From the
A key event for Ras transformation involves the direct physical
association between Ras and the Raf-1 kinase. This interaction promotes
both Raf translocation to the plasma membrane and activation of Raf
kinase activity. Although substantial experimental evidence has
demonstrated that Raf residues 51-131 alone are sufficient for
Ras binding, conflicting observations have suggested that the Raf
cysteine-rich domain (residues 139-184) may also be important for
interaction with Ras. To clarify the role of the Raf cysteine-rich
domain in Ras-Raf binding, we have compared the ability of two distinct
Raf fragments to interact with Ras using both in vitro Ras
binding and in vivo Ras inhibition assays. First, we
determined that both Raf sequences 2-140 and 139-186
(designated Raf-Cys) showed preferential binding to active, GTP-bound
Ras in vitro. Second, we observed that Raf-Cys antagonized
oncogenic Ras(Q61L)-mediated transactivation of Ras-responsive elements
and focus-forming activity in NIH 3T3 cells and insulin-induced
germinal vesicle breakdown in Xenopus laevis oocytes in
vivo. This inhibitory activity suggests that Raf-Cys can interact
with Ras in vivo. Taken together, these results suggest that
Ras interaction with two distinct domains of Raf-1 may be important in
Ras-mediated activation of Raf kinase activity.
Numerous biochemical and genetic studies have positioned Raf
downstream of Ras in Ras-dependent signal transduction pathways
(1) that lead to the activation of the mitogen-activated protein
kinases
(2) . Furthermore, several recent investigations have
used in vitro protein binding assays and the in vivo yeast two-hybrid system to demonstrate a direct physical
association between the Ras and Raf proteins, thus identifying Raf as a
key downstream target, or effector, of Ras-mediated signal transduction
(3, 4, 5, 6, 7, 8) .
This interaction requires the effector domain of Ras (residues
32-40) and a region in the amino-terminal regulatory domain of
Raf. The region encompassing Raf-1 amino acids 51-131 has been
shown to be sufficient for interaction with Ras
(6, 9, 10) . In addition, a single amino acid
mutation (R89L) in this domain of Raf disrupts the interaction with Ras
in vitro and prevents Ras-mediated activation of Raf in Sf9
insect cells
(11) .
While Raf residues 51-131 clearly
define a minimal Ras binding domain, other evidence has suggested that
the Raf cysteine-rich domain (residues 139-184) may also be
involved in Ras-Raf binding. First, a single point mutation (C168S) in
this domain was found to reduce Raf (residues 1-257) binding to
Ras in both two-hybrid and in vitro binding assays
(5) . Second, it was observed that renaturation of
Raf-(1-257) in the presence of zinc, which is required for
folding of the cysteine-rich domain, led to greater restoration of Ras
binding activity
(4) . Finally, we and others observed that Raf
residues 131-147, which are adjacent to and extend partially into
the cysteine-rich domain, are critically important for conferring high
affinity binding to Ras in vitro (9, 10) .
