(Received for publication, August 25, 1995)
From the
To study the function of the farnesyl modification of Ras, the farnesyl group and a variety of its structural analogs, which lack one or more double bonds and/or the methyl groups, were enzymatically incorporated into recombinant H-Ras in vitro. These proteins were used in a cell- and membrane-free, Ras-dependent mitogen-activated protein kinase (MAP kinase) activation system derived from Xenopus laevis eggs to examine the contribution of the farnesyl group toward the activation of the kinase. Whereas non-farnesylated H-Ras is unable to activate MAP kinase, farnesylation of H-Ras alone, in the absence of further processing, is sufficient to cause the activation of MAP kinase in this system. All of the analogs of the farnesyl group, when incorporated into H-Ras, support the activation of the kinase to variable extents. These results suggest a direct but fairly nonspecific interaction of the farnesyl moiety of H-Ras with a soluble upstream activator of MAP kinase.
The Ras GTP-binding proteins play a pivotal role in a variety of
signal transduction and differentiation
processes(1, 2) . Ras is also involved in the
generation of a number of human cancers, and several oncogenic point
mutations of Ras are known(3, 4) . Ras is activated by
the conversion of the GDP-bound inactive form to the GTP-bound active
form in response to various extracellular signals(5) . A
variety of extracellular signals can activate mitogen-activated protein
kinase (MAP kinase) ()(also known as extracellular
signal-regulated kinase (ERK)) through both Ras-dependent and
Ras-independent mechanisms(6) . A Ras-dependent pathway linking
the epidermal growth factor receptor to MAP kinase, through the protein
kinase Raf, has been elucidated(7, 8) . A
Ras-dependent, Raf-independent MAP kinase activation system has also
been identified(9, 10) .
Ras proteins are part of
the group of proteins that are post-translationally
prenylated(11) . In the case of Ras, this modification involves
the attachment of the farnesyl group to the protein through a thioether
linkage to a cysteine located four residues from the carboxyl terminus,
followed by removal of the three carboxyl-terminal amino acids and
methylation of the newly exposed -carboxyl group of the farnesyl
cysteine residue(12) . Additionally, H-Ras and N-Ras, but not
K-Ras, undergo palmitoylation at one or more upstream cysteine
residues(13) . Although necessary for the normal and oncogenic
functions of many proteins, including Ras(14) , the specific
properties imparted by these post-translational modifications have, to
a large extent, remained unclear. Prenylation of proteins has been
implicated in membrane binding (13, 15) and in
protein-protein recognition(16, 17) . Furthermore, the
relative contribution of each of the processing steps is unknown.
Previously we developed a cell-free assay system, derived from Xenopus laevis eggs, to identify a direct target molecule for Ras. In this system, Ras promotes the activation of MAP kinase through MAP kinase kinase/ERK kinase (MEK). Using this system, we have identified a Ras-dependent MEK kinase termed REKS (Ras-dependent ERK Kinase Stimulator)(18) . Subsequently, we have highly purified this protein and have determined that it is distinct from c-Raf-1, Mos, and mSte11, all of which are known to both phosphorylate and activate MEK (19) . Furthermore, c-Raf-1 partially purified by Mono-S chromatography from X. laevis eggs did not cause activation of MAP kinase either in the presence or the absence of Ras under these assay conditions(20) . We have previously shown that fully processed K-Ras (i.e. farnesylated, proteolysed, and methylated) is far more active than unmodified K-Ras in the REKS-dependent activation of MAP kinase(19, 21) . Similar results for the Ras-dependent activation of yeast adenylate cyclase have been reported(22, 23) . In the earlier reports, we could not exclude the possibility that the unmodified Ras was denatured, nor was it possible to examine the individual processing steps of Ras (see above) to determine which of these steps provides the critical modification.
