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INTRODUCTION |
Stimulation of growth factor receptors activates Raf kinases via
small GTP-binding proteins (Ras proteins) resulting in proliferation, differentiation, and cell survival (1, 2). The Raf kinase family
consisting of A-Raf, B-Raf, and C-Raf shares three highly conserved
regions: CR1, CR2, and CR3. The CR3 region represents the C-terminal
catalytic domain, whereas CR1 contains Ras-binding domain
(RBD)1 and a zinc-binding
domain called cysteine reach domain (CRD). While C-Raf is a
ubiquitously expressed protein with an apparent molecular weight
(Mr) of 72,000-74,000, B-Raf
(Mr 95,000) was found to be expressed
preferentially in neuronal tissues and testis (3, 4). A-Raf, the
smallest member with a Mr of 68,000, is limited
in expression mainly to urogenital tissue. Cytosolic C-Raf exists as a
300-500 kDa large complex including heat shock and 14-3-3 proteins
(5-7). Upon stimulation of cell surface receptors, C-Raf undergoes a
series of activation events at the inner side of plasma membrane
mediated by Ras and 14-3-3 proteins, including dephosphorylation and
phosphorylation events (1, 2, 6).
Although Ras proteins play a crucial role in the activation of Raf the
exact mechanism of Ras-Raf coupling is not completely understood. The
RBD of C-Raf comprises residues 51-131 and binds directly to the
so-called switch-I region of the activated Ras-GTP (8). A single point
mutation in the Raf-RBD (R89L) abrogates Ras binding and blocks Raf
activation (9). But not only RBD is involved in the Ras-Raf binding
process. The CRD of C-Raf encompassing residues 139-184 also appears
to be involved, although with lower binding affinity. Moreover, the
interactions between CRD and fully processed Ras proteins have been
proposed to be crucial for effective C-Raf activation (10, 11). Besides
the ability to interact with processed Ras proteins, C-Raf-CRD was
reported to possess binding sites for 14-3-3 proteins (12) in addition
to the established C-Raf/14-3-3-binding sites at phosphoserine 259 and
phosphoserine 621 (13, 14). The C-Raf-CRD was further proposed to bind
to phospholipids at the plasma membrane. The first evidence that C-Raf-CRD interacts with phosphatidylserine (PS) (15, 16) was not
unexpected, since C-Raf-CRD exhibits a structure very similar to the
C1-domain of protein kinase C family proteins (17, 18), although
C-Raf-CRD does not bind tumor promoting phorbol esters and binds only
weakly diacylglycerol (5). Taken together, C-Raf-CRD appears to have a
complex and multifunctional role in the regulation of C-Raf kinase
activity. Further investigations revealed (19) that the C-Raf kinase
interacts not only with PS but also with phosphatidic acid (PA). In
contrast to PS, the interaction sites for PA have been localized within
the CR3 region of C-Raf kinase. Whereas the agonist-induced hydrolysis
of phosphatidylcholine (PC) results in PA production, the cleavage of
sphingomyelin (SM) leads to generation of ceramides. Both PA and
ceramide are considered to act as intracellular lipid second
messengers. Interactions between ceramides and C-Raf kinases have been
observed (20, 21).
In the current study a quantitative approach for Raf associations with
lipids has been performed using Biomolecular Interaction Analysis, a technology based on surface plasmon resonance, which allows monitoring of biomolecular interactions in real time. The newly
developed L1-sensor chip permits capturing of intact lipid vesicles by
hydrophobic residues localized on the chip surface (22). The vesicles
captured in this way provide a chemically and physically stable
environment which resembles cellular membranes.
The interactions of a number of signaling molecules with the plasma
membrane depend not only on their association with particular lipids: a
special architecture of some lipid microdomains called rafts plays an
important role in the transduction of signals. Such lipid rafts form
liquid-ordered phases in the lipid bilayer and are dispersed in the
bulk of a liquid-disordered phase of the plasma membrane
(23-26). These microdomains have been found to be highly
enriched in cholesterol and sphingomyelin, making them resistant to
solubilization with non-ionic detergents (for example, Triton X-100)
thus yielding detergent-resistant membrane fractions (26). The special
features of rafts are probably due to the tight packing of highly
saturated fatty acid residues in sphingolipids with cholesterol. In
contrast to rafts, the non-raft regions are composed mainly of
glycerophospholipids (GPLs). Depending on cell type,
detergent-resistant membranes may contain caveolin, a 21-24-kDa
structural protein, which appears to mediate the formation of 60-100
nm flask-shaped invaginations (caveolae), which are distinct from the
much larger (250-300 nm) clathrin-coated pits (25, 27) and may thus
represent a subclass of rafts. Several other structural/integral
proteins have been found to be associated with rafts, such as
flotillins/reggies (28-30), stomatin (31), and the MAL/BENE
proteolipids (32). Additionally, rafts (and caveolae) are highly
enriched in components that mediate signal transduction and therefore
are thought to increase the efficiency and specificity of signal
transduction processes. Besides glycosylphosphatidylinositol-anchored proteins that associate with rafts at the extracellular side of the
plasma membrane, several acylated (and non-acylated) proteins such as
heterotrimeric and small G-proteins, Src kinases, eNOS, Shc, Nck, and
MAPK have been found to be attached to the rafts/caveolae microdomains
at the cytosolic side (25, 33-37). Moreover, several transmembrane
receptors (tyrosine kinase receptors, heptahelical receptors, and
T-cell antigen receptors) have been reported to be enriched in lipid
rafts (26, 33). Less is known about the precise association of Raf
kinases with lipid rafts. Although interactions of C-Raf with
raft/caveolae fractions have been observed (35, 38) it is still unclear
whether these interactions take place with lipid components or with
caveolin, which is normally present in such preparations. Because of
the small size (50 nm in average) and their flat structure, the
visualization of rafts, in contrast to caveolae, is very difficult and
was successfully performed only by fluorescence labeling, cross-linking
(clustering) of raft proteins by antibodies and photonic force
microscopy measurements (26, 39). Taken together, raft microdomains
have been characterized as membrane regions consisting of a unique
lipid environment and are believed to function in cellular signaling by
forming platforms for individual receptor signaling complexes.
In this study we examined the associations of purified and functional
full-length B- and C-Raf kinases with membrane lipids, particularly
with phosphatidylcholine (PC), phosphatidylethanolamine (PE), PS,
phosphatidylinositol (PI), PA, SM, ceramides, and cholesterol using
reconstitution techniques and biosensor measurements. We report
here that purified C-Raf binds tightly and specifically to PA, PS,
ceramides, and cholesterol. In contrast, the association with PC, PE,
PI, and SM was much less pronounced. Purified B-Raf exhibited binding
properties similar to C-Raf. The interactions of C-Raf with cholesterol
were examined under conditions which allow the formation of raft
microdomains. In this case we monitored a significant increase of Raf
association with cholesterol, indicating that raft formation
potentiates Raf binding to cholesterol. In general, binding of active
Raf kinases to liposomes led to a considerable decrease of kinase
activity and displacement of 14-3-3 proteins. Ras-Raf interaction
studies revealed that association of Raf with phospholipid vesicles
containing farnesylated Ras was only marginally increased compared with
vesicles without Ras. Based on these results we discuss an alternative
model for Raf activation in which Raf recruitment by Ras involves
primarily diffusion in the plane of the membrane.
