Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

RAF-1 (C-RAF)

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_591

Synonyms

Historical Background

Raf-1, also known as C-Raf-1 or C-Raf, was identified about 30 years ago as the oncogene (v-raf) in the murine sarcoma virus 3611 (3611-MSV) and, in parallel, in the naturally occurring avian retrovirus Mill Hill 2 (MH2). The gene was named after its enhancing effect on fibrosarcoma induction in newborn mice: Rapidly accelerated fibrosarcoma, or Raf. The sequences of the oncogenes, v-raf (derived from 3611-MSV) and v-mil (derived from MH2), were found to encode a serine/threonine protein kinase containing the catalytic, but not the N-terminal regulatory domain of the enzyme. This deletion rendered the protein constitutively active and was responsible for its transforming effect, making Raf the first oncogenic serine/threonine kinase discovered. A pseudogene (c-raf-2) and two paralogues of c-raf-1, named a-raf and b-raf, were subsequently identified (Wellbrock et al. 2004; Niault and Baccarini 2010). About 20 years ago, Raf-1 was reported to be phosphorylated in response to growth factor stimulation and was identified as the activator of the  MEK/ERK pathway, the first mitogen-activated protein kinase (MAPK) module discovered that acts downstream of receptor tyrosine kinases; and finally, the finding that Raf-1 could be recruited to the membrane and stimulated by active Ras, already then recognized as a human oncogene, made the picture complete and led to the “textbook” description of the pathway as it is known today. Briefly, pathway activation involves the growth factor–induced dimerization and tyrosine phosphorylation of cell surface receptors, which in turn triggers the binding of a complex containing a scaffolding protein, Grb2, and a nucleotide exchange factor for Ras, SOS. The interaction with SOS catalyzes the exchange of GDP for GTP on Ras; with the help of scaffolding proteins (e.g. KSR), active, GTP-bound Ras can now induce the formation of membrane-associated Raf/MEK/ERK complexes. Each protein in the cascade can activate its downstream target via phosphorylation, i.e., Raf can phosphorylate MEK and MEK can phosphorylate ERK. Active ERK can induce the expression of several genes involved in cell proliferation, differentiation, and survival. In addition, active ERK feeds back on SOS, Raf, and MEK, providing a mechanism for signal attenuation [Fig. 1; reviewed in (Niault and Baccarini 2010)]. Adaptor proteins other than KSR have been reported to direct pathway components to distinct subcellular compartments, with potentially different signaling outcomes (Kolch 2005; McKay and Morrison 2007), and inhibitors of the pathway can regulate it at different levels (Kolch 2005).
RAF-1 (C-RAF), Fig. 1

Outline of the ERK pathway. The Grb2-SOS complex is recruited via the binding of Grb2 to tyrosine phosphorylated residues in the cytoplasmic domain of activated Receptor Tyrosine Kinases (RTK). This brings Sos in the proximity of Ras, which is activated by the exchange of GDP for GTP. GTP-bound Ras recruits Raf to the membrane, where it is activated by phosphorylation (see Fig. 2 for more details on Raf-1 activation). From here, the signal is passed, in the form of phosphorylation, from Raf to MEK to ERK (solid arrows). ERK, in turn, quenches pathway activity at different levels by phosphorylating SOS, B-Raf, Raf-1, and MEK on negative regulatory residues (broken arrows)

The study of the essential functions of Raf-1 in conventional and conditional knockout mice [(Galabova-Kovacs et al. 2006) and references therein] has revealed that most of Raf-1’s essential functions are not linked to the activation of the MEK/ERK pathway and to proliferation, but rather to pathways that counteract apoptosis and promote migration and differentiation. These new roles of Raf-1 are based on its interaction with three other kinases: the Rho-dependent kinase Rok-α, also known as  ROCK2 (Ehrenreiter et al. 2005, 2009; Piazzolla et al. 2005), involved in cytoskeletal rearrangements; the mammalian Sterile-20-like kinase-2, MST2 (O’Neill et al. 2004), homolog of Drosophila’s Hippo; and the apoptosis signal-regulating kinase 1, ASK1 (Yamaguchi et al. 2004), upstream regulator of the p38 and JNK pathways.

