
transfer was much less efficient.
Although the two enzymes prenylate
each other's substrates inefficiently in vitro, this
cross-talk may be significant under certain conditions in
vivo. Yeast with defects in the -subunits of FTase are
partially viable because the
-subunit of GGTase-1 can compensate
for the defective
-subunit of FTase (13) . On the other
hand, yeast lacking either the common
-subunit or both
-subunits are nonviable. Moreover, overexpression of the
-subunit of GGTase-1 partially corrects the defective phenotype in
yeast lacking the
-subunit of FTase, and vice versa(13) .
In addition to the two CAAX prenyltransferases, cells
contain a third prenyltransferase designated Rab GGTase (also known as
GGTase-2)(3) . Instead of CAAX boxes, this enzyme
recognizes proteins that terminate in CysCys or CysXCys. The
protein substrates must also contain an upstream sequence, so far
unidentified, that is common to all members of the Rab family of
GTP-binding proteins (14) . The enzyme contains - and
-subunits that are related to, but distinct from, those of the
CAAX prenyltransferases(15, 16) , and it also
requires a third polypeptide, designated Rab escort protein (REP), that
binds the upstream Rab sequence and presents the Rab to the catalytic
heterodimer(17, 18) .
The complexity of prenylation in vivo is illustrated in a recent study of the p21 Rho
proteins(19) . These proteins, in the molecular mass range of
21 kDa, bind GTP and are believed to function in the coupling of
membrane and cytoskeletal events. RhoB, a member of this family,
terminates in the sequence CCKVL. According to the simple rules
outlined above, this protein should be geranylgeranylated by GGTase-1.
Adamson et al.(19) showed, surprisingly, that
preparations of this protein isolated from transfected simian COS cells
radiolabeled with [H]mevalonate contained roughly
equal portions of [
H]farnesyl and
[
H]geranylgeranyl residues. Moreover, incubation
of the nonprenylated protein with a crude reticulocyte lysate led to
prenyl transfer from both [
H]FPP and
[
H]GGPP. Incorporation of both prenyl groups was
abolished by mutation of the C of the CAAX box. Replacement of
the immediately upstream C (i.e. the fifth residue from COOH
terminus) reduced, but did not abolish, geranylgeranyl transfer, and it
did not affect the amount of farnesyl transfer. Adamson et al.(19) concluded that the prenylation of RhoB was
complicated and not consistent with any simple notion of CAAX box specificity. However, without the availability of purified
enzymes, it was impossible for them to determine which enzyme actually
attached farnesyl or geranylgeranyl to RhoB.
In the current studies we have used recombinant FTase and GGTase-1 to prenylate recombinant RhoB in vitro. The data show that this protein will accept either a farnesyl or a GG from GGTase-1. Surprisingly, the efficiency of farnesyl transfer is as great as that of GG transfer. On the other hand, RhoB is not a substrate for FTase.
E. coli cells containing
pET14b-RhoB were grown and lysed as recommended by the manufacturer
(Novagen). The His-tagged wild-type RhoB was purified by nickel column
chromatography as described previously (14) to greater than 95%
purity as judged by SDS-polyacrylamide gel electrophoresis. The protein
was dialyzed against a buffer containing 20 mM Tris-HCl (pH
7.5), 100 mM NaCl, 3 mM MgCl, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium
GDP, and stored in multiple aliquots at -70 °C.
Mutations in the COOH-terminal sequence of rat RhoB were introduced by PCR using 1 ng of the original pET-14b-RhoB clone. In all reactions the 5`-oligonucleotide was RH-1 as described above. The 3`-oligonucleotides were as follows: 5`-AGAGTCGACTCATAGCACCTTGCAAGAGTTGATGCAGC-3` for RhoB-SCKVL and 5`-AGAGTCGACTCATAGCACCTTAGAGCAGTTGATGCAGC-3` for RhoB-CSKVL. The PCR products were digested with NdeI-SalI and introduced into an NdeI-XhoI-digested pET14b vector. The mutant proteins were expressed in BL21 (DE3) E. coli cells and purified as described above. The COOH-terminal amino acid sequences of the mutant RhoB proteins were verified by DNA sequencing of the mutant plasmids.
