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FEBS Letters xxx (2014) xxxxxx
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journal homepage: www.FEBSLetters.org

Review

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RAF neighborhood: Proteinprotein interaction in the Raf/Mek/Erk

pathway

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Botond Cseh 1, Eszter Doma 1, Manuela Baccarini

Department of Microbiology, Immunobiology and Genetics, Center of Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Doktor-Bohr-Gasse 9, 1030 Vienna, Austria

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i n f o

Article history:
Received 14 May 2014
Revised 5 June 2014
Accepted 6 June 2014
Available online xxxx
Edited by Wilhelm Just

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Keywords:
Ras
Raf
Mek
Erk
Proteinprotein interaction
Kinase inhibitor
Cancer therapy

a b s t r a c t
The Raf/Mek/Erk signaling pathway, activated downstream of Ras primarily to promote proliferation, represents the best studied of the evolutionary conserved MAPK cascades. The investigation
of the pathway has continued unabated since its discovery roughly 30 years ago. In the last decade,
however, the identication of unexpected in vivo functions of pathway components, as well as the
discovery of Raf mutations in human cancer, the ensuing quest for inhibitors, and the efforts to
understand their mechanism of action, have boosted interest tremendously. From this large body
of work, proteinprotein interaction has emerged as a recurrent, crucial theme. This review focuses
on the role of protein complexes in the regulation of the Raf/Mek/Erk pathway and in its cross-talk
with other signaling cascades. Mapping these interactions and nding a way of exploiting them for
therapeutic purposes is one of the challenges of future molecule-targeted therapy.
2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical
Societies. This is an open access article under the CC BY license (http://creativecommons.org/licenses/
by/3.0/).

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1. Introduction

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The Raf/Mek/Erk signal transduction pathway is the best studied of the four mitogen-activated protein kinase (MAPK) cascades
present in vertebrates (Fig. 1). It is activated by growth factors, hormones and cytokines and has been shown to regulate proliferation
but also differentiation, survival, senescence, and migration [1].
Typically, ligand-binding to a cell surface receptor induces a wave
of tyrosine phosphorylation (autophosphorylation in the case of
receptor tyrosine kinases, or phosphorylation by receptor-associated kinases if the receptor itself lacks catalytic activity) resulting
in the generation of phosphotyrosine binding sites for adaptor proteins such as growth factor receptor-bound protein 2 (GRB2). GRB2
mediates the membrane translocation of the guanine nucleotide
exchange factor (GEF) son of sevenless (SOS), which in turn activates the membrane bound GTPase Ras [1]. Ras functions as a binary molecular switch that cycles between inactive GDP-bound and
active GTP-bound states with the help of GEFs and GTPase activating proteins (GAPs). The exchange of GTP for GDP by SOS changes
the conformation of Ras, allowing its interaction with effectors
such as Raf [2]. GTP-bound Ras recruits Raf to the plasma mem-

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Q2

Corresponding author. Fax: +43 1 4277 9546.

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E-mail address: manuela.baccarini@univie.ac.at (M. Baccarini).

These authors have contributed equally to the manuscript.

brane and enables it to phosphorylate its only substrates, Mek1

and Mek2 [3]. These dual specicity kinases mediate the activation
of Erk1 and Erk2, enabling them to phosphorylate a variety of
nuclear and cytoplasmic targets [4].
Mammals express three Raf isoforms, A-, B-, and C-Raf (the latter also called Raf-1) with distinct afnities for both the activator,
Ras, and the downstream target Mek. B-Raf is the isoform most
similar to Rafs expressed in lower organisms [5], and can therefore
be considered the archetypal mammalian Mek kinase. A-Raf and
C-Raf have evolved to fulll other, potentially Mek-independent
requirements [6,7]. Accordingly, growth factor-stimulated Erk activation is decreased in B-Raf-, but not A-Raf or C-Raf -decient cells
[812]. Similarly, the high occurrence of B-Raf but not A-Raf or
C-Raf mutations in human cancers implies a dominant role for
B-Raf in signaling to the Erk pathway [13,14].