Structure of Raf and Activation of Its Kinase Function

Structure: The structure of Raf consists of three conserved regions [Fig. 2a; see also (Baccarini 2005) and references therein]. The regulatory/autoinhibitory domain of Raf is composed of two Conserved Regions, CR1 and CR2. CR1 contains the RBD – Ras Binding Domain, which is required for membrane recruitment of the protein after activation by Ras; and the CRD – Cysteine-Rich Domain, which, besides being a secondary Ras binding site, is responsible for Raf-1 autoinhibition. CR2 is rich in Ser/Thr residues, whose phosphorylation can inactivate protein function (i.e., negative regulatory residues such as S259, whose dephosphorylation is prerequisite for Ras binding and Raf activation). CR3 is responsible for the catalytic activity and contains residues (S338, Y341) whose phosphorylation is involved in growth factor-induced kinase activation. The three-dimensional structure of the RBD [NMR structure, PDB 1RFA, (Emerson et al. 1995)], CRD [NMR, PDB 1FAR, (Mott et al. 1996)], and of the kinase domain [X-ray, PDB 3OMV, (Hatzivassiliou et al. 2010)] is known. The RBD domain fold resembles that of ubiquitin, while the CRD domain is an atypical C1 domain, which binds to phosphatidylserine and needs Zn2+ ions to preserve its folded structure (Fig. 2b, left panel). The structure of the kinase domain consists of a smaller (N-terminal) and a larger (C-terminal) lobe. The latter contains the activation segment (the region between the DFG…APE motif, from D486 to E515), including the P-loop and ß-strand 9 (ß9). The P-loop is responsible for the correct positioning of both the adenosine and the gamma-phosphate of ATP for catalysis. The inactive conformation of the enzyme (“DFG-out”, yellow) is stabilized by hydrophobic interactions between the P-loop and the activation segment (Fig. 2b, right panel). Kinase activation is mediated by phosphorylation of serine/threonine residues in the activation segment (T491 and S494), which results in a conformational change from the “DFG-out” to the “DFG-in” conformation (red). In the “DFG-in” conformation, the ß9 strand of the N-lobe interacts with the ß6 strand of the C-lobe of the kinase, closing the cleft between the two lobes and switching the enzyme to its active state. The ß9 strand contains the V482 residue which corresponds to the B-Raf residue frequently mutated to E in human melanoma [V600E; activating B-Raf mutation (Wellbrock et al. 2004)]. Interestingly, corresponding mutations in Raf-1, which would have the same activating effect, have not been reported.
RAF-1 (C-RAF), Fig. 2

Domain structure of Raf-1 and mechanism of activation. (a) Schematic representation of the domain structure of Raf-1. CR1, encompassing the RBD and the CRD, and CR2, containing some of the phosphorylation sites which restrain Raf-1 activity, comprise the regulatory domain; CR3 consists essentially of the kinase domain and contains the positive regulatory sites whose phosphorylation stimulates Raf-1 activity. (b) Three-dimensional structure of the RBD, with its ubiquitin fold, of the Zn2+-bound CRD, and of the Raf kinase domain. The inactive structure of the Raf-1 kinase domain (PDB: 3OMV, (Hatzivassiliou et al. 2010); ribbon representation, in green) is superimposed on the active structure of the B-Raf kinase domain (PDB: 2FB8, (King et al. 2006); cartoon representation, in white). The aminoacid numbering corresponds to the human Raf-1 protein. The inactive “DFG-out” conformation is shown in yellow, the active “DFG-in” conformation in red. Note the interaction of the ß9 strand of the N-lobe (red) with the ß6 strand of the C-lobe in the “DFG-in” conformation. The start of the DFG (D486) and the position of the V492 residue corresponding to the V600 in B-Raf frequently mutated in melanoma are indicated. (c) Mechanism of Raf-1 activation. In quiescent cells, intramolecular inhibition, stabilized by 14-3-3 binding to the phosphorylated S259 and S621 sites, prevents Raf-1 activation. The transition to the active state is mediated by the dephosphorylation of 259 and Ras binding, which recruits Raf-1 to the membrane, where phosphorylation of activating residues occurs. The Raf-1 signal is quenched by the phosphorylation of negative regulatory sites (in grey) mediated by active ERK, which results in kinase desensitization, followed by dephosphorylation of both positive and negative regulatory sites (resensitized state). Finally, rephosphorylation of S259 restores the close, inactive conformation of Raf-1. The residues phosphorylated at each step are shown at their approximate localization in the molecule. The kinases responsible for phosphorylation of the positive (PAK, Src) or negative (ERK, PKA, PKB) regulatory sites are shown