Purified recombinant CAAX FTase transferred
[H]farnesyl from [
H]FPP to
H-Ras with its normal CAAX box (CVLS), but not to a mutant
H-Ras in which X had been changed to leucine (Fig. 1A). The enzyme transferred a very small but
detectable amount of [
H]farnesyl to RhoB. It did
not transfer [
H]GG from
[
H]GGPP to any of the protein substrates (Fig. 1B).
Figure 1:
Inability of FTase to prenylate RhoB in
presence of [H]FPP (A) or
[
H]GGPP (B). Assays contained, in a
final volume of 50 µl, 100 ng of recombinant FTase and the
indicated amount of Ras-CVLS (
), Ras-CVLL (
), or RhoB
(
) in the presence of either 2 µM [
H]FPP (A) or 2 µM [
H]GGPP (B). After incubation for
15 min at 37 °C, the amount of [
H]farnesyl (A) or [
H]GG (B) transferred to
the indicated protein substrate was determined in duplicate. Blank
values carried out in parallel reactions in the absence of any protein
substrate (0.08-0.13 pmol/tube) were subtracted from each
value.
The results with recombinant GGTase-1
were quite different (Fig. 2). As expected, this enzyme
transferred [H]GG to the leucine-terminated
mutant form of H-Ras (H-Ras-CVLL), but there was no
appreciable transfer to wild-type H-Ras (H-Ras-CVLS) (Fig. 2B). GGTase-1 also transferred
[
H]GG to RhoB, which terminates in CCKVL (Fig. 2B). As expected, GGTase-1 did not transfer
[
H]farnesyl to H-Ras-CVLS, and it had only a
slight ability to farnesylate H-Ras-CVLL (Fig. 2A).
Surprisingly, however, GGTase-1 had a robust ability to farnesylate
RhoB (Fig. 2A). With RhoB as acceptor, the maximal
velocity for the transfer of [
H]farnesyl was more
than 3-fold greater than the maximal velocity of transfer of
[
H]GG (compare Panels A and B).
Figure 2:
Farnesylation (A) and
geranylgeranylation (B) of RhoB by recombinant CAAX GGTase-1. Assays contained, in a final volume of 50 µl, 100 ng
of recombinant GGTase-1 and the indicated amount of Ras-CVLS
(), Ras-CVLL (
), or RhoB (
) in the presence of
either 2 µM [
H]FPP (A) or 2
µM [
H]GGPP (B). After
incubation for 15 min at 37 °C, the amount of
[
H]farnesyl (A) or
[
H]GG (B) transferred to the indicated
protein substrate was determined in duplicate. Blank values carried out
in parallel reactions in the absence of any protein substrate
(0.15-0.23 pmol/tube) were subtracted from each
value.
Fig. 3shows the prenyl pyrophosphate saturation curves for
GGTase-1. With RhoB as acceptor, GGTase-1 showed a relatively high
affinity for [H]FPP (Fig. 3A).
The concentration of [
H]FPP giving half maximal
velocity (S
) was in the range of 0.5 µM. The
enzyme had a similar high affinity for [
H]GGPP
when H-Ras-CVLL was used as an acceptor (S
0.5
µM) (Fig. 3B). However, with RhoB as
acceptor, the affinity for [
H]GGPP was much
lower. Saturation was not approached at 5 µM, which was
the highest concentration tested (Fig. 3B). The enzyme
also showed a low affinity for [
H]GGPP when
H-Ras-CVLS was the acceptor (Fig. 3B).
Figure 3:
Prenyl pyrophosphate saturation curves for
recombinant CAAX GGTase-1. Assays contained, in a final volume
of 50 µl, 100 ng recombinant GGTase-1, 20 µM of the
indicated RhoB or H-Ras protein, and varying concentrations of either
[H]FPP (A) or
[
H]GGPP (B). After incubation for 15 min
at 37 °C, the amount of [
H]farnesyl (A) or [
H]GG (B) transferred to
the indicated protein substrate was determined in duplicate. Blank
values carried out in parallel reactions in the absence of any prenyl
pyrophosphate (0.005-0.01 pmol/tube) were subtracted from each
value.
