(Received for publication, September 26, 1995; and in revised form, December 21, 1995)
From the
Previous studies demonstrated that the cysteine-rich
amino-terminal domain of Raf-1 kinase interacts selectively with
phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G.
M., Strum, J. C., and Bell, R. M.(1994) J. Biol. Chem. 269,
10000-10007). Further analysis showed that full-length Raf-1
bound to both phosphatidylserine and phosphatidic acid (PA).
Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC;
residues 295-648 of human Raf-1) interacted strongly with
phosphatidic acid. The binding of RafC to PA displayed positive
cooperativity with Hill numbers between 3.3 and 6.2; the apparent K ranged from 4.9 ± 0.6 to 7.8
± 0.9 mol % PA. The interaction of RafC with PA displayed a pH
dependence distinct from the interaction between the cysteine-rich
domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at
high ionic strength. Of all the lipids tested, only PA and cardiolipin
exhibited high affinity binding; other acidic lipids were either
ineffective or weakly effective. By deletion mutagenesis, the PA
binding site within RafC was narrowed down to a 35-amino acid segment
between residues 389 and 423. RafC did not bind phosphatidyl alcohols;
also, inhibition of PA formation in Madin-Darby canine kidney cells by
treatment with 1% ethanol significantly reduced the translocation of
Raf-1 from the cytosol to the membrane following stimulation with
12-O-tetradecanoylphorbol-13-acetate. These results suggest a
potential role of the lipid second messenger, PA, in the regulation of
translocation and subsequent activation of Raf-1 in vivo.
The protooncogene product Raf-1 is a ubiquitously expressed Ser/Thr kinase which plays an essential role in signal transduction from a large number of growth factor receptors, cytokine receptors, several membrane-bound oncogenes, and certain mitogenic peptides with G-protein-linked receptors(1, 2, 3) . Truncation of an amino-terminal fragment (first 305 amino acids) renders Raf-1 highly oncogenic(4) . Based on in vitro direct binding assays and immunoprecipitation assays from cultured cells, the amino terminus of Raf-1 is now known to associate directly with another protooncogene product, p21Ras, and the regions on Raf-1 critical for high affinity interaction with p21Ras have been identified(5, 6, 7, 8, 9, 10) . This association between Raf-1 and p21Ras appears to be an essential prerequisite for the normal pathway of Raf-1 activation. As a result, the relative binding affinity of specific effector domain mutants of p21Ras protein for Raf-1 is directly correlated with their ability to transform murine fibroblasts(7) . Conversely, disruption of Raf-1:Ras interaction by a point mutation (R89L) in human Raf-1 prevents activation of Raf-1 kinase in Sf-9 insect cells (11) and also affects late zygotic functions of the Drosophila D-Raf protein(12) .
However, a simple
association between Raf-1 and p21Ras is not sufficient for the
activation of Raf-1 kinase. It appears that both p21Ras and Raf-1
require to be present at the plasma membrane for expression of their
biological activities. For example, the transforming activity of an
oncogenic variant of the p21ras (K-ras 4B), is dependent on
its membrane localization(13) . On the other hand, the
requirement for p21Ras in Raf-1 activation is overcome by targeting
Raf-1 to the plasma membrane by addition of a membrane localization
signal to Raf-1(14, 15) . These results suggest that
the function of p21Ras, at least for Raf-1 activation, is to recruit
inactive, cytosolic Raf-1 kinase to the plasma membrane where
additional protein-protein and/or protein-lipid interactions ()may regulate the activation of Raf-1.
The inner leaflet
of the plasma membrane of eukaryotic cells is composed of 30%
acidic and
70% zwitterionic phospholipids(16) . A growing
body of evidence suggests that several proteins enhance their plasma
membrane association by binding to the membrane lipids through
electrostatic as well as hydrophobic interactions(37) . Some
examples include protein kinase C(17, 18) , the
myristoylated, alanine-rich protein kinase C substrate,
MARCKS(19) , the human immunodeficiency virus Gag
protein(20) , and v- and c-Src(21) . For protein kinase
C, a zinc-containing cysteine-rich domain is believed to play a key
role in the activation of the enzyme by binding to the acidic
phospholipid, phosphatidylserine, and to diacylglycerol(22) . A
similar zinc-coordinating cysteine-rich region exists in the amino
terminus of Raf-1 and is capable of binding to PS (
)containing liposomes in vitro(23) . In
contrast to protein kinase C, the binding of the Raf-1 cysteine-rich
domain to PS is not modulated by either diacylglycerol or phorbol
esters.