Activation–Inactivation: In the inactive state of Raf, the N-terminal, regulatory domain of the protein binds to its kinase domain and inhibits its activity [Fig. 2c; see also (Wellbrock et al. 2004; Niault and Baccarini 2010)]. This conformation is stabilized by the binding of 14-3-3 proteins, which recognize two phosphorylated Raf-1 residues: S259 on the N-terminal and S621 on the C-terminal part of the protein. The current model of Raf-1 activation postulates that this binding must be disrupted to enable Raf-1 activation. This process is accomplished by protein phosphatases 1 and 2A, which dephosphorylate residue S259. 14-3-3 proteins remain bound to the phosphorylated S621 and maintain a productive conformation of the kinase domain. After S259 dephosphorylation, Raf-1 can be recruited to the membrane by binding to activated Ras, primarily via the RBD. Ras binding is followed by disruption of the inhibitory interaction between the regulatory and the kinase domain of the protein. The activation process is completed by the phosphorylation of the activating residues in the CR3 region (T491, S494), which stabilizes the active, “DFG-in” conformation. Inactivation of Raf-1 occurs via the phosphorylation on its negative regulatory residues by ERK, which is followed by the dephosphorylation of activating residues by PP2A. Finally, PKA/ PKB rephosphorylates the residue S259, making the rebinding of 14-3-3 possible (Wellbrock et al. 2004; Niault and Baccarini 2010).

Raf-1-Containing Complexes and Their Biological Functions

Raf-1 can also be activated by (Ras-dependent) homodimerization or by heterodimerization with other Rafs, particularly B-Raf. As part of this complex, Raf-1 can stimulate the MEK/ERK pathway and therefore regulate cell proliferation and several other biological functions (Fig. 3, pathway 1). In most cells and tissues, however, Raf-1 is not essential for MEK/ERK activation and proliferation. Instead, Raf-1 is required to promote survival, either through the inhibition of proapoptotic kinases such as MST2 and ASK1 (Fig. 3, pathways 2 and 3) or by restraining the cytoskeleton-based kinase Rok-α, which regulates the trafficking of the death receptor Fas (Fig. 3, pathway 4; and right panel). Interaction with, and inhibition of Rok-α is also the molecular basis of Raf-1’s role in cell migration, in keratinocyte differentiation, and in Ras-driven epidermal tumorigenesis (Niault and Baccarini 2010; Wimmer and Baccarini 2010; Kern et al. 2011).
RAF-1 (C-RAF), Fig. 3

Interactions of Raf-1 with different partners and their biological consequences. Left, As part of a Raf dimer, Raf-1 functions as a MEK/ERK activator and stimulates many cellular functions, in particular proliferation (1). Most of the essential Raf-1 functions rely on protein/protein interaction and are independent of Raf-1 kinase activity. Binding of Raf-1 to the MST2 (2) and ASK-1 (3) kinases promotes survival by reducing the strength of the downstream proapoptotic signal. Interaction with the cytoskeleton-based kinase Rok-α (4) promotes migration and survival and restrains differentiation. Right, molecular basis of Rok-α inhibition by Raf-1. Raf-1 and Rok-α share similar autoinhibitory domains, which, in quiescent cells, interact with the kinase domains and restrain their activity. When the kinases are activated by the respective upstream GTPases, autoinhibition is relieved, and the regulatory domain of Raf-1 is free to bind to the kinase domain of Rok-α and modulate its kinase activity

The following section describes the individual Raf-1-containing complexes.