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EXPERIMENTAL PROCEDURES |
Materials--
The phospholipids (PC, PE, PI, and PA), SM,
cholesterol, streptavidin, imidazole, benzamidine, leupeptin,
aprotinin, CHAPS, and n-octyl-
-D-glucoside
were obtained from Sigma. Nonidet P-40, GTP, GTP
S, GppNHp, GDP, and
GDP
S were from Roche Molecular Biochemicals. Ceramides (natural
sources) were purchased from Matreya (BIO-TREND) and biotin-PE was from
Molecular Probes. Sepharose CL-4B and glutathione-Sepharose were
obtained from Amersham Biosciences and Ni-NTA-agarose from Qiagen.
Monoclonal anti-pan-Ras antibodies were obtained from Transduction Laboratories, polyclonal anti-13-3-3 antibodies were from
Santa Cruz Biotechnology and monoclonal anti-phospho-ERK1/2 antibodies
were from New England Biolabs. Horseradish peroxidase-conjugated polyclonal anti-rabbit and anti-mouse IgG were purchased from Amersham Biosciences.
Cell Culture, Protein Purifications, SDS-PAGE, and Western Blot
Analysis--
For the production of recombinant Raf kinases,
Sf9 cells were infected with baculoviruses at the multiplicity
of infection of 5 and incubated for 48 h at 30 °C. The cells
were then washed with phosphate-buffered saline and pelleted at
230 × g. The Sf9 cell pellets (2 × 108 cells) were lysed in 10 ml of Nonidet P-40 lysis
buffer containing 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 10 mM Na-pyrophosphate, 25 mM -glycerophosphate, 25 mM NaF, 10%
glycerol, 0.75% Nonidet P-40, and a mixture of standard proteinase
inhibitors (1 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin) for 45 min with gentle rotation at 4 °C. The lysate was
centrifuged at 27,000 × g for 30 min at 4 °C. The
supernatants (10 ml) containing GST-tagged Raf kinases were incubated
with 0.5 ml GS beads for 2 h at 4 °C with rotation. After
incubation the GS beads were washed 3 times with Nonidet P-40 buffer,
whereby the third wash contained only 0.2% Nonidet P-40 instead of
0.75%. The Raf kinases bound to the beads were eluted 3 times with 0.5 ml of 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 25 mM -glycerophosphate, 25 mM NaF, 10%
glycerol, 0.1% Nonidet P-40, and 20 mM glutathione. The
purification procedure for His-tagged Raf kinases was similar as
described above with the exception that the Sf9 cell lysates (10 ml) were incubated with 0.5 ml of Ni-NTA-agarose. The bound proteins
were then eluted with imidazole using a step gradient. The purity of
the Raf kinase preparations was documented by SDS-polyacrylamide gel
electrophoresis (10% gels) and staining with Coomassie Blue (see Fig.
1A). For Western blot analysis the gels were transferred to
nitrocellulose membranes (Schleicher & Schuell) and probed with
antibodies specific for C-Raf (76), B-Raf (77), Ras, 14-3-3 proteins
and phospho-ERK. After washing, the membranes were incubated with
specific secondary horseradish peroxidase-conjugated antibodies and
detected by enhanced chemiluminescence (ECL, Amersham Biosciences).
Expression and purification of H-Ras was performed as described before
(40). Isoprenylation of full-length H-Ras protein was carried out with
Saccharomyces cerevisiae protein farnesyltransferase (41).
Briefly, 500 nmol of H-Ras was incubated with 20 nmol of
farnesyltransferase and 1 µmol of farnesyl pyrophosphate in 30 mM Tris-HCl, pH 7.4, 50 mM NaCl, 20 µM ZnCl2, 5 mM octylglucoside, and 2 mM dithioerythritol in a total volume of 5 ml for
3 h at 30 °C. Farnesylated product was separated from
non-farnesylated H-Ras by extraction with Triton X-114 (42), followed
by removal of the detergent with ion exchange chromatography
(DEAE-Sepharose column). Integrity of the product was verified by ESI
mass spectroscopy. The Ras-binding domain of C-Raf kinase was
synthesized as a GST fusion protein (GST-RBD) (43). K-Ras-4B protein
was cloned in the ptac vector (40) and expressed in Escherichia
coli AD202 cells after induction with
isopropyl-1-thio--D-galactopyranoside at 25 °C
overnight. K-Ras-4B was purified by ion exchange chromatography (Fractogel, Merck, Darmstadt) and gel filtration (HiLoad 26/60 Superdex
200, Amersham Biosciences, Freiburg).
Kinase Activity Measurements--
Kinase assays with Raf
proteins were performed using recombinant MEK and ERK-2 as substrates
in 25 mM Hepes, pH 7.6, 150 mM NaCl, 25 mM -glycerophosphate, 10 mM
MgCl2, 1 mM dithiothreitol, and 1 mM Na vanadate buffer (50 µl final volume). Following
additions of purified Raf kinases or Raf containing vesicles (5-10
µl) and ATP (500 µM), the samples were incubated for 30 min at 26 °C. The incubation was terminated by addition of Laemmli
sample buffer, the proteins were separated by 10% SDS-PAGE and
transferred to nitrocellulose membranes. The extent of ERK
phosphorylation was determined by anti-phospho-ERK antibodies and
scanning laser densitometry.
Ras-Raf Binding Assays with Full-length C-Raf Kinase--
To
measure binding of Ras to Raf in liposomes, the purified components
were mixed with vesicles (1.2 mM final lipid concentration) in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.2 mM dithiothreitol, 2 mM MgCl2, and
0.2 mg/ml BSA buffer as indicated in the figure legends. The proteins
not bound to the liposomes were removed by Sepharose CL-4B columns as
described above. To determine the Ras to Raf coupling efficiency in
solution, the GST-tagged C-Raf was first immobilized to GS and then
incubated with purified Ras proteins at 4 °C for 2 h in 25 mM Hepes, pH 7.6, 150 mM NaCl, 5 mM
MgCl2, 1 mM EDTA, 1% Nonidet P-40, 1 mg/ml
BSA, and 10% glycerol buffer (400 µl final volume) as depicted in
the figure legends. The GS beads were washed 3 times with 1 ml of the
binding buffer, omitting BSA, boiled for 5 min in the Laemmli sample
buffer, centrifuged, and the supernatant was applied to the SDS-PAGE
(12% gels). The visualization and quantification of Ras and Raf was
performed by ECL and scanning laser densitometry.
Binding of C-Raf-RBD to Farnesylated and Non-farnesylated
H-Ras--
The interactions of farnesylated and non-farnesylated H-Ras
with C-Raf-RBD were monitored by stopped-flow measurements using N-methylanthraniloyl (mant, m) derivatives of GppNHp. The
synthesis of mant-GppNHp was carried out as described (44). Loading of Ras proteins with nucleotides was performed according to John et
al. (45). Fluorescence kinetic assays were performed and analyzed
as described previously in a SX16MV stopped-flow system (Applied
Photophysics) (46). The system was equipped with a FP.1 accessory for
fluorescence anisotropy measurements. Briefly, for association kinetics
Ras proteins were mixed in 10 mM Hepes, pH 7.4, 150 mM NaCl, and 5 mM MgCl2 with
GST-RBD under pseudo-first order conditions at 20 °C with excitation
of the mant-fluorophore at 350 nm. Emission signals were measured using
a cut-off filter KV405 (Schott).
