The tumor suppressor DiRas3 forms a complex with H-Ras and C-RAF proteins and regulates localization, dimerization, and kinase activity of C-RAF.

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity.

Single substitution within the RKTR motif impairs kinase activity but promotes dimerization of RAF kinase.

The serine/threonine kinase RAF is a central component of the MAPK cascade. Regulation of RAF activity is highly complex and involves recruitment to membranes and association with Ras and scaffold proteins as well as multiple phosphorylation and dephosphorylation events. Previously, we identified by molecular modeling an interaction between the N-region and the RKTR motif of the kinase domain in RAF and assigned a new function to this tetrapeptide segment. Here we found that a single substitution of each basic residue within the RKTR motif inhibited catalytic activity of all three RAF isoforms. However, the inhibition and phosphorylation pattern of C-RAF and A-RAF differed from B-RAF. Furthermore, substitution of the first arginine led to hyperphosphorylation and accumulation of A-RAF and C-RAF in plasma membrane fraction, indicating that this residue interferes with the recycling process of A-RAF and C-RAF but not B-RAF. In contrast, all RAF isoforms behave similarly with respect to the RKTR motif-dependent dimerization. The exchange of the second arginine led to exceedingly increased dimerization as long as one of the protomers was not mutated, suggesting that substitution of this residue with alanine may result in similar a structural rearrangement of the RAF kinase domain, as has been found for the C-RAF kinase domain co-crystallized with a dimerization-stabilizing RAF inhibitor. In summary, we provide evidence that each of the basic residues within the RKTR motif is indispensable for correct RAF function.

BAD contributes to RAF-mediated proliferation and cooperates with B-RAF-V600E in cancer signaling.

BAD (Bcl-2 antagonist of cell death) belongs to the proapoptotic BH3-only subfamily of Bcl-2 proteins. Physiological activity of BAD is highly controlled by phosphorylation. To further analyze the regulation of BAD function, we investigated the role of recently identified phosphorylation sites on BAD-mediated apoptosis. We found that in contrast to the N-terminal phosphorylation sites, the serines 124 and 134 act in an antiapoptotic manner because the replacement by alanine led to enhanced cell death. Our results further indicate that RAF kinases represent, besides PAK1, BAD serine 134 phosphorylating kinases. Importantly, in the presence of wild type BAD, co-expression of survival kinases, such as RAF and PAK1, leads to a strongly increased proliferation, whereas substitution of serine 134 by alanine abolishes this process. Furthermore, we identified BAD serine 134 to be strongly involved in survival signaling of B-RAF-V600E-containing tumor cells and found that phosphorylation of BAD at this residue is critical for efficient proliferation in these cells. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation and its role in cancer signaling.

Pore-forming activity of BAD is regulated by specific phosphorylation and structural transitions of the C-terminal part.

BACKGROUND: BAD protein (Bcl-2 antagonist of cell death) belongs to the BH3-only subfamily of proapoptotic proteins and is proposed to function as the sentinel of the cellular health status. Physiological activity of BAD is regulated by phosphorylation, association with 14-3-3 proteins, binding to membrane lipids and pore formation. Since the functional role of the BAD C-terminal part has not been considered so far, we have investigated here the interplay of the structure and function of this region. METHODS: The structure of the regulatory C-terminal part of human BAD was analyzed by CD spectroscopy. The channel-forming activity of full-length BAD and BAD peptides was carried out by lipid bilayer measurements. Interactions between proteins and peptides were monitored by the surface plasmon resonance technique. In aqueous solution, C-terminal part of BAD exhibits a well-ordered structure and stable conformation. In a lipid environment, the helical propensity considerably increases. The interaction of the C-terminal segment of BAD with the isolated BH3 domain results in the formation of permanently open pores whereby the phosphorylation of serine 118 within the BH3 domain is necessary for effective pore formation. In contrast, phosphorylation of serine 99 in combination with 14-3-3 association suppresses formation of channels. C-terminal part of BAD controls BAD function by structural transitions, lipid binding and phosphorylation. Conformational changes of this region upon membrane interaction in conjunction with phosphorylation of the BH3 domain suggest a novel mechanism for regulation of BAD. GENERAL SIGNIFICANCE: Multiple signaling pathways mediate inhibition and activation of cell death via BAD.