In addition to the cysteine-rich domain, a detailed analysis of Raf-1-phospholipid interaction revealed a second site for association with acidic phospholipids, notably phosphatidic acid, in the carboxyl-terminal domain of the protein. In the present article, we have identified, by deletion mutagenesis, a 35-amino acid fragment of human Raf-1 kinase that is capable of interacting with PA. This finding suggests that, besides binding to p21Ras, Raf-1 also associates with plasma membrane lipids through multiple domains within the amino- and carboxyl-terminal regions of the protein, resulting in a firm anchor. One might postulate that these protein-lipid interactions result in a conformational switch in Raf-1 from an inactive to an activable state. Consistent with this postulate, we further show that an inhibition of the generation of phosphatidic acid in Madin-Darby canine kidney (MDCK) cells results in a decrease in translocation of Raf-1 kinase from the cytosol to the membrane following agonist stimulation.
Figure 1:
Schematic diagram of Raf-1 constructs.
The amino acid residues encoded by the various human Raf-1 constructs
used in this study are indicated. All proteins were fused to GST at
their NH termini. The circle represents GST, and
the hatched boxes represent Raf-1
sequences.
Figure 2: Binding of Raf-1 constructs to PA. A, binding of RafC to PC treated with phospholipases. One microgram of dipalmitoyl-PC was loaded onto individual wells of a microtiter plate and either left untreated or treated with 0.2 unit of phospholipase C (B. cereus) or phospholipase D (cabbage) for 1 h at room temperature. GST-RafC was then added to the wells in a 2-fold serial dilution from 50 nM to 0.78 nM and assayed for binding according to the ELISA format assay. B, binding to coated phosphatidic acid. RafFull, RafCys, and RafC (50 nM each in PBS, pH 7.2) were incubated with 1 µg of dioleoyl-PA and assayed for binding by the ELISA format assay. Results shown are the mean of assays performed in triplicate.
Figure 3: Binding of RafC to PA as a function of mole percent of PA in PA/PC mixtures. A, analysis by the ELISA format assay. Details of the experiment have been described in the text. A mixture of dioleoyl-PA and dipalmitoyl-PC was used where the mole percent of PA was varied from 0-30 mol %, maintaining the total lipid invariant at 10 µg. RafC was used at 150 nM. Results shown are the mean of assays performed in triplicate. Inset, the same experiment was repeated with PA varying from 0 to 50 mol % and a series of concentrations of RafC, 150 nM (solid circle), 75 nM (open square), 37.5 nM (solid square), 18.8 nM (open circle), and 9.4 nM (solid triangle). The data were fit to the modified Hill equation as described in the text. B, association of RafC to PA/PC liposomes. Liposome association experiments were performed as described previously(26, 27) . Liposomes of different PA/PC composition were incubated with RafC (4 µg of protein), and the extent of protein association with the liposomes was determined relative to the protein recovered in the absence of lipids. The protein bands obtained in the SDS-PAGE were quantitated by scanning densitometry, and the values are plotted as a function of PA concentration in the liposomes.
The sigmoidal nature of the association of RafC with PA was also confirmed by liposome association experiments. PA/PC liposomes containing varying mole fractions of PA were prepared as described previously (22) and incubated with RafC. The amount of RafC associated with the liposomes was quantitated as a function of the concentration of PA. Apparent association, in the absence of PA was taken as background (RafC does not bind significantly to PC as discussed later). The results of the liposome association experiment are summarized in Fig. 3B. Notably, an enhancement in RafC-PA association was observed between 10 and 20 mol % PA which is comparable to the results obtained in the ELISA format assay. The quantitative differences that exist between the ELISA format assay and the liposome association assay regarding the mole percent PA required for maximal Raf-1 association arise from two effects. First, in the ELISA format assay, the phospholipids are immobilized on the microtiter plates, thus providing a high, invariant, local concentration of PA which results in a high surface charge density even at a low mole percent. In the case of liposomes the total surface area is greatly increased owing to their spherical shape consequently requiring a higher mole percent PA to attain a similar surface charge density. Second, there is a gain of dimensionality in the liposome association assay compared to the ELISA format assay for Raf-lipid interactions. This additional degree of freedom increases the entropy of the system and consequently diminishes the chances of productive association between the liposomes and Raf-1. This effect is only counteracted to some extent when the concentration of the reagent (in this case, PA) is increased. Hence a greater mole percent PA is required in the liposome association assay to obtain saturable binding to Raf-1.