Preparation of Lipid Vesicles--
Large unilamellar vesicles
(LUVs) were prepared by the extrusion method (47) using a LiposoFast
Basic extrusion apparatus (Avestin, Inc., Canada). The lipids of
interest (in chloroform) were mixed and the solvent was removed by
nitrogen. After having kept the samples under high vacuum for 1 h,
the lipid mixtures were hydrated at a concentrations of 8 mM in 20 mM Tris-HCl, pH 7.4, and 50 mM NaCl for 1-2 h. The lipid suspensions were then vortexed intensively and after that, freeze and thawed 10 times in
liquid nitrogen. To obtain unilamellar vesicles the suspension was
passed 12 times through the extrusion apparatus using 100 or 200 nm
polycarbonate filters (Nucleopore, Pleasanton, CA).
Reconstitution of Purified Components into the Phospholipid
Vesicles and Liposome Binding Assays--
The purified Raf kinases
were associated with LUVs by incubation at 4 °C for 1 h. For
that purpose the lipid vesicles were diluted with 20 mM
Tris-HCl, pH 7.4, 100 mM NaCl, 0.2 mM
dithiothreitol, and 0.2 mg/ml BSA buffer to give a final volume of 200 µl and a lipid concentration of 1.2 mM. To remove the
proteins which were not associated with lipids, the reaction mixture
was applied to a small Sepharose CL-4B column (2.5 ml) and eluted with
the same buffer as above, omitting BSA. The opalescent lipid vesicle fraction was collected in the void volume (300 ml) and was further concentrated by Speed-vac apparatus. To visualize the associated Raf
kinases with phospholipid vesicles, the concentrated samples were
applied to SDS-PAGE (10% gels), transferred to nitrocellulose membranes, and incubated with specific antibodies. After detection of
lipid bound Raf by enhanced chemiluminescence (ECL) the quantification was performed by scanning laser densitometry.
Biosensor Measurements--
All biosensor measurements
were carried out on a BIAcore-X system (Biacore AB, Uppsala, Sweden) at
25 °C. Liposomes were captured onto the surface of a Pioneer L1
sensor chip (Biacore AB, Uppsala, Sweden). The surface of the L1 sensor
chip was first cleaned with 20 mM CHAPS at a flow rate of
10 µl/min followed by the injection of LUVs (30 µl, 0.4 mM lipid concentration) at a flow rate of 5 µl/min in
biosensor buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, and 0.1 mM dithiothreitol) which
resulted in a deposition of approximately 3000-5000 resonance units.
To prove whether reassociation of analyte occurs in the dissociation
phase, reduced concentrations of lipids (80 µM) were
used. To investigate the captured vesicles for their ability to
associate with proteins, the PC/PE/SM vesicles were supplemented with
5% PE which was covalently labeled by biotinyl residues (Biotin-PE)
and the biotin-streptavidin (SA) interaction was monitored (48). To
monitor the specific biotin-PE association with SA, the control flow
cell (flow cell-1) was loaded with vesicles omitting Biotin-PE, while
the flow cell-2 was immobilized with vesicles containing Biotin-PE. SA
(10 µg/ml) was then passed across flow cells 1 and 2. The resonance
unit differences derived from flow cell-2 and flow cell-1 (Fc2-Fc1)
resulted in specific binding of SA to Biotin-PE. SA associated
specifically to vesicles containing biotin-PE (data not shown). To
measure the association of Raf kinases with lipids, purified C-Raf
(20-320 nM diluted in biosensor buffer) was applied
to the captured LUVs at a flow rate of 10 µl/min. After binding of
Raf to lipids, the dissociation process was observed at the same flow
rate and at least for 30 min. At the end of the binding assay, the
sensor chip surface was regenerated by injection of 20 mM
CHAPS followed by washing with biosensor buffer before
reinjecting phospholipid vesicles for the next cycle. The evaluation of
the kinetic parameters for Raf binding to lipids was performed by
non-linear fitting of binding data using the BiaEvaluation 2.1 analysis
software. The apparent association (ka) and
dissociation rates constant (kd) were evaluated from
the differential binding curves (Fc2-Fc1) as shown in Fig. 7 assuming
a A+B = AB association type for the protein-lipid interaction. Two
independent sets of measurements were used for calculation of rate
constants. The affinity constant KD was calculated
from the equation KD = kd/ka.
| |
RESULTS |
Purification and Characterization of Raf kinases--
Analogous to
the binding of protein kinase C to lipids, such as PS and
diacylglycerol, C-Raf was shown to possess binding affinity for PS but
revealed almost no binding to diacylglycerol (5, 15, 16). Additionally
C-Raf was found to bind to PA (19). Interactions of B-Raf with membrane
lipids have not been reported so far. Since most published results,
including Ras-Raf binding assays (43, 49), were performed either with
fragments of C-Raf or with C-Raf expressed and purified from bacterial
origin, a material which turned out to be non-functional with regard to kinase activity (Fig.
1B),2
we expressed and purified diverse Raf kinases from Sf9 insect cells. Contrary to the bacterial expression system, Raf kinases expressed in Sf9 cells possess important co-translational
modifications such as phosphorylation of serine 621 and serine 259 of
C-Raf (1, 50), thus allowing associations with 14-3-3 proteins (13,
14). To obtain recombinant Raf kinases, Sf9 cells were infected
with recombinant baculoviruses containing cDNA coding for the
following Raf proteins: B-Raf wt, C-Raf wt, constitutively active C-Raf
mutant (Y340D/Y341D), a kinase dead mutant C-Raf R375W, and finally
C-Raf-BXB, which comprises the C-Raf kinase domain (residues 1-21 and
301-648). The Raf kinases were expressed as fusion proteins and the
purification was achieved by affinity chromatography using either
glutathione-Sepharose (GS) or Ni-NTA resin for hexa-histidine-tagged
proteins. The protein concentrations of the eluted Raf kinases were in
the range of 50-100 µg/ml. Thus, the single purification step
allowed purifications of about 0.3 mg of Raf proteins starting from
4 × 108 cells.
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Fig. 1.
Purification of Raf kinases and kinase
activity measurements. Sf9 insect cells were infected with
baculoviruses encoding either GST- or His-tagged Raf kinases. The
recombinant Raf was purified either by glutathione-Sepharose or nickel
chelate affinity chromatography and subjected to SDS-PAGE (10% gels).
A, Coomassie Blue staining of purified Raf proteins and
Western blot analysis (top) probed by monoclonal anti-C-Raf
(PBB-1) (76) and polyclonal anti-B-Raf antibodies (77).
B, kinase activity of purified Raf kinases. Kinase
assay was performed in the presence of purified MEK and ERK-2 and
detected by anti-phospho-ERK antibodies. The notation of 10x
indicates that the values of the kinase activity has to be multiplied
by factor 10.
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The purity of Raf protein preparations is documented in Fig.
1A. Most Raf proteins were purified as a complex with 14-3-3 proteins (not shown). The functional integrity of purified Raf kinases
was controlled by measuring the kinase activity (Fig. 1B) or
the ability to associate with Ras proteins. Preparations of wild-type
C-Raf possessed relatively low levels of kinase activity. The
constitutively active variant of C-Raf (Y340D/Y341D) had approximately 12-fold higher activity than C-Raf wt, whereas the kinase dead mutant
of C-Raf (K375W) and C-Raf wt from E. coli did not show any
kinase activity. The BXB form of C-Raf displayed elevated activity
relative to wild type C-Raf that was further increased (approximately
20-fold) in the BXB-Y340D/Y341D mutant. In comparison to C-Raf, the
B-Raf exhibited ~110-fold higher kinase activity.