H-Ras protein (1 nmol) was incubated with
recombinant PFT (0.25 nmol), obtained as described(29) , and 20
µM FPP or FPP analogs in 100 µl of buffer (16
mM Tris-HCl, pH 7.5, 0.34 M NaCl, 10 mM MgCl, 0.65 mM dithiothreitol, 12.6 µM ZnCl
, 12.5 mM EDTA) for 3 h at 30 °C.
Buffer exchange and removal of excess pyrophosphate ester were
performed using a spin column of P-6 gel (Bio-Rad) equilibrated in
column buffer (20 mM Tris-HCl, pH 8.0, 5 mM
MgCl
, 1 mM EDTA, 1 mM dithiothreitol).
The eluant was diluted with an equal volume of column buffer containing
1.2% CHAPS. Geranylgeranylated H-Ras was produced by incubation of
geranylgeranyl pyrophosphate and recombinant protein-geranylgeranyl
transferase-I (obtained from P. Casey, Duke University) with bacterial
H-Ras containing a modified carboxyl terminus (CVLL, obtained from P.
Casey, Duke University).
Eggs were obtained from fully mature X. laevis females and activated by electric shock (30) to drive them into interphase and inactivate endogenous MAP kinase and MEK activities(31, 32) . The cytosol of activated eggs was obtained by centrifugation as described (20) . REKS was partially purified by Mono-Q chromatography(19) .
It has been proposed that the role of the farnesyl group is primarily to bind proteins to membranes(13) . In the Ras- and Raf-dependent system, the primary function of Ras appears to be the recruitment of Raf to the plasma membrane, where this kinase is somehow activated through phosphorylation by an unknown kinase. Raf, in turn, activates the protein kinase MEK, which subsequently activates MAP kinase(7, 8) . Although Raf has been identified as one of the downstream targets of Ras, recent evidence indicates that other targets exist(9, 10, 38, 39) . In fact, Zheng et al.(40) have presented evidence that the Raf pathway plays only a relatively minor role in the growth factor-induced activation of MEK and, subsequently, of MAP kinase. Hence, an important aspect of the present system is that it is membrane free. To confirm this, the various components of the cell-free system were analyzed for their membrane content. This was done by searching for the common fatty acid linolenic acid by negative chemical ionization mass spectrometry. Only a trace amount of this fatty acid was found, representing 1 nM or less in the final assay mixture, which is well below the concentration of H-Ras added to the assays, indicating that the assay mixture was virtually membrane free.
For the present studies, we used REKS that was partially purified from X. laevis eggs by ion exchange chromatography to a completely soluble form to study the role of the farnesyl group of Ras in promoting the activation of MAP kinase. We have been able to quantitatively farnesylate recombinant H-Ras produced in Escherichia coli by incubation with FPP and recombinant PFT (Fig. 1). The unmodified H-Ras was prepared in such a way as to produce an intact carboxyl-terminal CVIS sequence, necessary for farnesylation. Lower than maximal incorporation of the farnesyl group into H-Ras is observed when using other procedures for isolating this protein, probably due to proteolytic damage at the carboxyl terminus.
Figure 1: A, incorporation of the farnesyl group and its analogs into H-Ras as monitored by SDS-polyacrylamide gel electrophoresis (15% acrylamide, 0.087% bisacrylamide)(28) . Arrows indicate the positions of unmodified (U) and lipidated (L) H-Ras. Lane 1, unmodified H-Ras produced in E. coli; lane 2, complete reaction mixture containing H-Ras, FPP, and PFT; control farnesylation reactions containing all ingredients except FPP (lane 3), PFT (lane 4), or H-Ras (lane 5); lipidation reactions containing all ingredients except that FPP is replaced with (Z)-3-methyl-2-dodecenyl pyrophosphate (lane 6), (7S)-6,7-dihydrofarnesyl pyrophosphate (lane 7), or 6,7,10,11-tetrahydrofarnesyl pyrophosphate (lane 8). The incorporation of the other analogs into H-Ras was also quantitative (not shown). B, structures of the farnesyl group and its analogs.