However, it remains to be clarified whether the Raf cysteine-rich
domain enhances Ras association with Raf residues 51-131 or
independently interacts with Ras. To address this question, we have
utilized both in vitro and in vivo analyses to
characterize Ras interaction with the Raf cysteine-rich domain. We
observe that the isolated Raf cysteine-rich domain shows high affinity,
guanine nucleotide-dependent binding to Ras in vitro and can
function as a dominant inhibitor of Ras signaling and transformation
in vivo. Thus, the Raf NH
Fig. 2
shows data
obtained from an ELISA using various purified recombinant glutathione
S-transferase-Raf fragments and Ras. For these experiments we
prepared stoichiometric complexes of Ras bound to GMPPCP. This
nonhydrolyzable GTP analog eliminates GTP hydrolysis during the course
of the experiment and is useful for determining whether various Raf
fragments bind to Ras in a GTP-dependent fashion. Binding curves were
generated by serially diluting Ras complexed to either GMPPCP or GDP
into wells containing 100 pmol of each glutathione
S-transferase-Raf protein. We observed that both Raf-Cys and
Raf-N2 showed high affinity binding to Ras. In contrast, a Raf fragment
lacking the NH
We first evaluated the ability of each Raf-1 fragment
to inhibit oncogenic Ras-mediated stimulation of transcription from a
Ras-responsive reporter plasmid (Fig. 3 A). As shown
previously
(22, 23) , Raf301 reduced Ras-induced
transcriptional activation, whereas wild type Raf further stimulated
Ras transcriptional activation (data not shown). Additionally, we
observed that both Raf-Cys and Raf-N1 reduced activation by oncogenic
Ras in vivo. Furthermore, Raf constructs containing both the
minimal Ras binding sequence (residues 51-131) and sequences from
the cysteine-rich domain (residues 139-184) showed the strongest
inhibition of transcriptional activity, possibly due to the cooperative
binding of the two independent binding sites.
Finally, we observed
that Raf-Cys and Raf-Cys+ could block insulin-induced germinal
vesicle breakdown in X. laevis oocytes
(Fig. 3 C). Previous studies have shown that this insulin
response is dependent on Ras activity
(24, 25, 26) . This inhibitory activity could be
reversed by co-injection of excess oncogenic Ras protein (data not
shown). Thus, we have observed that, like the truncated Raf fragment
containing the residues corresponding to the minimal Ras binding
sequence (residues 51-131), the Raf cysteine-rich domain can
inhibit oncogenic Ras transcriptional activation, transforming
activity, and oocyte maturation. Taken together, these observations
in vivo are consistent with our demonstration that Raf-Cys can
bind directly to Ras in vitro and support the possibility that
Raf contains a second distinct Ras binding site, which can promote
Ras-Raf interaction in vivo.
Although
previous studies have implicated the Raf cysteine-rich domain in
facilitating Ras binding, the precise nature of this role was unclear.
Our observations that Raf-Cys shows high affinity, GTP-dependent
binding to Ras in vitro and can antagonize Ras function in
vivo provide evidence that the cysteine-rich domain constitutes a
second Ras binding site in the Raf NH
Our observation that the cysteine-rich domain
can bind Ras can be reconciled with the report that a single amino acid
substitution at Raf residue 89 is sufficient to abolish Ras-Raf
interaction
(11) . While it is possible that expression of the
isolated cysteine-rich domain may have unmasked a nonspecific binding
activity for Ras that is not a property of full-length Raf, our
observation that this domain preferentially interacts with the active,
GTP-complexed form of Ras argues that this interaction is specific.
Instead, we propose that the Ras binding site in the cysteine-rich
domain is protected in the intact, unstimulated protein, possibly due
to negative regulatory contacts with the COOH-terminal domain. We
propose that Ras may initially interact with Raf via contacts with
residues between positions 51 and 131. The initial Ras-Raf interaction
and/or the resultant membrane translocation of Raf may promote exposure
of the cysteine-rich domain for interaction with Ras and possibly other
activating molecules. Thus, Ras interaction with these two binding
sequences may be necessary to induce the removal of the negative
regulatory action of the Raf NH
We thank Elaine S. G. Bardes for performing the oocyte
injections and John Carpenter for help in generating reagents. We thank
Geoff Clark, Adrienne Cox, John O'Bryan, and Lawrence Quilliam
for critical comments and Ashley Overbeck for excellent assistance in
manuscript preparation.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
terminus contains two
distinct Ras binding domains that may be important for Ras-mediated
activation of Raf kinase activity.