Ras-Raf Binding Studies--
The Ras-Raf binding data so far
available have been obtained exclusively by using C-Raf-fragments,
mostly C-Raf-RBD (43, 49). Therefore, it was of interest to investigate
the full-length C-Raf preparations presented in Fig. 1. The Ras-Raf
binding studies shown in Fig. 2 have been
carried out with GST-C-Raf which was immobilized to GS. We observed
effective Ras binding to the full-length C-Raf but we did not detect
significant differences in the binding properties between farnesylated
and non-farnesylated forms of Ras. The Ras binding to C-Raf was clearly
GTP-dependent. Yields of H-Ras bound to Raf at equilibrium
conditions were rather high (60-70%), reflecting a high efficiency
for Ras binding to C-Raf. The lack in discrimination of full-length
C-Raf kinase for farnesylated and non-farnesylated Ras was also
observed for the interactions between the isolated Ras-binding domain
of C-Raf (C-Raf-RBD) and either non-modified or farnesylated Ras
protein in a quantitative analysis determined by stopped-flow
fluorescence anisotropy. The association and dissociation experiments
performed with RBD and Ras proteins complexed with fluorescent
non-hydrolysable GTP analogue mant-GppNHp showed no significant
differences in rate constants for non-modified and farnesylated H-Ras.
The association rate constants (ka) were 10.2 × 106 M
1 s1 and
11.3 × 106 M1
s1 for Ras·mGppNHp and Rasfar·mGppNHp,
respectively. Dissociation rate constants were 2.0 s1 for
both Ras·mGppNHp and Rasfar·mGppNHp leading to affinity
constants (KD values) of 200 and 180 nM,
respectively.
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Fig. 2.
Ras binds to full-length C-Raf in
GTP-dependent manner. GST-C-Raf wt immobilized to GS
(60 nM) was incubated with purified Ras proteins at 40 nM (lanes 1 and 3) and 200 nM (lanes 2 and 4) final
concentrations, respectively. Immobilized GST-C-Raf-BXB was incubated
with 200 nM H-Ras. Ras proteins were loaded either with
GTPS (depicted as T) or GDP (depicted as D).
Following SDS-PAGE and immunoblotting, Ras and Raf were visualized by
specific antibodies and ECL.
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Next we investigated the Ras-Raf binding properties in the presence of
lipid environment. For that purpose we reconstituted purified C-Raf
into the LUVs containing PS and PA to obtain a high degree of
C-Raf association (see also Fig. 5). As depicted in Fig.
3 the Ras binding to Raf was clearly
GTP-dependent and the binding ratio of GTP-loaded Ras to
GDP-loaded Ras was approximately 8 (see Fig. 3 for the case that Raf
was present in excess over Ras). In contrast, when the amount of added
Ras exceeded Raf, the Ras-GTP/Ras-GDP binding ratio was considerably
reduced due to the increased nonspecific binding of Ras to the lipid
vesicles. The yields for Ras-Raf coupling under the equilibrium
conditions were 10-20% compared with 60-70% of that measured in
solution. To evaluate the possible C-Raf recruitment by Ras in
vitro, we reconstituted farnesylated H-Ras into the phospholipid
vesicles and measured the Raf recruitment by Ras. As reported (51),
farnesylated Ras reveals much higher association rates to phospholipid
monolayers than non-farnesylated Ras. We have obtained similar results
using our LUV preparations. As shown in Fig.
4A the association of
farnesylated Ras to liposomes was 4-fold higher compared with
non-farnesylated Ras. In these experiments the molar ratio of Ras to
phospholipids was 1:2,400 corresponding to a weight ratio of 1:240.
Next we investigated the association of purified full-length C-Raf and farnesylated-Ras in the presence of liposomes, thus mimicking the
normal constellation of these interactions (Fig. 4, B and C). For these experiments we have chosen a high excess of
farnesyl-Ras over C-Raf and omitted PS and PA in the lipid composition
of vesicles. But even under such conditions the recruitment of Raf by
Ras was marginal, indicating that the recruitment of Raf by lipids
plays a primary role. In the experiments using LUVs which contained additionally PS and PA, i.e. vesicles which correspond to
the composition of the inner leaflet of the plasma membranes, it was difficult to register any additional recruitment of Raf by Ras (data
not shown).
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Fig. 3.
Ras-Raf binding assay in the presence of
lipid environment. Purified GST-C-Raf wt (10 nM) was
incubated with PC/SM/PE/PS/PA-containing lipid vesicles (1.2 mM lipid concentration) for 1 h at 4 °C. H-Ras
loaded with GTPS (hatched bars) or GDPS (open
bars) were added as indicated and the mixture was incubated again
for 1 h at 4 °C. Proteins not bound to the liposomes were
separated from those bound to the liposomes by Sepharose CL-4B columns
(2.5 ml). Following SDS-PAGE and immunoblotting, the liposome-bound
proteins were visualized by specific antibodies and ECL and quantified
by scanning laser densitometry. Data shown represent the mean ± standard deviation of two independent experiments.
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Fig. 4.
C-Raf recruitment by farnesylated H-Ras in
phospholipid vesicles. A, incorporation of
farnesylated (black bar) and non-farnesylated (open
bar) H-Ras into PC/SM/PE-containing vesicles. H-Ras (500 nM) was incubated with lipid vesicles and applied to
Sepharose CL-4B columns (2.5 ml). The vesicle-bound Ras was visualized
as described in the legend to Fig. 3. Each bar represents
the mean ± standard deviations from three independent
experiments. B and C, farnesylated H-Ras
loaded with GTPS (lanes 1-3) or GDPS (lanes
4-6) was incubated with PC/SM/PE-containing vesicles at 500 nM final concentration for 1 h at 4 °C and passed
through Sepharose CL-4B columns to remove the free Ras. The vesicle
fractions were pooled and incubated with increasing amounts of purified
GST-C-Raf wt (5, 10, and 20 nM). The proteins bound to
lipid vesicles were separated from non-bound proteins by Sepharose
CL-4B chromatography. Following SDS-PAGE and immunoblotting, Ras and
Raf bound to the liposomes were visualized by specific antibodies and
ECL (C). The bars shown in B represent
the quantification of data depicted in C.
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Association of Raf Kinases with Phospholipids and Lipid Second
Messengers--
Since the extent of Raf recruitment by Ras under
in vitro conditions described in Fig. 4 was comparable with
that caused by simple Raf-lipid associations, we hypothesized that Raf
translocation to the lipid surfaces might be independent of activated
Ras. To examine this we characterized the interactions of purified Raf kinases with phospholipids such as PC, PE, PS, PI, SM, and cholesterol and lipid second messengers PA and ceramide. To assess whether these
associations take place with purified full-length and functional B- and
C-Raf kinases, we developed a lipid binding assay using phospholipid
vesicles prepared by the extrusion method (47) and tested Raf kinases
such as B-Raf, C-Raf, and constitutively active forms of C-Raf. The
results from Raf-lipid association experiments are depicted in Fig.
5. Since we originally
observed that Raf kinases do not exhibit significant association to
vesicles composed of PC, PE, and SM we used vesicles with a lipid ratio of PC/SM/PE = 50:13:37 (mol %) as a basic lipid mixture. Vesicles with other lipid combinations containing additionally PS, PA, PI, or
ceramides (20 and 30% for PS, 10 and 20% for PA, PI, and ceramides,
respectively) have been prepared from basic PC/PE/SM vesicles by
substitution of PC content with lipids of interest. The interactions of
C-Raf with cholesterol are shown in Fig. 9. The basic lipid mixture
enriched by 20-30% PS represents the typical lipid composition of the
inner leaflet of plasma membranes (52). Additionally, the PS content
may vary depending on the structure of membrane microdomains such as
rafts or caveolae (25, 26, 53). The results shown in Fig. 5,
A and B, provide direct evidence that C-Raf
preferentially associates with PS and PA and to somewhat lower extent
with ceramide. The constitutively active mutant C-Raf-Y340D/Y341D exhibited the same binding pattern as C-Raf wt (Fig. 5C).