The Raf-1/B-Raf complex (Fig. 3, pathway 1): All three Raf isoforms,  A-Raf, B-Raf, and Raf-1, can mediate MEK/ERK activation. Conditional mutagenesis has revealed that B-Raf is the essential MEK activator in various cells and tissues (Galabova-Kovacs et al. 2006; Niault and Baccarini 2010). A-Raf and Raf-1 can heterodimerize with B-Raf, yielding a MEK kinase more potent than the individual monomeric forms. Whether A-Raf or Raf-1 are preferred B-Raf partners in the context of the heterodimer is yet unknown; however, both A-Raf and Raf-1 must be ablated to reduce MEK/ERK phosphorylation in fibroblasts (Mercer et al. 2005), consistent with an interchangeable role of these two kinases as dimer subunits. The heterodimerization of Raf has been in the limelight since the discovery that B-Raf inhibitors currently in the process of being approved for the treatment of melanoma patients activate the Raf/MEK/ERK pathway, instead of stopping it (see below; and Fig. 4).
RAF-1 (C-RAF), Fig. 4

Paradoxical activation of MEK/ERK pathway by B-Raf inhibitors. Upper panel, in wild type (WT) cells, Raf dimer formation and MEK/ERK activation are stimulated by extracellular signals acting through RTKs and Ras. In melanoma cells harboring constitutively active B-RafV600E, MEK/ERK activation is Ras and Raf-1-independent; while melanoma cells harboring mutated N-Ras activate MEK/ERK by stimulating the formation of B-Raf-Raf-1 dimers. Lower panel, B-Raf inhibitors will block B-RafV600E kinase activity, reducing ERK activation and inducing a proliferation block and subsequent tumor regression. However, in cells harboring NRAS mutations, inhibitors can induce Raf dimerization and ERK activation. This is due to the fact that dimers containing only one functional kinase subunit are active as MEK kinases. Such dimers may arise if the inhibitors used have fast off-rates, or if low concentrations of inhibitors are used. In both situations, the conformational change induced by the inhibitors would suffice to stimulate dimerization, but not to completely inhibit the resulting dimeric kinase. This mechanism may be linked to the development of drug-related tumors observed in melanoma patients treated with B-Raf inhibitors

Raf-1 and MST2 (Fig. 3, pathway 2): In mammalian cells, MST2 is activated by stress signals and causes apoptosis acting upstream of the mammalian Hippo signaling pathway (Pan 2010). MST2 was identified as a Raf-1 interacting partner using mass spectrometry (O’Neill et al. 2004). In quiescent cells, MST2 is phosphorylated on two negative regulatory sites in its N-terminal and C-terminal region (Romano et al. 2010). This diphosphorylated form can interact with Raf-1 (region between amino acids 150–303) and this inhibits MST2 activation by preventing homodimerization. This interaction is disrupted by proapoptotic stimuli, enabling MST2 activation by the tumor suppressor RASSF1.

Raf-1 and ASK1 (Fig. 3, pathway 3): ASK1 is a Ser/Thr kinase which acts upstream of JNK and p38 and promotes apoptosis induced by stress or death receptors. Cardiac-specific ablation of Raf-1 induces apoptosis in the cardiac muscle in vivo and leads to a transient increase in ASK1, JNK, and p38 activity during postnatal heart development, without affecting the MEK/ERK pathway (Yamaguchi et al. 2004). Concomitant ablation of ASK1 rescues the phenotype of Raf-1 conditional knockout mice, indicating a causal role of ASK1 in the abnormalities observed in Raf-1 knockout hearts and suggesting that Raf-1 limits ASK1 activity in cardiomyocytes. How exactly Raf-1 inhibits ASK1 has not yet been established, but it is known that this function of Raf-1 is independent of the MEK/ERK pathway, although the phosphorylation of the activating Raf-1 regulatory residues S338 and S339 is a prerequisite, at least in endothelial cells (Alavi et al. 2007). Raf-1 physically interacts with the N-terminal autoinhibitory domain of ASK1; it is therefore possible that by doing so it will promote and/or stabilize an inactive ASK1 conformation (Chen et al. 2001).