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2. Homo and heterodimers in Raf activation

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Homo- and heterodimerization play an important role in the

Erk pathway, whether by allowing the propagation of the signal
to downstream effectors, by orchestrating feedback loops within
the pathway, or by enabling communication with parallel signaling
circuits [15]. Dimerization of pathway components can result in
their activation (Raf) or inhibition (Mek). Furthermore, binding to
different scaffolds can inuence the localization of the components

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http://dx.doi.org/10.1016/j.febslet.2014.06.025
0014-5793/ 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

Please cite this article in press as: Cseh, B., et al. RAF neighborhood: Proteinprotein interaction in the Raf/Mek/Erk pathway. FEBS Lett. (2014), http://
dx.doi.org/10.1016/j.febslet.2014.06.025

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Fig. 1. The Raf/Mek/Erk Pathway. (A) Schematic wiring of the pathway the Raf/Mek/Erk pathway is a three-tiered kinase cascade that operates downstream of the small
GTPase Ras. The three Rafs bind Ras with different afnities, which determine their sensitivity to activated Ras. Rafs, in particular B-Raf and C-Raf, form homo- and heterodimers which phosphorylate and activate Meks, which in turn transfer the signal to Erks. Erks have many substrates whose activation leads to a variety of biological
responses. Knockout studies have revealed that B-Raf is essential for Mek/Erk activation downstream of Ras; A-Raf and C-Raf can also activate Erk upon heterodimerization
with B-Raf. Raf and Mek1 are the recipients of negative feedback phosphorylation by Erk, which determines the strength and duration of the Erk signal. (B) Cross-talk with
other pathways A-Raf and C-Raf can transmit signals in a Mek-independent manner, by communicating with parallel pathways. Both of them bind to and inhibit the
proapoptotic kinase Mst2. In addition, C-Raf can bind and inhibit another proapoptotic kinase, Ask1, and the cytoskeleton-based Rok-a. An intact C-Raf:Rok-a complex is
required for cell shape and motility, it impacts on angiogenesis and it is essential for preventing differentiation in Ras-driven epidermal tumors. Similar to C-Raf, Mek1
impacts a parallel pathway leading to Akt phosphorylation, by preventing PTEN-Mediated PIP3 turnover in the context of a Mek1/Magi1/PTEN ternary complex. (C)
Phosphatases interacting with Erk pathway components phosphatases play a dual role in Erk pathway regulation: a positive role, by facilitating C-Raf activation (PP2A, PP1C;
green arrows) and a negative role (red lines) by dephosphorylating Shc, Mek and Erk (PP2A), C-Raf (PP5) or Erk (DUSPs). In Fig. 1B, line thickness is proportional to the
strength and signicance of the interactions.

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to different cellular compartments, increasing signal delity and

strength [16]. One of these scaffolds, the pseudokinase Ksr, interacts with Raf, Mek and Erk and localizes to the plasma membrane
in a Ras-dependent manner [17].
Activation of Raf occurs via a complex, yet incompletely understood mechanism requiring membrane translocation, regulatory
phosphorylation/dephosphorylation events [16] and, crucially,
allosteric activation in the context of a side-to-side dimer comprising two Raf molecules or a Raf and a Ksr molecule [1822]. Raf:Raf
and Raf:Ksr dimerization depends on the dimer interface, a region
located in the kinase domain, and in particular on a cluster of basic
residues comprising B-RafR509, C-RafR401 and KsrR615 [21].
When these critical arginine residues are mutated to histidine
(B-RafR509H, C-RafR401H, KsrR615H), activation does not take
place. Conversely, the B-RafE586K mutation, which enhances
dimerization and possibly allosteric transactivation, increases Erk
signaling [23]. Growth factor-induced Raf dimerization can also
be inhibited by an 18 amino acid peptide able to bind C-Raf and
B-Raf, resulting in decreased Mek activation [23].
Of the three Raf kinases, only B-Raf is able to function as an
allosteric activator in the context of the Raf heterodimers, a role
independent of B-Raf kinase activity [14,19,24]. The molecular
basis for this has recently been elucidated by the Shaw lab [22],
who has shown that the ability of acting as an activator depends
on the presence of negative charges in the Raf N-terminal acidic
motif. In B-Raf, this motif is negatively charged due to the constitutive phosphorylation of Ser446 and/or 447, and to the presence
of two aspartates at position 448/9 [25] (Fig. 2A). Allosteric activation by B-Raf induces cis-autophosporylation in the activation loop
of the receiver kinase, i.e. C-Raf, and renders it able to phosphorylate Mek. Mek, in turn, phosphorylates the N-terminal acidic motif
in C-Raf, converting it to an allosteric activator of other Rafs [22,26]
(Fig. 2B and C). This model explains why C-Raf mutants devoid of
kinase activity cannot function as activators, and why B-Raf can
activate Mek directly as a homodimer [23]. Phosphorylated Ksr