Identification of novel in vivo phosphorylation sites of the human proapoptotic protein BAD: pore-forming activity of BAD is regulated by phosphorylation.

BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-X(L) proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.

Regulation of RAF activity by 14-3-3 proteins: RAF kinases associate functionally with both homo- and heterodimeric forms of 14-3-3 proteins.

Mammalian 14-3-3 proteins play a crucial role in the activation process of RAF kinases. However, little is known about the selectivity of the mammalian 14-3-3 isoforms with respect to RAF association and activation. Using mass spectrometry, we analyzed the composition of the 14-3-3 isoforms attached to RAF kinases and found that B-RAF associates in vivo with 14-3-3 at much higher diversity than A- and C-RAF. We also examined in vitro binding of purified mammalian 14-3-3 proteins to RAF kinases using surface plasmon resonance techniques. While B- and C-RAF exhibited binding to all seven 14-3-3 isoforms, A-RAF bound with considerably lower affinities to epsilon, tau, and sigma 14-3-3. These findings indicate that 14-3-3 proteins associate with RAF isoforms in a pronounced isoform-specific manner. Because 14-3-3 proteins appear in dimeric forms, we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. For that purpose, the budding yeast Saccharomyces cerevisiae, possessing only two 14-3-3 isoforms (BMH1 and BMH2), served as testing system. By deletion of the single BMH2 gene, we found that both homo- and heterodimeric forms of 14-3-3 can participate in RAF activation. Furthermore, we show that A-, B-, and C-RAF activity is differentially regulated by its C-terminal and internal 14-3-3 binding domain. Finally, prohibitin, a scaffold protein that affects C-RAF activation in a stimulatory manner, proved to interfere with the internal 14-3-3 binding site in C-RAF. Together, our results shed more light on the complex mechanism of RAF activation, particularly with respect to activation steps that are mediated by 14-3-3 proteins and prohibitin.

X-linked and cellular IAPs modulate the stability of C-RAF kinase and cell motility.

Inhibitor of apoptosis proteins (IAP) are evolutionarily conserved anti-apoptotic regulators. C-RAF protein kinase is a direct RAS effector protein, which initiates the classical mitogen-activated protein kinase (MAPK) cascade. This signalling cascade mediates diverse biological functions, such as cell growth, proliferation, migration, differentiation and survival. Here we demonstrate that XIAP and c-IAPs bind directly to C-RAF kinase and that siRNA-mediated silencing of XIAP and c-IAPs leads to stabilization of C-RAF in human cells. XIAP binds strongly to C-RAF and promotes the ubiquitylation of C-RAF in vivo through the Hsp90-mediated quality control system, independently of its E3 ligase activity. In addition, XIAP or c-IAP-1/2 knockdown cells showed enhanced cell migration in a C-RAF-dependent manner. XIAP promotes binding of CHIP (carboxy terminal Hsc70-interacting protein), a chaperone-associated ubiquitin ligase, to the C-RAF-Hsp90 complex in vivo. Interfering with CHIP expression resulted in stabilization of C-RAF and enhanced cell migration, as observed in XIAP knockdown cells. Our data show an unexpected role of XIAP and c-IAPs in the turnover of C-RAF protein, thereby modulating the MAPK signalling pathway and cell migration.

Positive regulation of A-RAF by phosphorylation of isoform-specific hinge segment and identification of novel phosphorylation sites.