To explore
the roles of the two cysteine residues in the CCKVL sequence of RhoB,
we prepared recombinant RhoB with serine residues replacing either of
the cysteines (Fig. 4). Replacement of the first cysteine
(SCKVL) reduced slightly the ability of the protein to be farnesylated (Fig. 4A) and produced a 2-fold increase in the amount
of [H]GG incorporated (Fig. 4B).
Replacement of the second cysteine (CSKVL) eliminated the
acceptance of [
H]farnesyl (Fig. 4A) and severely reduced the ability of the
protein to accept [
H]GG (Fig. 4B).
Figure 4:
Farnesylation (A) and
geranylgeranylation (B) of COOH-terminal mutants of RhoB by
recombinant CAAX GGTase-1. Assays contained, in a final volume
of 50 µl, 100 ng of recombinant GGTase-1, either 2 µM [H]FPP (A) or 2 µM [
H]GGPP (B), and varying
concentrations of the indicated RhoB protein. After incubation for 15
min at 37 °C, the amount of [
H]farnesyl (A) or [
H]GG (B) transferred to
the protein substrate was measured in duplicate. Blank values carried
out in parallel reactions in the absence of any protein substrate
(0.12-0.17 pmol/tube) were subtracted from each
value.
Fig. 5shows an experiment
designed to compare the carrier function of GGTase-1 with respect to
the two prenyl pyrophosphates. The enzyme was incubated with either
[H]FPP (Fig. 5A) or
[
H]GGPP (Fig. 5B), and the
complexes were isolated by gel exclusion chromatography. We then
incubated the complexes with RhoB and measured the amount of
[
H]prenyl that was transferred. GGTase-1 bound
comparable amounts of [
H]FPP and
[
H]GGPP. During the subsequent incubation,
approximately 50% of both prenyl groups were transferred to RhoB.
Figure 5:
Prenyl pyrophosphate saturation curves for
formation of [H]prenyl pyrophosphate/CAAX GGTase-1 complex and transfer of [
H]prenyl
from isolated complex to RhoB. Assays contained, in final volume of 50
µl, 1 µg of recombinant GGTase-1, the indicated amount of
[
H]FPP (A) or
[
H]GGPP (B), and buffer components as
described under ``Experimental Procedures.'' After incubation
for 10 min at 37 °C, the [
H]prenyl
pyrophosphate/enzyme complex was isolated on a Sephadex G-50 column as
described under ``Experimental Procedures'' and divided into
equal aliquots. One aliquot was used to determine the amount of
isolated [
H]prenyl pyrophosphate/enzyme complex
(
). The second aliquot was incubated with 20 µM RhoB
for 5 min at 37 °C, after which the amount of
[
H]farnesyl or [
H]GG
transferred to RhoB was determined (
) as described under
``Experimental Procedures.'' Blank values carried out in
parallel reactions in the presence of 20 µM unlabeled FPP
or GGPP (0.002-0.04 pmol/tube) were subtracted from each value.
Each value is the average of duplicate
reactions.
The binding of [H]FPP to GGTase-1 was
competitively inhibited by unlabeled FPP and by unlabeled GGPP (Fig. 6A). Surprisingly, the binding of
[
H]GGPP was not inhibited by unlabeled FPP,
although it was inhibited by unlabeled GGPP (Fig. 6B).
This striking result was repeated on several occasions with the same
results.
Figure 6:
Inhibition of formation of
[H]FPP/GGTase-1 complex (A) and
[
H]GGPP/GGTase-1 complex (B) by
unlabeled prenyl pyrophosphates. One µg of recombinant CAAX GGTase-1 was incubated in 50 µl of a solution containing 75
nM [
H]FPP (A) or
[
H]GGPP (B) plus the indicated
concentration of unlabeled FPP or GGPP. After incubation for 10 min at
37 °C, the [
H]prenyl pyrophosphate/enzyme
complex was isolated, and the amount of
H radioactivity was
determined as described under ``Experimental Procedures.''