can also function as a transactivator; however, since Raf binding

to Ksr induces limited kinase activity [27], in quiescent cells the
constitutive association of Ksr with B-Raf may serve to prevent
C-Raf binding to B-Raf, safeguarding against undue activation of
the pathway [28].
Some naturally occurring mutants of B-Raf can bypass the
requirement for dimerization-mediated activation. These mutations (i.e. G469A and V600E, 599insT) disrupt the interaction
between the P-loop and activation-loop [14], resulting in a constitutively active B-Raf kinase largely resistant to the disruption of
the dimer interface [29]. B-Raf V600E mutants require homodimerization for activation only when their binding to Ras is impaired
[30]. Oncogenic Ras has also been shown to promote the binding
of B-RafV600E-to wild-type C-Raf, which results in a weakening
of V600E activity and of Erk activation [31]. This work implies that
the B-RafV600E mutant is unable to transactivate C-Raf, which
may explain why oncogenic Ras mutations and B-RafV600E appear
to be mutually exclusive.

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3. Signaling through Mek:Erk activation, negative feedback and

pathway cross-talk

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Although loss of function or conventional knockout studies

have revealed distinct roles for all three kinases, embryonic- (BRaf and C-Raf) [9,32] or post natal lethality (A-Raf) [33] has hindered the analysis of the role of Rafs in vivo. Conditional knockout
models have provided an opportunity to bypass this difculty, and
to test the role of specic isoforms in the activation of the ERK
pathway in different organs. In good agreement with the pivotal
role of B-Raf in Raf activation, B-Raf has been identied as the
essential Mek/Erk activator in the placenta during vascular development [34] and in oligodendrocyte differentiation and myelination [35]. In the context of cancer, keratinocyte-restricted B-Raf
deletion reduces Ras-driven carcinogenesis, which is consistent
with its importance in this type of skin tumors [36]. B-Raf is also

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Fig. 2. A Model of Raf transactivation. (A) Conserved domains all Rafs share N-terminal Ras-binding domains (RBD) and cysteine-rich domains (CRD), both required for
membrane recruitment. The Kinase Domain is located in the C-terminus, the activation loop is highlighted in green. Upstream of the Kinase Domain, the N-terminal Acidic
motif (NtA; red boxes) contains phosphorylatable tyrosine residues (YY301/2 in A-Raf, Y340/41 in C-Raf), whereas B-Raf features aspartates in the corresponding region
(D448/9). One Serine residue is conserved in all Raf proteins (S299 in A-Raf, S338 in C-Raf and S445 in B-Raf), but is constitutively phosphorylated only in B-Raf. (B) Raf
transactivation activated Ras recruits B-Raf to the plasma membrane. Ras binding allows a further Raf monomer to bind and dimerize. In the dimer interface, the
constitutively phosphorylated NtA of B-Raf (green dot) induces a conformational change that allows the cis-phosphorylation of the receiver kinase (here C-Raf), enabling it to
phosphorylate Mek. Mek, in turn induces the phosphorylation of S338 in the C-Raf NtA, converting it to a transactivator. (C) As a transactivator, C-Raf can dissociate from BRaf and dimerize with, and transactivate, further Raf molecules. This cycle results in signal amplication.