Using Mono-Q purified REKS, the GTPS-bound form of
non-prenylated H-Ras, even at high concentrations, does not detectably
activate MAP kinase (Fig. 2, B and C). In
marked contrast, farnesylated but otherwise unprocessed
GTP
S
H-Ras, produced by the in vitro farnesylation
of recombinant H-Ras, is as active as fully processed H-Ras or K-Ras in
activating MAP kinase. Fully processed K-Ras was used as a standard to
compare the activity of the modified H-Ras proteins due to the
difficulty in purifying and handling fully processed H-Ras. However,
fully processed H-Ras supports activation of MAP kinase, through REKS,
to roughly the same extent as fully processed K-Ras (Fig. 2A).
Figure 2:
A,
activation of MAP kinase, as measured by incorporation of P into MBP, by the indicated concentrations fully
processed GTP
S
K-Ras (
) or fully processed
GTP
S
H-Ras (
). 100% MBP phosphorylation is the
activation caused by 50 nM fully processed
GTP
S
K-Ras. B, activation of MAP kinase, as measured
by incorporation of
P into MBP, by the indicated
concentrations of GTP
S
Ras containing prenyl chains of
different length.
, fully processed K-Ras;
, farnesylated
H-Ras;
, geranylgeranylated H-Ras;
, geranylated H-Ras;
, non-prenylated H-Ras, prepared by in vitro prenylation with all of the reaction components except FPP.
Control prenylations with H-Ras in the absence of PFT or H-Ras (CVLL)
and protein geranylgeranyl transferase-I in the absence of
geranylgeranyl pyrophosphate also caused no activation. 100% MBP
phosphorylation is the activation caused by 50 nM fully
processed GTP
S
K-Ras; in this case, 100% = 43.3 pmol. C, activation of MAP kinase, as measured by the incorporation
of
P into MBP by the indicated concentrations of
GTP
S
Ras modified with farnesyl analogs.
, fully
processed K-Ras;
, farnesylated H-Ras;
,
10,11-dihydrofarnesylated H-Ras;
,
racemic-6,7,10,11-tetrahydrofarnesylated H-Ras;
,
(7S)-6,7-dihydrofarnesylated H-Ras;
,
3-methyl-2-dodecenylated H-Ras;
, non-prenylated H-Ras prepared
by in vitro prenylation with all of the reaction components
except FPP). 100% MBP phosphorylation is the activation caused by 50
nM fully processed GTP
S
K-Ras; in this case, 100%
= 43.3 pmol. The results shown are representative of three
independent experiments. The error for each individual point is less
than ±10%.
This result clearly shows that inactive unmodified H-Ras can be converted to a fully active form solely by farnesylation, and thus further processing (carboxyl-terminal proteolysis and methylation and palmitoylation) are not required.
To determine if the precise structure of the farnesyl group is necessary for REKS-dependent activation of MAP kinase, a variety of farnesyl analogs (Fig. 1B) were also incorporated enzymatically into recombinant H-Ras (Fig. 1A). Using this method, the activity of these differently modified proteins toward the activation of MAP kinase in the cell-free system described above was examined (Fig. 2, B and C). H-Ras proteins containing the farnesyl analogs were able to support the activation of MAP kinase to variable extents. A significant dependence on the size of the prenyl group was observed (Fig. 2B); H-Ras bearing the longer 20-carbon geranylgeranyl group was the best activator, while H-Ras containing the shorter 10-carbon geranyl group was the least potent activator. With regard to the structurally modified farnesyl analogs, saturation of either of the more distal double bonds of the farnesyl group caused a small decrease in activity (Fig. 2C). H-Ras containing the (Z)-3-methyl-2-dodecenyl side chain was one of the least potent activators; this group, while the same length as the farnesyl group, structurally bears the least resemblance to the native lipid. The trend observed indicates that there is a component of specificity based on the prenyl structure of the side chain as well as a component based on hydrophobicity. Therefore, it appears that H-Ras, in this system, is interacting at least in part through its prenyl group with some soluble component or components of the MAP kinase activation system.