Each value is the average of duplicate
incubations.
As expected from the steady state kinetic analysis, the
[H]FPP bound to GGTase-1 was transferred
efficiently to RhoB, but not efficiently to H-Ras-CVLL or H-Ras-CVLS (Fig. 7A). The bound [
H]GGPP was
transferred efficiently to RhoB and to H-Ras-CVLL, but not to
H-Ras-CVLS (Fig. 7B).
Figure 7:
Transfer of CAAX GGTase-1 bound
[H]FPP (A) or
[
H]GGPP (B) to RhoB and Ras protein
substrates. One µg of recombinant GGTase-1 was incubated with
either 0.5 µM [
H]FPP (A) or
[
H]GGPP (B) in a final volume of 50
µl for 10 min at 37 °C. The resulting
[
H]prenyl pyrophosphate/enzyme complex was
isolated as described under ``Experimental Procedures.'' The
isolated complex was divided into two equal aliquots. One aliquot was
used to measure the amount of complex formed, and the second aliquot
was incubated for 45 s at 37 °C with 20 µM of the
indicated RhoB or Ras protein, after which the percentage of
[
H]farnesyl or [
H]GG
transferred to the indicated protein substrate was determined as
described under ``Experimental Procedures.'' Each value is
the average of duplicate incubations.
The results in this study, considered together with previous data, reveal that the CAAX prenyltransferases are highly unusual enzymes. In the absence of a protein acceptor, both enzymes form highly stable noncovalent complexes with FPP and with GGPP (5, 11, 12) (Fig. 5). Farnesyltransferase will efficiently transfer only the farnesyl group(11, 12) . GGTase-1 will transfer either the farnesyl or the GG group depending on which protein acceptor is presented (Fig. 7).
The peculiar properties of GGTase-1 are particularly apparent when RhoB is the substrate. In this case the enzyme will transfer either the farnesyl or the GG group. Farnesyl transfer is more efficient than GG transfer in terms of the maximal velocity of the enzyme (Fig. 2). Moreover, its affinity for FPP is greater than for GGPP (Fig. 3). We believe that these findings explain the previous observation of Adamson et al.(19) that RhoB can be farnesylated as well as geranylgeranylated in animal cells and in reticulocyte lysates.
What are the properties of RhoB that make it an efficient acceptor of farnesyl groups? RhoB terminates in CCKVL. The C at the fifth position from the COOH terminus is not crucial in directing the prenylation specificity. When this C was substituted with a serine, RhoB remained susceptible to farnesylation as well as geranylgeranylation by GGTase-1 (Fig. 4). As expected, the C at the fourth position from the COOH terminus was crucial for the acceptance of both prenyl groups. Based on these observations, we believe that each RhoB molecule accepts only a single prenyl group, either a farnesyl or a GG, which is attached to the cysteine of the CAAX box.
A prominent
feature of the RhoB CAAX box is the lysine in place of the
usual aliphatic amino acid at the first A position. This is
not likely to be important in dictating prenylation specificity since
Adamson et al.(19) changed this lysine to a leucine
without any effect on either farnesylation or geranylgeranylation in
crude systems. Thus, the CAAX box and the adjacent C are
unlikely to account for the ability of RhoB to be farnesylated by
GGTase-1. The responsible sequence must be on the
NH-terminal side of the CAAX box.
One other
small GTP binding protein, K-RasB, is known to have a regulatory
sequence upstream of a CAAX box. Recently, this protein was
shown to be geranylgeranylated by GGTase-1 even though its CAAX box terminates in methionine, which is not the preferred substrate
for geranylgeranylation. Studies with chimeric proteins
indicated that the high affinity of K-RasB for GGTase-1 was
attributable in part to a polylysine sequence immediately upstream of
the CAAX box.
Inasmuch as RhoB does not have a
polylysine sequence, this explanation cannot suffice for this protein.
The unusual ability to transfer different prenyl groups to different protein acceptors renders GGTase-1 highly unusual among transferase enzymes. Further studies are clearly needed to define the mechanism for this selectivity, and eventually to find the structural basis.