Molecular Constructs
To generate mammalian
expression vector constructs encoding different Raf fragments, we
isolated BamHI- EcoRI DNA fragments from pGEX- raf constructs that encoded the different Raf sequences indicated in
Fig. 1(12) and introduced each into the BamHI
site of the pCGN-hyg mammalian expression vector
(13) (generously provided by M. Ostrowski, Duke). Raf301 encodes
a full-length mutant human Raf-1 sequence that contains a single amino
acid substitution (K to W) in the ATP binding site, which inactivates
its kinase activity
(14) .
Figure 1:
Molecular constructs of Raf-1. The
amino acid residues encoded by the various human Raf constructs are
indicated as well as the locations of the Raf cysteine-rich and kinase
domains. The pGEX- raf bacterial expression and pCGN- raf mammalian expression constructs were generated as described under
``Experimental Procedures.''
Expression and Purification of Ras and Glutathione
S-Transferase-Raf Proteins
The pAT- rasH bacterial
expression plasmid and procedures for expression and purification have
been described previously
(15) . Ras protein complexed to
GMPPCP,(
)
a nonhydrolyzable GTP analog
(Boehringer Mannheim), was prepared as described elsewhere
(16) by replacement of GDP bound to Ras. Glutathione
S-transferase-Raf proteins were purified as described
previously
(12) .
Enzyme-linked Immunosorbent Assay (ELISA) for Measuring
Ras-Raf Interaction in Vitro
Purified glutathione
S-transferase and glutathione S-transferase-Raf
proteins were plated onto 96-well microtiter plates (Costar) coated
with 0.025 mg/ml poly-L-lysine and allowed to bind overnight.
Wells were blocked for 1 h at room temperature with a
phosphate-buffered saline solution containing 130 mM NaCl, 10
mM NaHPO
, and 3 mM KCl, pH
7.40 (PBS), which was supplemented with 0.5% gelatin, 0.05% Tween 20,
and 0.2% sheep serum (PBSGTS). H-Ras complexed to either GDP or GMPPCP
was captured in PBSGTS for 1 h at concentrations ranging from 31
nM to 2 µM. The plates were washed 3 times in PBS
with 0.1% Tween 20 and then incubated for 1 h with an anti-H-Ras
antibody (LAO69) (Quality Biotech) diluted 2000-fold in PBSGTS. The
wash step was repeated, and the plates were incubated with a 1:1000
dilution of sheep anti-mouse IgG-alkaline phosphatase conjugate (Sigma)
in PBSGTS for 30 min, followed by development with the chromogen,
p-nitrophenyl phosphate (Sigma). Optical densities (405 nm)
were read after 30 min in a Biotech microtiter plate reader. The
intensity of the absorbance was directly related to the amount of Ras
bound. The concentration of Ras at half-maximal binding was determined
as described previously
(12) .
NIH 3T3 Transcription Activation and Transformation
Assays
NIH 3T3 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% calf serum. DNA
transfections were performed as described previously using the calcium
phosphate precipitation technique
(17) . The pB4X-CAT reporter
plasmid contains the CAT gene, and CAT is driven by a minimal promoter
that contains the ets/AP-1 Ras-responsive promoter element
(18) . Cells were co-transfected with the
pZIP- rasH(Q61L) plasmid DNA encoding H-Ras(Q61L) (50 ng/dish)
and either the empty pCGN-hyg vector or the appropriate pCGN- raf construct (5 µg/dish) along with 1 µg of the pB4X-CAT
reporter. Forty-eight h after transfection, total cell lysates were
prepared, and CAT activity was determined as described previously
(19) . A similar co-transfection analysis was done to assess the
ability of each Raf mutant (5 µg/dish) to block Ras(Q61L) (10
ng/dish) transforming activity. Transfections were performed in
triplicate, and transformed foci were quantitated after 14-16
days.