The interaction of Raf-1 with PA was next characterized with respect to pH and ionic strength. RafFull, RafCys, or RafC were allowed to bind to PA at acidic, neutral or alkaline pH and the binding was quantitated by the ELISA format assay (Fig. 4A). RafFull and RafC bound to PA with the same profile; lower binding was observed at acidic (4.5) or alkaline (9.0) pH values, with maximal binding occurring at near neutral pH values (6.0 and 7.5). In contrast, RafCys displayed maximal PA binding at acidic pH which was drastically reduced with increases in pH. This difference in pH dependence between RafC and RafCys explains why, under the standard conditions of the ELISA format assay (pH 7.2), RafCys binds with about 10-fold lower affinity to PA compared to RafC (Fig. 2A). The identical pH profiles observed for RafC and RafFull further support the hypothesis that the binding of full-length Raf-1 to PA is mediated primarily by its COOH-terminal domain.
Figure 4: Effect of pH and ionic strength on RafC-PA interaction. A, effect of pH on the binding of Raf-1 fusion proteins to PA. The ELISA format assay was carried out with 1 µg of dioleoyl-PA and 50 nM each of RafFull, RafCys, and RafC. The incubations were performed at pH values of 4.8 (solid bar), 6.0 (hatched bar), 7.5 (stippled bar), and 9.0 (open bar). Results were normalized for each fusion protein by setting the binding value at pH 4.8 to be 100. Results shown are the mean of assays performed in triplicate. B, effect of ionic strength. Dioleoyl-PA (1 µg/well) was incubated with RafC in the presence of different concentrations of NaCl (0-1000 mM) and then assayed for binding by the ELISA format assay. The experiments were carried out at RafC concentrations of 25 (solid square), 50 (open circle), and 100 (solid circle) nM. Results are the mean of assays performed in triplicate.
A possible mechanism for the RafC-PA interaction may be postulated as arising from electrostatic interactions where the negatively charged PA nonspecifically binds to positively charged amino acids on the protein. If such were the case, then the interaction would be highly dependent on ionic strength and would eventually be competed out at high ionic strength. We therefore determined the effect of increasing ionic strength on RafC-PA interaction and the results are shown in Fig. 4B. At all RafC concentrations tested (25, 50 and 100 nM), the binding to PA was weak in the absence of salt. Binding was progressively increased up to 250 nM NaCl after which additional increases in salt concentration, up to 1 M, had little effect on binding. The requirement for a certain concentration of salt for maximal binding may be attributed to shielding effects provided by counter ions between adjacent negatively charged lipid molecules or between negatively charged regions of RafC and PA. Since the binding cannot be competed out even at 1 M NaCl, it suggests that the interaction is not entirely electrostatic in nature. Other forces (e.g. hydrophobic, hydrogen bonding, and van der Waals) are therefore inferred to play a part in stabilizing the RafC-PA interaction.
We next tested different potential competitors of the RafC-PA interaction. In all cases, RafC was first incubated with the test reagent in solution for 30 min and then the entire mixture was added to the wells of microtiter plates coated with PA and allowed to incubate for an additional 30 min. The results of the experiment are shown in Fig. 5. None of the reagents tested competed effectively for the binding of RafC to PA. At all concentrations of RafC tested, again a weaker binding was observed in the absence of salt. Addition of all reagents at 50 mM (10 mM for Ser-O-phosphate and ATP) enhanced the binding of RafC to PA by varying extent. Importantly, 10 mM ATP did not inhibit RafC-PA interaction, which suggested that the site within RafC involved in PA binding was distinct from the site involved in binding to ATP.
Figure 5: Effect of anions and cations of RafC-PA interaction. RafC, at concentrations of 25 (open bar), 50 (hatched bar), and 100 (solid bar) nM, was preincubated with anions (as sodium salts) or cations (as chloride salts) for 30 min before addition to dioleoyl-PA (1 µg/well). The amount of RafC bound to PA was subsequently assayed by the ELISA format assay. All compounds were tested at 50 mM excepting O-phosphoserine and ATP which were used at 10 mM. Results shown are the mean of assays performed in triplicate.