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the main Mek/Erk-activator in a RIP1Tag2 tumor model in which

the Erk pathway is not mutationally activated. In this model, the
B-Raf/Mek/Erk axis was proven to be the key determinant of the
communication between tumor and microenvironment, promoting
the secretion of proangiogenic factors [37].
Rafs transmit the signal downstream by phosphorylating Mek1
and Mek2 on Ser218/Ser222 and Ser222/Ser226, respectively.
Mek1 and Mek2 form homo- and heterodimers in vitro and
in vivo [38,39], but unlike Raf heterodimers, the Mek1-Mek2 heterodimer is stable and its amount does not depend on growth factor stimulation [39]. Although they share common targets, Mek1
and Mek2 have unique biological properties. While disruption of
Mek1 in mice is recessive embryonic lethal [40], the Mek2 knockout mice are viable, fertile and have no apparent abnormalities
[41]. This predicts that on the systemic level, Mek1 homodimers

can compensate for the loss of heterodimers and Mek2 homodimers, while Mek2 homodimers cannot. Mek1 and Mek2 mediate
the phosphorylation of Erk1 and Erk2, which also form dimers
[42,43]. Akin to the situation with Meks, only one of the isoforms,
Erk2, is necessary during embryonic life, more specically for trophoblast development [44]. In contrast, Erk1 knockout mice show
only some minor defects in T-cell development, decreased adiposity and facilitated learning [4547]. Both Erk1 and Erk2 are positive
regulators of proliferation and are thought to be largely redundant
in this context [48]. The dimerization of Erks does not inuence
their translocation to the nucleus, but is essential for signaling
[43,49]. To reach specic cellular compartments, Erk rather interacts with scaffold proteins such as Ksr1, paxillin, IQGAP, Sef, MP1
or MORG [50]. These scaffolds contact the same hydrophobic site
on Erk that is also needed for its substrate binding. Therefore, for

Please cite this article in press as: Cseh, B., et al. RAF neighborhood: Proteinprotein interaction in the Raf/Mek/Erk pathway. FEBS Lett. (2014), http://
dx.doi.org/10.1016/j.febslet.2014.06.025

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their proper functioning, Erks have to form dimers to be able to

simultaneously bind the scaffold and their substrates. Interfering
with either Erk-dimerization or Erk binding to their scaffolds
results in loss of proliferation and transformation [43].
In addition to mediating part of the biological response to Raf
activation, Erk exerts negative feedback on the pathway through
the phosphorylation of SOS [51,52], C-Raf [5355], B-Raf
[20,56,57] and Mek1 [39] (Fig. 1A). In this most proximal feedback,
Erk phosphorylates a specic residue in Mek1, T292, which is
absent in Mek2; disruption of the Mek1-Mek2 dimer, either by
Mek1 deletion or by introducing a mutation in the dimer interface,
eliminates this feed-back loop resulting in increased and prolonged
Mek2 and Erk phosphorylation [39]. How the regulatory phosphorylation of T292 on Mek1 controls Mek2 activation still remains
elusive.
Inactivating Mek by inhibition or silencing also enhances
growth factor-induced Akt activation [5860]. Intriguingly, phosphorylation of the same residue responsible for the negative feedback loop described above, T292, by Erk, enables Mek1 to form a
ternary complex with the 100 kDa isoform of the scaffold protein
Magi1 and the lipid phosphatase PTEN. The Mek1/Magi1/PTEN
complex mediates the translocation of PTEN to the membrane,
where it affects the amount of PIP3 and ultimately Akt activity
(Fig. 1B). Thus, Mek1 phosphorylation by Erk limits the activation
of both the Erk and the Akt pathway. In vivo, this chain of events
lead to changes in peripheral self-tolerance and myeloproliferation
in the Mek1-decient mice [60].

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4. Interacting phosphatases a double-edged sword

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The onset and duration of the Raf/Mek/Erk signal is regulated by