Raf-1 and Rok-α (Fig. 3, pathway 4): In mammalian cells, the Rho effector Rok-α is responsible for cytoskeletal rearrangements essential for cell adhesion and motility. Conditional gene ablation studies have revealed that Rok-α is hyperactive and mislocalized in the absence of Raf-1 (Ehrenreiter et al. 2005; Piazzolla et al. 2005). Importantly, chemical or genetic inhibition of Rok-α rescues all phenotypes of Raf-1 knockout mouse embryonic fibroblasts, defining Rok-α hyperactivity as the rate-limiting factor in this context (Piazzolla et al. 2005). In addition, both the cellular phenotypes and Rok-α hyperactivity could be rescued by complementation with Raf-1 mutants devoid of kinase activity, as well as by mutants featuring the isolated Raf-1 regulatory domain, indicating that the physical presence of at least part of Raf-1 is necessary for the inhibition of Rok-α activity (Ehrenreiter et al. 2005; Piazzolla et al. 2005). The molecular basis of Raf-1 interaction with Rok-α is understood in some detail. Both Raf-1 and Rok-α are modular kinases featuring a similar domain structure. In quiescent cells, the activity of Raf-1 and Rok-α is restrained by intramolecular inhibition. The negative regulatory domain (cysteine-rich region, CRD) of each protein can interact with its own kinase domain and inhibit its activity, likely by preventing substrate binding. Upon mitogenic stimulation, binding to activated small G-proteins (Ras for Raf-1 and Rho for Rok-α) relieves autoinhibition and, at the same time, makes the interaction between the regulatory domain (CRD) of Raf and the kinase domain of Rok possible (Niault et al. 2009). In this situation, the autoinhibitory domain of Raf-1, much like an ill-fitting lego brick, can restrain the activity of the Rok-α kinase domain without blocking it completely (Fig. 3, right panel). This mechanism of inhibition in trans is the first example of kinase regulation mediated by physical interaction rather than phosphorylation on negative regulatory residues.

Raf-1 and Cancer

A wealth of reports has implicated Raf isoforms, particularly B-Raf, in different aspects of tumor development (Niault and Baccarini 2010; Maurer et al. 2011). The next chapters will focus on two recently described functions of Raf-1 in melanoma and squamous cell carcinoma, both of which relay on Raf-1’s ability to form physical complexes with other kinases.

Raf-1-B-Raf interaction and melanoma: MEK/ERK signaling is particularly important in melanoma. Somatic mutations occur in B-Raf and N-Ras in ~50% and ~15% of cutaneous melanomas, respectively (www.sanger.ac.uk/genetics/CGP/cosmic/). The most frequent mutation in B-Raf is the V600E mutation, which causes constitutive activation of the kinase and thus of the MEK/ERK pathway. After this discovery, tremendous efforts were made to find inhibitors targeting the mutated form of B-Raf. Surprisingly, these inhibitors could inactivate the enzyme in vitro, but activated the RAF/MEK/ERK pathway in cells not harboring the V600E mutation. The molecular basis of the Raf-inhibitor paradox is the ability of Raf enzymes to heterodimerize and form a potent MEK kinase, even when only one dimer subunit is enzymatically active [Fig. 4; reviewed in (Cichowski and Janne 2010; Wimmer and Baccarini 2010)]. Selective inhibitors cause a conformational change in the structure of B-Raf, which promotes the dimerization of this “inhibited” form of B-Raf with Raf-1 or A-Raf. This leads to the stabilization of an active MEK kinase and to the stimulation of the MEK/ERK pathway. Inhibitor-induced stabilization of Raf hetero- or homodimers is predicted to be particularly dangerous in cells containing mutations that stimulate dimer formation, such as melanoma cells with an N-Ras mutation, as indicated by a recent animal study (Heidorn et al. 2010), and also in any other tumor-prone cell. This mechanism might be the reason for the appearance of keratoacanthomas and squamous cell carcinomas in about 30% of patients treated with Raf inhibitors in clinical studies (Arkenau et al. 2011).