Control proteins such as purified GST alone and ERK-1 (see Fig.
5B) displayed only marginal binding to the vesicle samples
tested without any specificity for particular lipids. The same was true
when Raf proteins were added in the absence of lipid vesicles. The
C-Raf-BXB, a protein which represents the C-terminal half of C-Raf, was
still capable of association with PS and PA, but failed to interact with ceramides (Fig. 5D). Of particular interest are the
experiments with regard to associations of B-Raf with membrane lipids,
since such interactions have not been investigated previously. We found that B-Raf-like C-Raf possesses high binding affinity for PS and PA
(Fig. 5E), yielding 60-70% lipid-associated Raf. In
contrast, unlike C-Raf, B-Raf showed increased association with
vesicles containing PI, indicating that B-Raf does not discriminate
between the acidic phospholipids tested. In parallel to the specific
binding effects of Raf proteins to lipids, we observed that
interactions of active Raf kinases with liposomes resulted in a
considerable reduction of initial kinase activity. As documented in
Fig. 5, C and D, the reconstitution of
constitutively active C-Raf-Y340D/Y341D and C-Raf-BXB into liposomes
resulted in more than 90% reduction of initial kinase activity.
However, the decrease of B-Raf kinase activity in the presence of
liposomes was not as dramatic as that detected for C-Raf (Fig.
5E). Only in the presence of PS containing vesicles was a
strong activity decrease (down to 3% of initial) observed, in the
presence of other lipid combinations the residual activity remained at
about 30% of initial values. Taken together, the experiments shown in
Fig. 5 illustrate clearly that the functionally intact and full-length
B- and C-Raf kinases associate with membrane lipids, preferentially
with PS and PA and to somewhat lower degree with ceramide.
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Fig. 5.
Specific associations of Raf kinases with
phospholipid vesicles. Purified Raf kinases (5-10 pmol) were
incubated with phospholipid vesicles (1.2 mM) consisting of
lipid compositions indicated in graphs A-E for 1 h at
4 °C. The lipid composition of basic vesicles consisting of
PC:SM:PE = 50:13:37 (mol %) was modified by substitution of PC
content by PS (20%), PA (10%), PI (10%), and ceramides (10%), thus
resulting in vesicles with following lipid compositions:
PC:SM:PE:PS = 30:13:37:20, PC:SM:PE:PA = 40:13:37:10,
PC:SM:PE:PI = 40:13:37:10, and PC:SM:PE:CER = 40:13:37:10
(mol %). To remove Raf proteins which were not associated with
vesicles, the samples were passed through Sepharose CL-4B columns (2.5 ml). Subsequently, the opalescent vesicle fractions containing the
lipid-associated Raf were applied to SDS-PAGE and immunoblotted. The
amounts of Raf associated with lipids were visualized by incubation
with anti-Raf antibodies used in Fig. 1. Quantification of the
associated Raf was performed by scanning laser densitometry. The
insets in graphs A-E depict the representative
immunoblots of Raf proteins associated with liposomes used. The inputs
of Raf proteins represent 10, 20, and 50% of added Raf in particular
experiments and were used for quantification. Kinase activity of Raf
proteins was measured as described under "Experimental Procedures."
Following Raf, kinases were tested for their ability to associate with
liposomes: A, GST-C-Raf wt; B,
C-Raf-His wt; C, GST-C-Raf-Y340D/Y341D mutant;
D, GST-C-Raf-BXB form of C-Raf; and E,
B-Raf-His wt. Bar graphs represent mean ± standard
deviations from three independent experiments.
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Raf-Lipid Interactions Monitored by Biosensor
Technology--
To derive kinetics and equilibrium parameters for
the interactions between Raf kinases and lipid vesicles, we applied
Biomolecular Interaction Analysis technology using the newly developed
L1 sensor chip, which permits capturing of lipid vesicles by
hydrophobic anchors. The injection of phospholipid vesicles at 0.4 mM resulted in approximately 4,000 resonance units. In
general, there were no significant differences in the binding
properties of vesicles tested to the sensor chip surface. To
characterize the Raf interactions with lipids in terms of quantitative
kinetic studies, we re-examined the results presented in Fig. 5 using
biosensor technology. To assess kinetic parameters and
equilibrium constants for Raf-lipid interactions, it was necessary to
monitor the specific ingredients of this protein-lipid association
process. For that purpose the control flow cell was loaded with the
basal lipid mixture (PC/PE/SM vesicles) while the second flow cell was
loaded with vesicles containing additionally PS, PA, PI, ceramide, or
cholesterol (for cholesterol binding results, see Fig. 9). As shown in
Fig. 6 the binding pattern of C-Raf with
lipids was very similar to those obtained by the simple lipid-binding
assay depicted in Fig. 5. However, the extent of C-Raf binding to PS
and PA using biosensor methodology was considerably greater
compared with results obtained by standard lipid binding assays. A
site-specific peptide derived from the sequence of C-Raf (amino acids
389-408) representing the putative binding domain for PA was
synthesized and revealed high affinity for PA containing vesicles in
biosensor measurements, in contrast to numerous control peptides
tested (data not shown). These data confirm the assumption that PA
binding by C-Raf indeed occurs within the kinase domain (CR3).
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Fig. 6.
Surface plasmon resonance analysis of the
interactions between Raf kinases and lipids. L1 sensor chip was
loaded with phospholipid vesicles as described under "Experimental
Procedures." The control (reference) flow cell (Flow
cell-1) was loaded with vesicles consisting of basic lipid mixture
(PC:SM:PE = 50:13:37 mol %), whereas Flow cell-2 was
loaded with vesicles which contained additionally PS (20%), PA (10%),
PI (10%), or ceramides (10%) as indicated. Purified GST-C-Raf wt (160 nM) was injected at a flow rate of 10 µl/min. The
bar graph represents the maximal responses measured for
specific C-Raf associations with lipids. Data represent mean ± standard deviations from two independent experiments.
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Kinetic Analysis--
To evaluate the kinetic and equilibrium
parameters for associations between C-Raf kinases and lipids, the
measurements have been performed in the presence of increasing
concentrations of C-Raf. The apparent association
(ka) and dissociation rate constants
(kd) were evaluated from the differential binding
curves (Fc2-Fc1). Representation association-dissociation curves for
interactions of C-Raf to lipids are illustrated in Fig.
7. The corresponding data for
ka, kd, and KD
values are summarized in the Table I. The
association of C-Raf to PA appear to be most tight with an apparent
KD value of approximately 0.5 nM
followed by PS with KD = 12 nM and
ceramide with KD = 52 nM. These results
document the high affinity and specificity of C-Raf-lipid interactions under the conditions used in our binding studies. The very slow dissociation of bound Raf kinase to lipids may be explained in part by
re-association events and in this case the calculated kd values should be considered as a first
approximation. Such effects can be reduced by increasing the flow rate
or lowering the lipid density. However, neither elevation of flow rate
to 200 µl/min nor injection of highly concentrated NaCl buffer (3 M) or reduction of lipid concentrations from 0.4 mM to 80 µM changed significantly the
dissociation properties. Injections of 10 mM NaOH yielded
only incomplete removal of bound Raf. Therefore the bound lipid
vesicles were cleared by injection of CHAPS or octylglucoside after
each measurement.
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Fig. 7.