Preparation and Experimental Manipulation of
Oocytes
Oocytes were isolated from primed Xenopus laevis ovaries by procedures described previously
(20) . Isolated
stage VI oocytes were injected with 200 µg of the indicated
glutathione S-transferase-Raf protein (20 oocytes/Raf protein)
in a volume of 40 nl. Subsequent to the injection, the oocytes were
treated with 1 µM insulin and maintained in modified
Barth's solution with Ca, between 18 and 20
°C, for maturation. Insulin-induced maturation was scored at
definite time intervals by counting the number of oocytes undergoing
germinal vesicle breakdown manifested by the appearance of a whitish
spot at the pigmented animal pole of the oocytes.
The Raf Cysteine-rich Domain Shows High Affinity,
GTP-dependent Association with Ras in Vitro
Although extensive
analyses have clearly shown that Raf residues 51-131 alone are
sufficient for interaction with Ras, it is presently unclear what the
precise role of the cysteine-rich domain (residues 139-184) is in
mediating Ras-Raf binding. To address the possibility that this domain
can bind Ras, we have conducted both ELISA and direct binding assays
(12) to determine if Raf-Cys binds directly to Ras in vitro and to assess the dependence of this association on the guanine
nucleotide-bound state of Ras. For these assays, we compared the
binding activities of a glutathione S-transferase fusion
protein containing Raf-Cys with the binding properties of glutathione
S-transferase-Raf-(2-140) (designated Raf-N2), which
contains the minimal Raf sequence required for Ras binding
(51-131) and includes only the first two residues from the
cysteine-rich domain (Fig. 1).
-terminal regulatory domain (containing Raf
residues 273-648; designated Raf-C) did not bind to Ras.
Furthermore, the concentration dependence of the binding curves
demonstrates that much higher concentrations of Ras-GDP relative to
Ras-GMPPCP were required to saturate binding to either Raf-N2 or
Raf-Cys, and both fragments showed
7-fold preferential binding to
Ras-GTP relative to Ras-GDP. Therefore, these results indicate that two
distinct regions within the NH
-terminal regulatory domain
of Raf-1 are capable of specific interaction with GTP-Ras in
vitro.
Figure 2:
The Ras cysteine-rich domain
preferentially binds to Ras-GTP. Three distinct Raf fragments, prepared
as glutathione S-transferase-Raf fusion proteins, were tested
for their ability to bind Ras-GDP and Ras-GMPPCP by antigen capture
ELISA as described under ``Experimental Procedures.'' Both
Raf-N2 and Raf-Cys exhibited preferential binding to
Ras-``GTP,'' whereas Raf-C did not bind to either form of
Ras. Raf-N2 and Raf-Cys bound to Ras-GTP with 130 and 48 nM
Cvalues compared with 870 and 310 nM for
Ras-GDP, respectively. All experiments were performed in triplicate
with glutathione S-transferase (control) absorbance values
subtracted from the absorbance values of glutathione
S-transferase-Raf proteins.
Two Independent Amino-terminal Domains of Raf Block
Oncogenic Ras Signaling and Transformation in Vivo
Previous
studies have yielded discrepancies between the ability of different Ras
and Raf mutants to bind when performed by in vitro versus in vivo two-hybrid analyses
(21) . Therefore, we
employed three biological assays to determine if Ras-Cys could
associate with Ras in vivo and consequently block Ras
signaling and transforming activity. We and others have previously
shown that kinase-deficient mutants of Raf can block Ras function by
antagonizing Ras interaction with its downstream effectors
(14, 22, 23) . Thus, an inhibitory activity of a
Raf mutant is a strong indication of an in vivo interaction
with Ras that prevents Ras association with full-length, endogenous Ras
effectors. We included the well characterized, kinase-deficient Raf301
dominant inhibitory protein as a control for these studies
(14) .