Figure 6: Specificity of RafC-PA interaction. A, different lipids (1 µg each) were coated on the wells of microtiter plates in the presence of dipalmitoyl-PC (9 µg). RafC (200 nM) was added to the lipids and the extent of association was quantitated by the ELISA format assay. The lipids used were PC, dipalmitoyl phosphatidylcholine; PS, bovine brain phosphatidylserine; PI, pig liver phosphatidylinositol; PA, dioleoyl phosphatidic acid; PE, bovine heart phosphatidylethanolamine; Bis-PA, bis-tetrapalmitoyl phosphatidic acid; PMt, dipalmitoyl phosphomethanol; PPt, dipalmitoyl phosphopropanol; PG, dioleoyl phosphatidylglycerol; DG, dioleoyl glycerol; ganglioside, bovine brain type II ganglioside; cardiolipin, bovine heart cardiolipin; sphingomyelin, pig brain sphingomyelin; ceramide, bovine brain type IV ceramide; sphingosine, bovine brain D-sphingosine. Results shown are the mean of two independent experiments. B, schematic representation of phosphatidic acid, cardiolipin, and bis-phosphatidic acid. The ionized oxygen atoms are depicted in bold type.
Figure 7: Identification of a PA binding minimal domain on RafC by deletion mutagenesis. A, ELISA analysis of all GST-Raf-1 fusion proteins with anti-GST polyclonal antiserum. The fusion proteins were serially diluted (2-fold) in coupling buffer (0.05 M carbonate/bicarbonate, pH 9.6) and coated on the wells of a microtiter plate, from a starting concentration of 100 nM. Anti-GST polyclonal antiserum was used at 1:2000 dilution. Results shown represent mean antibody binding to the range of concentrations of each GST-fusion protein. B-D, ATP-binding mutant and deletion mutants of different regions of RafC were generated as GST-fusion proteins as described in the text. All fusion proteins were serially diluted and tested for their ability to bind PA (1 µg/well) in the ELISA format assay.
Scheme 1: Scheme 1A, multiple sequence alignment of the PA binding segment of human Raf-1 (amino acids 390-426) to other Raf polypeptides. Conserved regions of charged and hydrophobic residues are shown in boxes. B, secondary structure prediction for the PA binding segment of Raf-1. The graph represents a Kyte-Doolittle hydrophilicity profile of the segment with hydrophilic regions lying on the (+) side. The top panel indicates the predicted secondary structure according to the profile network prediction algorithm. The letters E, H, and L stand for ``strand,'' ``helix,'' and ``neither strand nor helix,'' respectively. The bottom panel shows the amino acid sequence of the PA binding domain of Raf-1.
It has been shown that phorbol esters such
as TPA activate phospholipase D via a PKC--dependent mechanism in
MDCK cells(48) , resulting in the generation of PA, a potential
second messenger. Since in vitro studies indicated a specific
interaction between PA and Raf-1, we next determined if this was also
the case in vivo. In order to determine if the PA generated by
TPA-activated PLD has a role in Raf-1 translocation, we blocked the
production of PA derived from PLD with ethanol. In the presence of
ethanol, PLD catalyzes a transphosphatidylation reaction generating PEt
at the expense of PA. MDCK cells were stimulated with 10 nM TPA with varying concentrations of ethanol for 15 min. Cytosol and
membrane fractions were prepared and analyzed for Raf-1 as described
under ``Experimental Methods.'' The translocation of Raf-1
from the cytosol to the membrane in response to TPA was inhibited by
blocking the formation of PA by PLD with ethanol (Fig. 8A). Inhibition of translocation of Raf-1 was
dependent upon the concentration of ethanol. To determine if ethanol
had an effect on the translocation of PKC-
, the nitrocellulose
membrane was stripped and reprobed for PKC-
(Fig. 8B). The translocation of PKC-
in response
to TPA was not significantly affected by ethanol. In addition,
increasing the concentration of ethanol correlated with the increase in
PEt (Fig. 8C), and with a decrease in Raf-1
translocating to the membrane. These results suggest a role of PA
derived from PLD in Raf-1 translocation in vivo. It is
conceivable that PKC-
activates PLD, leading to the generation of
PA which aids in associating Raf-1 to the membrane. Once positioned at
the membrane, Raf-1 can be phosphorylated and activated by other
kinases such as PKC-
.