In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. Activation of RAF kinases involves a complex series of phosphorylations. Although the most prominent phosphorylation sites of B- and C-RAF are well characterized, little is known about regulatory phosphorylation of A-RAF. Using mass spectrometry, we identified here a number of novel in vivo phosphorylation sites in A-RAF. In particular, we found that Ser-432 participates in MEK binding and is indispensable for A-RAF signaling. On the other hand, phosphorylation within the activation segment does not contribute to epidermal growth factor-mediated activation. Furthermore, we show that the potential 14-3-3 binding domains in A-RAF are phosphorylated independently of its activation status. Of importance, we identified a novel regulatory domain in A-RAF (referred to as IH-segment) positioned between amino acids 248 and 267 that contains seven putative phosphorylation sites. Three of these sites, serines 257, 262, and 264, regulate A-RAF activation in a stimulatory manner. The spatial model of the A-RAF fragment, including residues between Ser-246 and Glu-277, revealed a switch of charge at the molecular surface of the IH-region upon phosphorylation, suggesting a mechanism in which the high accumulation of negative charges may lead to an electrostatic destabilization of protein-membrane interaction resulting in depletion of A-RAF from the plasma membrane. Together, we provide here for the first time a detailed analysis of in vivo A-RAF phosphorylation status and demonstrate that regulation of A-RAF by phosphorylation exhibits unique features compared with B- and C-RAF.

B- and C-RAF display essential differences in their binding to Ras: the isotype-specific N terminus of B-RAF facilitates Ras binding.

Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.

Unique N-region determines low basal activity and limited inducibility of A-RAF kinase: the role of N-region in the evolutionary divergence of RAF kinase function in vertebrates.

In mammals the RAF family of serine/threonine kinases consists of three members, A-, B-, and C-RAF. A prominent feature of RAF isoforms regards differences in basal and inducible kinase activities. To elucidate the nature of these differences, we studied the role of the nonconserved residues within the N-region (Negative-charge regulatory region). The nonconserved amino acids in positions -3 and +1 relative to the highly conserved serine 299 in A-RAF and serine 338 in C-RAF have so far not been considered as regulatory residues. Here we demonstrate the essential role of these residues in the RAF activation process. Substitution of tyrosine 296 in A-RAF to arginine led to a constitutively active kinase. In contrast, substitution of glycine 300 by serine (mimicking B- and C-RAF) acts in an inhibitory manner. Consistent with these data, the introduction of glycine in the analogous position of C-RAF (S339G mutant) led to a constitutively active C-RAF kinase. Based on the three-dimensional structure of the catalytic domain of B-RAF and using the sequences of the N-regions of A- and C-RAF, we searched by molecular modeling for the putative contact points between these two moieties. A tight interaction between the N-region residue serine 339 of C-RAF and arginine 398 of the catalytic domain was identified and proposed to inhibit the kinase activity of RAF proteins, because abrogation of this interaction contributes to RAF activation. Furthermore, tyrosine 296 in A-RAF favors a spatial orientation of the N-region segment, which enables a tighter contact to the catalytic domain, whereas a glutamine residue at this position in C-RAF abrogates this interaction. Considering this observation, we suggest that tyrosine 296, which is unique for A-RAF, is a major determinant of the low activating potency of this RAF isoform.

Reversible membrane interaction of BAD requires two C-terminal lipid binding domains in conjunction with 14-3-3 protein binding.

BAD is a Bcl-2 homology domain 3 (BH3)-only proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Binding of BAD to mitochondria is thought to be exclusively mediated by its BH3 domain. We show here that BAD binds to lipids with high affinities, predominantly to negatively charged phospholipids, such as phosphatidylserine, phosphatidic acid, and cardiolipin, as well as to cholesterol-rich liposomes. Two lipid binding domains (LBD1 and LBD2) with different binding preferences were identified, both located in the C-terminal part of the BAD protein. BAD facilitates membrane translocation of Bcl-XL in a process that requires LBD2. Integrity of LBD1 and LBD2 is also required for proapoptotic activity in vivo. Phosphorylation of BAD does not affect membrane binding but renders BAD susceptible to membrane extraction by 14-3-3 proteins. BAD can be removed efficiently by 14-3-3zeta, -eta, -tau and lesxs efficiently by other 14-3-3 isoforms. The assembled BAD.14-3-3 complex exhibited high affinity for cholesterol-rich liposomes but low affinity for mitochondrial membranes. We conclude that BAD is a membrane-associated protein that has the hallmarks of a receptor rather than a ligand. Lipid binding is essential for the proapoptotic function of BAD in vivo. The data support a model in which BAD shuttles in a phosphorylation-dependent manner between mitochondria and other membranes and where 14-3-3 is a key regulator of this relocation. The dynamic interaction of BAD with membranes is tied to activation and membrane translocation of Bcl-XL.