Raf-1-Rok-α interaction and Ras-driven epidermal carcinogenesis: To date, Raf-1 is the only Ras effector that has been shown to be essential for the maintenance of Ras-driven tumors. This has been achieved by conditional gene ablation experiments conducted in mice with epidermis-restricted Raf-1 ablation. Besides showing defects in wound healing, keratinocyte adhesion and migration (Ehrenreiter et al. 2005), these animals are refractory to epidermal tumors caused by Ras activation; more importantly, Raf-1 ablation causes the complete regression of established tumors. Thus, Ras-driven tumors are addicted to endogenous Raf-1, constituting a prime example of non-oncogene addiction. Mechanistically, Ras drives the formation of a complex between Raf-1 and Rok-α, ultimately resulting in Rok-α inhibition. In the absence of Raf-1, hyperactive Rok-α drives keratinocyte differentiation and tumor regression via a pathway involving phosphorylated cofilin, the inhibition of STAT3 phosphorylation, and of Myc expression (Fig. 5). Thus, inhibiting Raf-1-Rok-α complex formation, either by silencing the Raf-1 gene or by using small molecule inhibitors which can disrupt the complex, may be a viable strategy for the (co-)therapy of Ras-driven epidermal tumors (Ehrenreiter et al. 2009).
RAF-1 (C-RAF), Fig. 5

Raf-1-mediated Rok-α inhibition is essential for the establishment and maintenance of Ras-induced epidermal tumors. Activated Ras stimulates the interaction between Raf-1 and Rok-α. The resulting attenuation of Rok-α leads to decreased expression of the epidermal differentiation cluster genes (EDC) and reduces the phosphorylation of cofilin (via  LIMK). Since phosphocofilin, in turn, inhibits the pro-proliferative STAT3/myc pathway, Raf-1-mediated Rok-α attenuation supports Ras-driven tumorigenesis

Summary

Born as the first serine/threonine kinase oncogene and intensively studied as the link between Ras and the mitogenic MEK/ERK pathway, Raf-1 is coming of age as a versatile signal transducer with multiple partners impinging on cell motility, differentiation, and survival. The basis for this is its modular structure, featuring a kinase domain kept in check by an autoinhibitory domain. This autoinhibition is relieved by intricate regulatory mechanisms involving dephosphorylation of negative regulatory sites, Ras binding, and phosphorylation of activating sites. Once autoinhibition is relieved, Raf-1 can function as a MEK kinase, in the context of homodimers or of Raf heterodimers, but it can also exert kinase-independent functions by binding to, and directly regulating, serine/threonine kinases operating in distinct pathways. These functions in pathway cross-talk are the essential ones, as revealed by conventional and conditional gene ablation studies. One obvious unresolved question in this context is how extracellular cues direct Raf-1 (or, for that matter, other signal transducers) to the appropriate signaling complex in order to implement the correct biological response. One of the kinase-independent functions of Raf-1, the inhibition of the cytoskeleton-based kinase Rok-α, is essential for the development and maintenance of Ras-driven epidermal tumors. Will other Raf-1 interactions prove similarly essential in tumorigenesis, possibly in the context of other cell types/tissues? And if yes, will it be possible to design inhibitors for molecule-based therapy? The key to these questions lies in the further investigation of Raf-1’s role in tumor models in vivo and in obtaining structural information on the complexes between Raf-1 and its interacting proteins.

Notes

Acknowledgments

The authors wish to thank all the members of the Baccarini group for helpful discussions. Dr. Andrea Varga is supported by a FEBS long term fellowship. Work in the Baccarini lab is supported by funds of the Austrian National Research Fund (FWF), the Austrian Society for the Advancement of Research (FFG), the Obermann Foundation, and the European Community.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Microbiology and Immunobiology, Center for Molecular BiologyUniversity of Vienna, Max F. Perutz LaboratoriesViennaAustria