Quantitative biosensor
analysis of C-Raf interactions with PA. L1 sensor chip was
loaded with phospholipid vesicles as described under "Experimental
Procedures." Purified GST-C-Raf wt was injected at increasing
concentrations as indicated in the diagram and
inset. The reference cell (Flow cell-1, Fc1) was loaded with
vesicles containing PC, SM, and PE (50:13:37 mol %), whereas the Flow
cell-2 (Fc2) was loaded with vesicles which contained additionally 10%
PA. The diagram shows differential binding curves (Fc2-Fc1 values),
which represent the specific associations of C-Raf to PA. The
inset depicts the values of maximal responses measured for
C-Raf interactions with PA-containing vesicles ( ) and for
associations with vesicles containing only PS, SM, and PE ( ).
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Association of Raf Kinases with Liposomes Leads to Displacement of
14-3-3 Proteins--
In parallel to the specific Raf-lipid
interactions and loss of kinase activity as demonstrated in Fig. 5, we
observed in the presence of liposomes an almost complete dissociation
of 14-3-3 proteins. The displacement of 14-3-3 proteins upon Raf
association with LUVs was observed with both C-Raf and B-Raf. A typical
experiment is shown in Fig. 8. The
preparations of both C-Raf wt and B-Raf wt contained 14-3-3 proteins
(lanes 6 and 7 in Fig. 8). After addition and
incubation with PS/PA-containing vesicles, the liposome-bound Raf was
separated from the free Raf. As evident from Fig. 8 the Raf fraction
associated with liposomes has almost quantitatively lost the 14-3-3 proteins (lane 1 in Fig. 8). Preincubation of phospholipid
vesicles with H-Ras proteins (both farnesylated and non-farnesylated
forms were used) did not prevent 14-3-3 displacement (lanes
2-5 in Fig. 8). Based on these data we propose that the displacement of 14-3-3 proteins may be a consequence of lipid interactions with the putative third 14-3-3-binding site within the CRD
region of Raf. These observations on 14-3-3 displacement together with
the observed loss of kinase activity of lipid-bound Raf are in
agreement with results of Tzivion et al. (54), who reported
that displacement of 14-3-3 proteins from activated C-Raf by addition
of a synthetic phosphopeptide corresponding to the pS-621-binding
region was also accompanied by significant reduction of Raf kinase
activity.
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Fig. 8.
Association of Raf·14-3-3 complex with
liposomes leads to displacement of 14-3-3 proteins. The
phospholipid vesicles (1.2 mM) consisting of PC, SM, PE,
PS, and PA (20:13:37:20:10 mol %) were incubated with farnesylated and
non-farnesylated H-Ras (120 pmol), which were loaded either with
GTPS or GDP (lanes 2-5) or without addition of Ras
(lane 1). Purified C-Raf (10 pmol, A) or B-Raf
(10 pmol, B) were added and the mixture was further
incubated for 30 min at 4 °C. The lipid-bound Raf was separated from
the free Raf by Sepharose CL-4B columns (2.5 ml). The opalescent
vesicle fractions were applied to SDS-PAGE and immunoblotted. The
amounts of Raf and 14-3-3 proteins associated with vesicles were
visualized by specific antibodies and quantified by scanning laser
densitometry. The lanes 6 and 7 were loaded
directly with 5 and 10 pmol of C-Raf and B-Raf, respectively. Similar
results have been obtained from five independent experiments.
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Raf Associates with Cholesterol and Lipid Rafts--
To mimic the
association of signaling molecules with rafts, we prepared liposomes
containing variable amounts of cholesterol, SM, and GPLs. To test the
general binding properties of Raf for cholesterol, we first took
advantage of the Raf-lipid binding assay presented in Fig. 5 using the
Sepharose CL-4B column method. We created two sets of lipid vesicles
(in the presence of either 13 or 26% SM) and with increasing amounts
of cholesterol (2, 10, 20, and 40 mol %). The concentration of PE was
kept constant while PC was substituted for cholesterol as indicated. As
depicted in Fig. 9A, C-Raf wt
exhibited moderate and concentration-dependent binding for cholesterol. In the presence of 13% SM (left
panel), 35-40% of added C-Raf was retained by cholesterol (a
10% PA control showed retention of about 60% C-Raf). To achieve lipid
compositions favoring raft formation (55, 56), SM content was increased to 26% (right panel). In the presence of 10 and 20%
cholesterol, the extent of Raf association with liposomes was
comparable with those observed in the left panel.
Strikingly, in the presence of 40% cholesterol Raf association with
liposomes increased up to 75-80% of added Raf (Fig. 9A).
In preliminary experiments B-Raf exhibited similar binding properties
for cholesterol (data not shown). Next we evaluated the same
interactions depicted in Fig. 9A using the biosensor
technique, since the background binding of analyte (Raf) to the
captured control vesicles amounts to less than 0.1% of injected Raf
using this technique. The results performed by the surface plasmon
resonance technique correlated very well with results obtained by
column binding assays with the exception that Raf binding in the
presence of 10% cholesterol was almost not detectable. This could be
explained by the fact that the surface plasmon resonance technique
records the initial association of Raf in contrast to the equilibrium
conditions used in the Sepharose CL-4B experiments. Under the
conditions of raft formation (Fig. 9, C and D,
liposomes containing 26% SM and 40% cholesterol) the association of
Raf increased dramatically from 2-fold to almost 9-fold over the
background binding. These results indicate (i) that a special
composition and proportion of lipids (cholesterol/SM/GPL) is required
for raft formation and (ii) that full-length wild type Raf exhibits
high association tendency for cholesterol in raft-like microdomains.
Additionally, we examined the effects of simultaneous incorporation of
cholesterol and ceramides into the vesicles, since metabolical
formation of ceramide from SM is supposed to occur within the lipid
rafts (57). Interestingly, already small amounts of ceramide (5 mol %)
considerably stabilized the raft structure (Fig. 9). These results
indicate that ceramide may substitute for cholesterol and that C-Raf
binds to both cholesterol and ceramides in rafts. The
association-dissociation behavior of C-Raf binding to liposomes
containing cholesterol is shown in Fig. 9, B and
C. Similar to the results obtained with PS- and PA-containing vesicles (Fig. 7), C-Raf exhibits a very slow
dissociation, indicating a tight association of Raf to cholesterol. The
functional consequences of Raf interactions with cholesterol (and
rafts) were examined by measuring the kinase activity of C-Raf wt
(basal activity) and constitutively active mutant C-Raf-Y340D/Y341D in the presence of liposomes listed in Fig. 9. We observed, similar to the
results shown in Fig. 5, that Raf kinase activity was considerably decreased in the presence of cholesterol containing vesicles (data not
shown). These data are in accordance with the observation that diverse
signaling molecules such as Ras, Src kinases, and eNOS are maintained
in their inactive form during the association with rafts/caveolae (25,
35).
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Fig. 9.
Raf associates with cholesterol and lipid
rafts. A, association of GST-C-Raf (10 pmol) with
cholesterol containing vesicles was monitored exactly as described in
the legend to Fig. 5 using the Sepharose CL-4B column method. SM and PE
amounts were kept constant: in the left panel, 13 and
37%; and in the right panel, 26 and 24%,
respectively. PC was substituted for PA, cholesterol, and ceramide as
indicated. The insets show the representative Western blots
developed by anti-C-Raf antibodies quantified by scanning laser
densitometry. The binding data correspond to the bars shown
in the left and right panels. B and
C, biosensor analysis of GST-C-Raf interactions
with cholesterol containing vesicles. L1 sensor chip was loaded with
lipid vesicles as described under "Experimental Procedures." The
reference cell was loaded with vesicles containing PC, SM, and PE
(50:13:37 mol % in B and 50:26:24 mol % in C).