B-Raf and C-Raf signaling investigated in a simplified model of the mitogenic kinase cascade.

Signaling pathways based on the reversible phosphorylation of proteins control most aspects of cellular life in higher organisms. Extracellular stimuli can induce growth, differentiation, survival and the stress response through a number of highly conserved signaling pathways. We discuss how the intensity and duration of signals may have dramatic consequences on the way cells respond to stimuli. Picking the central Ras-Raf-MEK-ERK signal cascade, we developed a mathematical model of how stimuli induce different signal patterns and thereby different cellular responses, depending on cell type and the ratio between B-Raf and C-Raf. Based on biochemical data for activation and dephosphorylation, as well as the differential equations of our model, we suggest a different signaling pattern and response result for B-Raf (strong activation, sustained signal) and C-Raf (steep activation, transient signal). We further support the significance of such differential modulatory signaling by showing different Raf isoform expression in various cell lines and experimental testing of the predicted kinase activities in B-Raf, C-Raf and mutated versions.

Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration.

Ras proteins control the signalling pathways that are responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins that initiate the mitogen-activated protein kinase (MAPK) cascade, which mediates diverse biological functions such as cell growth, survival and differentiation. Here we show that prohibitin, a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf-MEK-ERK pathway by Ras. The membrane targeting and activation of C-Raf by Ras needs prohibitin in vivo. In addition, direct interaction with prohibitin is required for C-Raf activation. C-Raf kinase fails to interact with the active Ras induced by epidermal growth factor in the absence of prohibitin. Moreover, in prohibitin-deficient cells the adhesion complex proteins cadherin and beta-catenin relocalize to the plasma membrane and thereby stabilize adherens junctions. Our data show an unexpected role of prohibitin in the activation of the Ras-Raf signalling pathway and in modulating epithelial cell adhesion and migration.

Novel C-Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation.

The C-Raf kinase is regulated by numerous phosphorylation steps. To quantify the most prominent phosphorylation sites of C-Raf, we performed mass spectrometry analysis of wild-type C-Raf and the constitutively active C-Raf mutant C-Raf-Y340D/Y341D. We confirmed phosphorylation of most of the sites reported in the literature with the exception that we did not detect phosphorylation of threonine 268/269 (autophosphorylation sites) and threonine 491/serine 494 (kinase activation loop). Importantly, we detected novel phosphorylation sites at the positions of serine 296 and 301. The degree of phosphorylation in these positions depends on the level of activation of C-Raf. Furthermore, we show here, using point mutant forms of C-Raf kinases with serine to alanine and serine to aspartic acid substitution, that serines 296 and 301 contribute to negative regulation of C-Raf.

Crystal structure of the p14/MP1 scaffolding complex: how a twin couple attaches mitogen-activated protein kinase signaling to late endosomes.

Signaling pathways in eukaryotic cells are often controlled by the formation of specific signaling complexes, which are coordinated by scaffold and adaptor proteins. Elucidating their molecular architecture is essential to understand the spatial and temporal regulation of cellular signaling. p14 and MP1 form a tight (K(d) = 12.8 nM) endosomal adaptor/scaffold complex, which regulates mitogen-activated protein kinase (MAPK) signaling. Here, we present the 1.9-A crystal structure of a biologically functional p14/MP1 complex. The overall topology of the individual MP1 and p14 proteins is almost identical, having a central five-stranded beta-sheet sandwiched between a two-helix and a one-helix layer. Formation of the p14/MP1 heterodimer proceeds by beta-sheet augmentation and yields a unique, almost symmetrical, complex with several potential protein-binding sites on its surface. Mutational analysis allowed identification of the p14 endosomal adaptor motif, which seems to orient the complex relative to the endosomal membrane. Two highly conserved and hydrophobic protein-binding sites are located on the opposite "cytoplasmic" face of the p14/MP1 heterodimer and might therefore function as docking sites for the target proteins extracellular regulated kinase (ERK) 1 and MAPK/ERK kinase 1. Furthermore, detailed sequence analyses revealed that MP1/p14, together with profilins, define a protein superfamily of small subcellular adaptor proteins, named ProflAP. Taken together, the presented work provides insight into the spatial regulation of MAPK signaling, illustrating how p14 and MP1 collaborate as an endosomal adaptor/scaffold complex.