As shown in Fig. 3, the GDPS-bound form of
farnesylated H-Ras is weakly stimulatory but does inhibit the
stimulation of MAP kinase by farnesylated GTP
S
H-Ras, giving
50% inhibition at approximately 30 nM. Non-farnesylated
GTP
S
H-Ras neither activates MAP kinase nor inhibits the
activation of MAP kinase by farnesylated GTP
S
H-Ras. This
result also suggests that the farnesyl group is needed for the binding
of H-Ras to some protein component of the MAP kinase activation system.
Figure 3:
Competition of the farnesylated
GTPS
H-Rasdependent activation of MAP kinase by farnesylated
GDP
S
H-Ras or non-farnesylated GTP
S
H-Ras. Effect
of the indicated concentrations of farnesylated GDP
S
H-Ras
(
) or non-farnesylated GTP
S
H-Ras (
) on the
activation of MAP kinase in the presence (solid line) or
absence (dashed line) of 6.25 nM farnesylated
GTP
S
H-Ras. 100% MBP phosphorylation is the activation caused
by 6.25 nM farnesylated GTP
S
H-Ras alone. The
results shown are representative of three independent experiments. The
error for each individual point is less than
±10%.
Based on the current studies it is concluded that inactive bacterially produced H-Ras can be converted to an active form solely by farnesylation using FPP and recombinant PFT. Our results indicate that farnesylation of Ras is the critical modification needed for the Ras- and REKS-dependent activation of MAP kinase and that further processing (proteolysis, methylation, and palmitoylation) is not necessary for this aspect of Ras function. This is demonstrated by the results showing that the farnesylated but otherwise unprocessed H-Ras is comparable to fully processed K-Ras or H-Ras in its ability to stimulate MAP kinase in this system. This is further illustrated by the inability of non-farnesylated H-Ras to either stimulate the activity or to inhibit the activity caused by the farnesylated H-Ras. It appears that the dependence of REKS-mediated stimulation of MAP kinase by Ras on the farnesyl group arises from a fairly nonspecific hydrophobic interaction of the prenyl group with some soluble component of the system. The various analogs of the farnesyl group, when incorporated into H-Ras, cause only relatively small changes in the extent of activation.
We have recently purified a REKS activity from bovine brain cytosol. The bovine REKS was found to be a complex of three proteins, one of which was B-Raf(41) . While the Xenopus REKS did not cross-react with anti-B-Raf antibody(18) , we cannot rule out that Xenopus REKS is an isoform of Raf. Clearly, the interaction of this protein with Ras is different than that previously described for Raf(7, 8) . Unlike the membrane binding function of Ras described previously, the effects of farnesylation in this system are not the result of membrane binding, as no membranes are present in the assay. Moreover, if the role of the farnesyl group in the H-Ras- and REKS-dependent activation of MAP kinase is only to bind H-Ras to membranes or other possible interfaces in this in vitro system, the level of maximal activation caused by all of the lipidated H-Ras proteins should be the same; the results in Fig. 2clearly show that this is not the case. Taken together, the data strongly suggest that there is a direct interaction of the farnesyl group of H-Ras either with REKS, or with an as yet undetermined soluble component of the system, and that the activation of MAP kinase, in this system, is not dependent on the binding of Ras to membranes. Alternatively, prenylation may cause a structural change in Ras, which allows it to interact with its target protein. In any case, these experiments demonstrate a role for the prenyl group of Ras, which is distinct from that of a simple membrane anchor and is more similar to the prenyl protein-protein interaction model described for heterotrimeric G proteins in which the prenyl group appears to play a crucial role in subunit interaction(16) .