Cholesterol concentrations were as indicated. Purified GST-C-Raf (160 nM) was injected at a flow rate of 10 µl/min.
D, the values of maximal responses monitored by
biosensor technique for C-Raf interactions with increasing
amounts of cholesterol, 10% PA, and 5% ceramides at different SM
concentrations (13% SM in the left panel and 26% SM in the
right panel) are illustrated as fold increase of C-Raf
association over the reference values. The results presented here have
been obtained from two independent experiments.
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DISCUSSION |
In this report we provide evidence that purified B- and C-Raf
kinases possess a high tendency for associations with phospholipid vesicles and that Raf translocation to membranes may occur by direct
association with phospholipids, lipid second messengers, and
cholesterol. Specific interactions have been observed with lipid
vesicles containing PS, PA, or ceramides, even in the absence of
activated Ras proteins, but the most effective Raf associations with
liposomes were observed with cholesterol under the conditions where
lipid raft formation is favored. In all instances, lipid association of
Raf was accompanied by inhibition of kinase activity.
Interactions of Ras Proteins with Raf Kinases--
The binding of
processed Ras-GTP to C-Raf occurs through two relatively independent
interactions (8). Whereas the coupling mechanism of Ras to Raf-RBD are
understood in great detail, numerous reports have provided conflicting
data with regard to the interactions of Raf-CRD with Ras. Initial
studies (49, 58) demonstrated a considerably decreased interaction of
Ras with the N-terminal part of Raf when a zinc binding cysteine at
position 168 was mutated to serine. These findings have been further
confirmed with full-length C-Raf expressed in mammalian cells (10, 11)
showing that Raf-CRD is not required for membrane recruitment by Ras
but is critical for Raf activation.
In the study presented here we compared the Ras-Raf binding properties
in solution and in lipid environment. The full-length C-Raf kinase
tested in solution associated with purified Ras proteins in a
GTP-dependent manner, as already observed for C-Raf
fragments containing RBD (43, 49, 58, 59), but did not discriminate between farnesylated and non-farnesylated Ras and between H- and K-Ras
(Fig. 2). The same was true for the interactions of the isolated RBD
region of C-Raf with farnesylated and non-modified H-Ras (data not
shown). The binding of Ras to C-Raf in the lipid vesicle environment
was also GTP-dependent (Figs. 3 and 4), but with
significantly lower binding efficacy (10-20% compared with 60-70%
of coupling efficacy measured in solution). A plausible explanation for
this finding might be the fact that lipids (preferentially PS and PA)
bind simultaneously with Ras to Raf and compete for the same or
neighboring domains, thus reducing the apparent Ras-Raf binding
affinity. As shown in Fig. 4, even in the absence of Ras, C-Raf shows
high binding tendency for the liposomes consisting of PC, PE, and SM.
Using vesicles containing additionally PS and PA, thus mimicking the
lipid composition of the inner side of native plasma membrane, C-Raf
recruitment by Ras was not detectable, since almost 70% of Raf was
bound to lipids in the absence of Ras. These results suggested strongly
that Raf kinases might translocate to membranes in the absence of Ras
and that Raf possesses a high tendency to associate with lipid
environment. Therefore we investigated in more detail, using the
reconstitution technique, the putative interactions of Raf kinases with lipids.
Specific Associations of B- and C-Raf Kinase with PS, PA, and
Ceramides--
Evidence that fragments of Raf proteins, particularly
C-Raf-CRD, interact with phospholipids have been presented (15, 16). The amino acids in C-Raf-CRD interacting with PS have been mapped as
Arg-143, Lys-144, and Lys-148, since substitution of all three basic
residues to alanine resulted in a C-Raf variant that failed to interact
with PS. Additionally, further interactions between Raf and membranes
might occur by insertions of hydrophobic residues of C-Raf-CRD into the
lipid bilayer. A similar mechanism for membrane attachment has been
proposed for Rabphilin-3A, which is an effector of the Ras-related
small GTPase Rab3A (17, 60). Based on x-ray crystal structure, the zinc
finger of Rabphilin-3A, which is closely related to the FYVE domain (a
zinc-containing membrane-binding motif), was shown to penetrate the
lipid membrane which might result in the optimal conformation for
coupling to G-proteins (61).
PA has also been shown to interact with C-Raf (19). The interaction
sites for PA have been localized within the C-terminal part of the
C-Raf kinase. Deletion mutagenesis of C-Raf revealed that the
PA-binding sites are positioned between residues 389 and 423 (19). This
region contains a positively charged tetrapeptide sequence RKTR
(residues 398-401), which seems to be involved in an initial
electrostatic interaction with PA. Additional interactions such as
association with the hydrophobic segment ILLFM (residues 405-409)
might then stabilize this interaction. In vivo experiments (62) confirmed these preliminary in vitro results. Using
mutational analysis it was demonstrated that a single mutation in the
putative PA-binding region of C-Raf (R398A) prevented the translocation of C-Raf to endosomal membranes, whereas disruption of Ras-Raf interaction by C-Raf mutant (R89L) did not affect
agonist-dependent translocation (62). These results are in
agreement with our findings demonstrating that both B- and C-Raf
interact with PA, and support a model in which agonist-induced C-Raf
recruitment to the membranes could be mediated solely by lipids such as
PA and PS. With regard to Raf localization and linkage to other
signaling pathways, several observations were reported recently that
support the view that Raf might be associated also with intracellular vesicles and activated by alternative mechanisms. In the course of the
activation and internalization of G-protein-coupled receptors, C-Raf
was found to be complexed with -arrestin and to be attached to the
endosomal vesicles via clathrin-coated pits (63, 64).
Phosphatidic acid and ceramide are considered to act as intracellular
lipid second messengers. Interactions of ceramides with C-Raf
kinase have been described (20, 21). Our results obtained with purified
Raf kinases reconstituted into phospholipid vesicles enriched with
ceramides support the findings that ceramides bind to Raf kinases. But
contrary to results of Müller et al. (21), we observed
that ceramides bind to the regulatory domain of Raf (presumably to CRD)
since the BXB form of C-Raf, which corresponds to the catalytic domain,
did not exhibit any binding to ceramides (Fig. 5). Taken together, our
results with respect to lipid binding performed with purified and
functional full-length C-Raf kinases using in vitro
reconstitution assay are consistent with observations previously
reported (15, 16, 19). We extended these investigations to include
B-Raf kinase, an active mutant of C-Raf (C-Raf-Y340D/Y341D) and the BXB
form of C-Raf. We show that the binding specificities of B-Raf and
C-Raf for lipids investigated in this study are similar, with the
exception that B-Raf also possesses binding affinities for PI. This
behavior might be explained by the amino acid composition of the
corresponding domains proposed to participate in the Raf binding to PS
and PA. Alignment of the PA binding segment of the human C-Raf to that
of B-Raf reveals identical amino acid sequences. Similarly, two
proposed basic PS-binding residues Arg-143 and Lys-144 are present in
both C- and B-Raf. In addition to data presented in Fig. 5 performed by
a lipid binding assay, we evaluated kinetic parameters and affinity
constants for Raf binding to PS, PA, and ceramides using
biosensor technology. According to these data the interactions
of C-Raf with PS, PA, and ceramides were shown to be specific with
apparent affinity constants in the range of 0.5-50 nM
(Table I). In the meantime, the applications of L1 sensor chips for
capturing of intact vesicles became widespread: direct interactions
between drugs and liposomes (65) and measurements of protein-protein
associations in the presence of liposomes (22) have been described. A
quantitative approach for the interactions of FYVE zinc finger domains
with phosphatidylinositol 3-phosphate has been reported by Gaullier
et al. (66). Interestingly, they measured also slow
dissociation rates and high affinity binding between analyte and
liposomes with a KD value of 45 nM comparable with our findings.