Figure 8:
PEt formation and its effect on
translocation of Raf-1 and PKC-. A, translocation of
Raf-1 in TPA-stimulated MDCK cells in the presence of ethanol. Details
of the experiment have been described under ``Experimental
Methods.'' Controls represent cells not stimulated with TPA.
Duplicates are shown for the controls not treated with ethanol. B, the Western blot used to probe for Raf-1 in A was
stripped and reprobed with antibodies to PKC-
to determine
translocation of the protein under identical conditions of cell
stimulation by 10 nM TPA in the absence or presence of
ethanol. C, phosphatidylethanol formation in MDCK cells. Cells
were labeled with [
H]-20:4-arachidonic acid as
described under ``Experimental Methods'' and then stimulated
with 10 nM TPA in the presence of varying concentrations of
ethanol. Cellular lipids were extracted and the amount of PEt formed
was quantitated by scintillation counting.
Our results demonstrate that RafC is capable of interacting with phosphatidic acid. This interaction between RafC and PA displayed a distinct pH dependence when compared to another lipid-binding motif within Raf-1, RafCys. At physiological pH, RafCys bound PA with markedly reduced affinity compared to RafC. Also, the binding of RafC to PA was not abolished in the presence of high salt concentrations, suggesting that the interaction was not purely electrostatic. Binding of RafC to PA exhibited a highly sigmoidal dependence on the mole % PA with Hill numbers between 4 and 6, under the experimental conditions described. This observed positive cooperativity in RafC-PA interaction may arise from two sources, (i) as a consequence of cooperative sequestering of PA molecules by RafC such as been reported for protein kinase C binding to PS and PA (31) and (ii) an electrostatics driven accumulation of positively charged regions of RafC to the negatively charged PA surface, followed by a reduction in dimensionality. Interactions of the second kind result in an apparent positive cooperativity(49) .
Through deletional mutagenesis, we identified a segment within RafC (residues 389-423) that was competent to bind PA. This region did not contain any characteristic motifs with the exception of a positively charged tetrapeptide sequence, RKTR (residues 398-401), which could be involved in an initial electrostatic interaction with PA; additional forces would then stabilize this interaction which consequently becomes relatively insensitive to high ionic strengths.
The physiological relevance of
the interaction of Raf-1 with PA is unclear. Raf-1 is translocated to
the plasma membrane before it is activated. However, the events at the
membrane that trigger the activation of Raf-1 are yet unknown. Although
the highly conserved family of acidic proteins termed 14-3-3 was shown
to associate with Raf-1 and implicated in Ras-dependent activation of
Raf-1 (50, 51, 52) , Raf-1 mutants unable to
stably interact with 14-3-3 could still be biologically activated in a
Ras-dependent manner (53) , suggesting that 14-3-3 is not a
necessary agent for the activation of Raf-1. Our observation that
inhibition of PA formation in intact cells can inhibit the
translocation of Raf-1 from the cytosol to the membrane suggests that
the presence of PA may facilitate the translocation and stabilization
of Raf-1 at the plasma membrane. It is interesting to note that many of
the signals that activate the mitogen-activated protein kinase pathway
through Ras also activate phospholipase D leading to a transient
generation of PA(54, 55, 56) . The role of PA
as a second messenger has been documented in several systems (57, 58) . In the present context, one might postulate
that a simultaneous activation of Ras (from RasGDP to
Ras
GTP) and phospholipase D would create an environment at the
membrane where translocated Raf-1 kinase can be firmly anchored by
interaction with Ras
GTP and with the membrane lipids, notably PS
(via the cysteine-rich domain) and PA (via RafC). Conversely, the
hydrolysis of PA to diacylglycerol by phosphatidate phosphohydrolase,
and the deactivation of Ras
GTP to Ras
GDP would sufficiently
weaken the affinity of Raf-1 for the membrane causing a net return of
Raf-1 to the cytosol. Recent evidence also suggests that Raf-1 may be
deactivated at the membrane by the action of membrane-associated
phosphatases(59) . Whether lipids would function as additional
activators of Raf-1 in a manner analogous to protein kinase C is at
present
unclear(60, 61, 62, 63, 64) .
Thus, the exact role of membrane lipids in the regulation of Raf-1
kinase activity remains to be elucidated.