Dynamic changes in C-Raf phosphorylation and 14-3-3 protein binding in response to growth factor stimulation: differential roles of 14-3-3 protein binding sites.

Phosphorylation events play a crucial role in Raf activation. Phosphorylation of serines 259 and 621 in C-Raf and serines 364 and 728 in B-Raf has been suggested to be critical for association with 14-3-3 proteins. To study the functional consequences of Raf phosphorylations at these positions, we developed and characterized phosphospecific antibodies directed against 14-3-3 binding epitopes: a monoclonal phosphospecific antibody (6B4) directed against pS621 and a polyclonal antibody specific for B-Raf-pS364 epitope. Although 6B4 detected both C- and B-Raf in Western blots, it specifically recognizes the native form of C-Raf but not B-Raf. Contrary to B-Raf, a kinase-dead mutant of C-Raf was found to be only poorly phosphorylated in the Ser-621 position. Moreover, serine 259 to alanine mutation prevented the Ser-621 phosphorylation suggesting an interdependence between these two 14-3-3 binding domains. Direct C-Raf.14-3-3 binding studies with purified proteins combined with competition assays revealed that the 14-3-3 binding domain surrounding pS621 represents the high affinity binding site, whereas the pS259 epitope mediates lower affinity binding. Raf isozymes differ in their 14-3-3 association rates. The time course of endogenous C-Raf activation in mammalian cells by nerve growth factor (NGF) has been examined using both phosphospecific antibodies directed against 14-3-3 binding sites (6B4 and anti-pS259) as well as phosphospecific antibodies directed against the activation domain (anti-pS338 and anti-pY340/pY341). Time course of Ser-621 phosphorylation, in contrast to Ser-259 phosphorylation, exhibited unexpected pattern reaching maximal phosphorylation within 30 s of NGF stimulation. Phosphorylation of tyrosine 340/341 reached maximal levels subsequent to Ser-621 phosphorylation and was coincident with emergence of kinase activity. Taken together, we found substantial differences between C-Raf.14-3-3 binding epitopes pS259 and pS621 and visualized for the first time the sequence of the essential C-Raf phosphorylation events in mammalian cells in response to growth factor stimulation.

Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts.

The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras proteins in their GTP-bound state. In this study we provide evidence that both purified B- and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. In the presence of phosphatidylserine and lipid second messengers such as phosphatidic acid and ceramides these associations were very specific with affinity constants (K(D)) in the range of 0.5-50 nm. Raf association with liposomes was accompanied by displacement of 14-3-3 proteins and inhibition of Raf kinase activities. Interactions of Raf with cholesterol are of particular interest, since cholesterol has been shown to be involved, together with sphingomyelin and glycerophospholipids in the formation of specialized lipid microdomains called rafts. We demonstrate here that purified Raf proteins have moderate binding affinity for cholesterol. However, under conditions of lipid raft formation, Raf association with cholesterol (or rafts) increased dramatically. Since ceramides also support formation of rafts and interact with Raf we propose that Raf may be present at the plasma membrane in two distinct microdomains: in raft regions via association with cholesterol and ceramides and in non-raft regions due to interaction with phosphatidylserine and phosphatidic acid. At either location Raf kinase activity was inhibited by lipid binding in the absence or presence of Ras. Ras-Raf interactions with full-length C-Raf were studied both in solution and in phospholipid environment. Ras association with Raf was GTP dependent as previously demonstrated for C-Raf-RBD fragments. In the presence of liposomes the recruitment of C-Raf by reconstituted Ras-farnesyl was only marginal, since almost 70% of added C-Raf was bound by the lipids alone. Thus Ras-Raf binding in response to activation of Ras-coupled receptors may utilize Raf protein that is already present at the membrane.