Interactions of Raf with Cholesterol and Lipid Rafts--
Lipid
Rafts represent liquid-ordered microdomains distributed into the plasma
membrane whose lipid composition differs from the rest of the membrane.
The main function of such microdomains would be to concentrate
particular signal transduction components into small regions, thus
allowing more efficient communication between the outer and inner
leaflet of plasma membrane. The high cholesterol levels in rafts enable
the tight packing of sphingolipid molecules by occupying the spaces
between the saturated fatty acid chains of the sphingolipids. Since
rafts could become easily aggregated, detergent-resistant membrane
preparations contain not only individual rafts but also clustered rafts
and caveolae. Caveolae, exhibiting morphologically defined cell surface
invaginations, are supposed to represent a subtype of rafts containing
caveolin, a scaffold- and cholesterol-binding structural protein (25, 27, 34, 67). The list of signaling proteins apparently associated with
caveolin has increased and includes, among others, Src family kinases,
heterotrimeric G-proteins, small G-proteins, adenylyl cyclase, eNOS,
nNOS, PKA, PKCa, MEK, ERK, and some receptor proteins (25, 27, 34).
Direct interactions of caveolin with signaling molecules led to their
inactivation (68). Interactions of caveolin with Raf kinases have not
been observed so far. However, association of C-Raf with isolated
caveolae/raft fractions prepared by sucrose gradient centrifugation
have been reported (35, 38). While Mineo et al. (38) found
that C-Raf was accumulated in caveolae/raft fractions after epidermal
growth factor stimulation (and Ras activation), Prior et al.
(35) located Raf (in the presence of active Ras) to non-raft fractions.
In the same article, Prior et al. (35) observed that only
non-active H-Ras (Ras-GDP) remains associated with raft/caveolae
fractions and that K-Ras, contrary to H-Ras was found to be localized
almost completely in non-raft fractions. However, very recently
Kranenburg et al. (36) reported, in contrast to Prior
et al. (35), that K-Ras co-localized largely with caveolin, whereas N-Ras (which exhibits the same acylation pattern as H-Ras) co-localized to both caveolar and non-caveolar regions of plasma membrane. This discrepancy might be explained by the fact that different cell lines were used.
Having in mind that in vivo isolated raft preparations
represent aggregated material, including caveolae and sometimes rafts from subcellular compartments, we decided to investigate Raf
interactions with rafts under well defined conditions using in
vitro experiments. We prepared liposomes with fixed lipid
compositions and monitored Raf association with cholesterol and
possibly raft structures (Fig. 9). Recently, the formation and
visualization of rafts in model membranes has been described (55, 56).
It was shown that lipid mixtures consisting of cholesterol, SM, and
GPLs in the proportion of 1:1:1 could form unilamellar vesicles
exhibiting properties expected for raft microdomains, as proved by
fluorescent labeled phospholipid analogues (55, 56). To monitor Raf
association with cholesterol (and rafts), we used lipid vesicle
preparations with a defined diameter of 200 nm prepared by extrusion
method (47). The lipid compositions and proportions were similar to those described by Dietrich et al. (55, 56) and are
indicated in Fig. 9. By using increasing amounts of cholesterol we
demonstrate here that Raf associates with cholesterol. Two to 3-fold
increase over basal association have been observed by using both column binding assay and biosensor technology. A dramatic increase in Raf binding was observed only in the case that both SM and cholesterol amounts were elevated (Fig. 9C). Since the proportions of
lipids were similar to those described by Dietrich et al.
(55, 56), we assume that formation of raft microdomains is responsible
for this dramatic increase of Raf association with liposomes.
Additionally, as shown in Fig. 9 we found that ceramides clearly
influence the raft structure and formation and that C-Raf binding to
ceramides increased in the presence of cholesterol. This observation is in accordance with data recently published by Xu et al. (69) showing that small amounts of ceramides stabilize raft formation. Since
caveolin is not present in our experiments we suggest that Raf
association with rafts could take place outside of caveolae. Furthermore, we propose (see also Fig.
10) that Raf association with lipid
rafts occurs by interaction with both ceramides and cholesterol. A
limitation of this proposal stems from the lack of knowledge about raft
structure. For example, it is currently unknown to what degree the
inner and outer leaflet of raft microdomains might differ in lipid
composition (26).
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Fig. 10.
Current model for the
activation-inactivation cycle of Raf kinases. Interactions of Raf
with cholesterol and ceramides (raft microdomains) may result in
translocation of the cytosolic Raf (locked Raf) to the membranes
followed by displacement of 14-3-3 proteins and marked decrease of
kinase activity. Transition from the inactive to the active
membrane-associated form of Raf requires the conversion of Ras from
GDP- to GTP-bound state (a), binding of Ras-GTP to Raf
(b), dephosphorylation of the serine at position 259 (c), several Raf phosphorylation events (d),
re-association of 14-3-3 proteins (e), and Raf dimerization
(f). Targeting of Raf to the membranes and binding to
activated Ras re-orients Raf molecules and induces structural
modifications which allows phosphorylation-dephosphorylation events and
assembly of a Raf signalosome containing different adaptor/scaffold
proteins and perhaps the Raf substrate MEK. For further details, see
text.
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Production of ceramides occurs metabolically from SM, which is highly
enriched in rafts. Therefore it is possible that ceramide synthesis
regulates the Raf translocation and further activation process.
Preliminary experiments that involve addition of SMase to SM-containing
liposomes support this possibility (data not shown). The interactions
of Raf with PS and PA probably take place in non-raft regions, since
these regions represent GPL-rich domains. Our next effort will focus on
investigating other signaling molecules such as Ras, heterotrimeric
G-proteins, MEK/ERK, and Src kinases (in the presence and absence of
Raf) with respect to their interactions with rafts and to delineate
conclusively the constellation of components sufficient for a complete
in vitro re-activation of Raf kinases.
Conclusions for the Regulation of Raf Activation-Inactivation
Cycle--
The currently accepted model for Raf translocation to
membranes is based on the observation of Ras-dependent
recruitment of Raf induced by external stimuli or constitutive
activation by oncogenic mutations (1, 70-72). Since experiments
demonstrating Ras-Raf binding at the plasma membrane generally involved
overexpression of Ras and/or Raf, it has to be considered that
unphysiologically high levels of Ras in the plasma membranes may be
responsible for the apparent Raf translocation. According to data
presented here and by others (19, 62), the direct and Ras-independent Raf association with membranes should be considered as the basal Raf
translocation pathway. Fig. 10 depicts a model of a Raf
activation-inactivation cycle that is consistent with our data. In this
model we propose Raf to be targeted to the plasma membrane primarily by
its association with cholesterol and ceramides (raft microdomains). The
activation process utilizes membrane-prebound Raf that exhibits low
basal activity due to the lipid interactions and loss of 14-3-3 proteins (see Figs. 5 and 8). The dissociation of 14-3-3 may facilitate the dephosphorylation of serine at the position Ser-259 (73). Removal
of the phosphate at this position by specific phosphatases may be a
prerequisite for the effective activation of C-Raf (74). Upon
conversion of Ras to its GTP-bound state by cell surface receptors, the
membrane-bound Raf interacts
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ABSTRACT
INTRODUCTION