Figure 3:
Multiple, independent domains of Raf-1
inhibit Ras-dependent signaling in vivo. Panel A, Raf fragments block Ras(Q61L) stimulation of
transcriptional activation of the ets/AP-1 Ras-responsive
element. Results from one of four experiments performed in duplicate
are shown. CAT assays were performed as described under
``Experimental Procedures.'' Panel B,
co-transfection of various Raf fragments significantly reduces
Ras(Q61L) focus-forming activity. Two experiments were performed in
triplicate, and results of one are presented. Relative focus-forming
units ( FFU) shown are normalized to the activity of Ras(Q61L)
(8.97 10
foci/µg of transfected DNA). Panel C, stage VI oocytes from X. laevis were isolated
and injected with 200 µg each of the indicated proteins. The
oocytes were then treated with insulin and scored for germinal vesicle
breakdown ( GVBD), as described under ``Experimental
Procedures.'' The data are presented as the percent of oocytes
undergoing germinal vesicle breakdown as a function of time after
treatment with insulin (compare 15-h time point). GST, glutathione
S-transferase.
We next determined
whether Raf-Cys could also inhibit oncogenic Ras(Q61L) focus-forming
activity. Co-transfection of Raf-Cys as well as other Raf fragments
that contained overlapping sequences showed >50% inhibition of
oncogenic Ras(Q61L) focus-forming activity (Fig. 3 B).
One possible explanation for the inhibitory action of these Raf
fragments is that they inhibit cell growth in a nonspecific manner.
However, we observed that for NIH 3T3 cells transfected with each
raf construct, equivalent numbers of hygromycin-resistant
colonies are obtained following drug selection of cells transfected
with vector only or with vector constructs encoding each of the Raf
fragments (data not shown), thus arguing against this possibility.
Additionally, we have isolated stably transfected cells that co-express
both oncogenic Ras(Q61L) and certain Raf fragments, and these cells
show a flatter morphology, which is more characteristic of
untransformed NIH 3T3 cells (data not shown).
A Noncatalytic Carboxyl-terminal Raf Mutant Blocks Ras
Signaling in Vivo
Although Raf-C showed no Ras binding activity
in vitro (Fig. 2 A), we did observe that this
Raf fragment could inhibit oncogenic Ras-mediated transcription
activation (Fig. 3 A), focus-forming activity
(Fig. 3 B), and insulin-induced oocyte maturation
(Fig. 3 C). Raf-C (residues 273-648) lacks both
NH-terminal Ras binding domains and contains the
serine-threonine kinase domain (residues 333-625). However, a
previous study has demonstrated that Raf-C lacks transforming activity
(27) . Thus, we suggest that the inhibitory activity of Raf-C is
not a consequence of complex formation with Ras but instead that it
inhibits Ras transforming activity by complex formation with the Raf
substrate, MEK. MEK has been shown to bind to Raf COOH-terminal
sequences that include the kinase domain
(7) . Consistent with
this possibility, we found that exogenously introduced wild type MEK
reversed the inhibitory action of Raf-C but not of Raf-N1 (data not
shown). Therefore, Raf-C may function as a dominant inhibitor of MEK
activity, thereby inhibiting signaling downstream of Ras.
-terminal regulatory
domain. These results provide an explanation for the reduction in Ras
binding, observed both in vitro and in vivo, to an
amino-terminal Raf fragment, Raf-(1-257), containing a mutation
(C168S) that disrupts the integrity of the cysteine-rich domain
(5) . Our finding is also consistent with the observation that
Raf NH
-terminal fragments containing residues 131-147
(which includes residues 139-147 of the Raf cysteine-rich domain)
display increased affinity for binding Ras
(9, 10) . A
recent investigation has demonstrated partially reduced coprecipitation
and in vitro binding between Ras and a Raf mutant lacking the
cysteine-rich domain
(28) , supporting our finding of two Ras
binding sites in Raf.
terminus and consequently
may facilitate Raf activation by additional events. These may include
interaction with 14-3-3 proteins
(29) , tyrosine kinases
(30) , or lipids
(12) .
,
-methylenetriphosphate); CAT,
chloramphenicol acetyltransferase; ELISA, enzyme-linked immunosorbent
assay; MEK, mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.