protein serine/threonine phosphatases (PSPs) and by the dual-specicity phosphatase family (DUSPs/MKPs) (Fig. 1C). Specically,
computational modeling of the pathway has shown that kinases
control signal amplitude and phosphatases both signal amplitude
and duration [61,62].
PSPs dephosphorylate phosphoserine and phosphothreonine
residues. One of the most abundantly expressed PSPs, protein
phosphatase 2A (PP2A) [63], can regulate the Raf/Mek/Erk pathway
both positively and negatively [64]. As a positive regulator, PP2A
associates with C-Raf [6569] and Ksr1 [68] and dephosphorylates
negative regulatory sites on both proteins, allowing their recruitment to the membrane [6669] and leading to Mek and Erk activation [6568]. A similar role in C-Raf activation has been described
for the catalytic subunit of PP1C, which associates with C-Raf in
Ras- and growth factor-stimulated cells [70]. In addition to promoting C-Raf activation, PP2A is also able to dephosphorylate
Erk-dependent sites on C-Raf [53]. Since the sites have been
described alternatively as negative regulatory [53,71] or activating
[72], the signicance of these dephosphorylation events for Raf
activation is unclear.
As a negative regulator of the pathway, PP2A can dephosphorylate the adapter protein Shc [73], required downstream of some
tyrosine kinase receptors for the activation of the Raf/Mek/Erk
module; and it can dephosphorylate Mek and Erk proteins [74
76], thus inhibiting the cascade directly. A further negative regulator of the cascade, at the level of C-Raf, is Protein phosphatase 5
(PP5), which associates with C-Raf via its N-terminal tetratricopeptide (TPR) domain in growth factor stimulated cells. This interaction leads to the activation of PP5 catalytic activity and to the
selective dephosphorylation of the activating serine residue at
position 338, terminating the signal [77].
While PSPs dephosphorylate the Raf-dependent phosphoserine
site on Mek and the Mek-dependent phosphothreonine sites on
Erk, DUSPs dephosphorylate both threonine and tyrosine residues

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of the MAP-Kinases Erk, p38 and Jnk [78]. The family consists of
highly similar phosphatases with distinct substrate preference.
These are dictated by the interaction of a modular binding domain
in the N-terminus of the DUSPs, consisting of a kinase interaction
motif (KIM) and of an additional stretch of positively charged residues anked by hydrophobic amino acids, with a common docking (CD) site on the MAPK. As in the case of PP5, substrate
binding is required to stimulate the activity of DUSP1, DUSP2,
DUSP6 and DUSP9. DUSPs also differ in their subcellular localization. While DUSP1, DUSP2, DUSP3, DUSP4, DUSP5 are localized in
the nucleus and dephosphorylate Erk, p38 and Jnk, the Erk-selective DUSP6, DUSP7 and DUSP9 are localized in the cytoplasm,
and at least in the case of DUSP6, anchor inactive Erk in the cytosol
[79] and transport dephosphorylated Erk from the nucleus back to
the cytosol [80]. Thus, phosphatases serve multiple functions in the
Raf/Mek/Erk pathway.

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5. Mek-independent Raf-signaling

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In addition to activating Mek downstream of B-Raf, C-Raf can

communicate with other parallel pathways (Fig. 1B). C-Raf is able
to bind and inhibit the proapoptotic proteins Ask-1 [81] and Mst2 [82,83], counteracting apoptosis; however, C-Rafs best dened
interaction partner outside the Mek/Erk pathway is the Rok-a
kinase, which controls cytoskeletal dynamics downstream of the
small GTPase Rho [84]. The C-Raf:Rok-a interaction has various
consequences, depending on the cell type in which it takes place.
It regulates the migration of keratinocytes and broblasts in culture, and wound healing in vivo [85]. In Ras-induced skin carcinogenesis, C-Raf-mediated inhibition of Rok-a blocks keratinocyte
differentiation and promotes proliferation through the colin/
STAT3/Myc axis. If C-Raf is ablated, Ras-induced epidermal tumors
do not form, and established ones regress [86]. In endothelial cells,
C-Raf is needed to translocate Rok-a to VE-cadherin-based cell
junctions. Here, Rok-a signaling stabilizes nascent adherens junctions, allowing the collective migration of endothelial cells
required for sprouting angiogenesis [87]. In broblasts and
embryonic liver, inhibition of Rok-a by C-Raf controls the trafcking of the death receptor Fas, counteracting apoptosis by setting
the threshold of Fas sensitivity [88].
Finally, a Mek-independent function of C-Raf was also demonstrated in K-Ras-driven lung cancer models, but its molecular basis
is currently unclear [89,90].
Similar to C-Raf, A-Raf has been reported to regulate Mst2 independently of its kinase activity [91]. B-Raf-dependent cross-talk
with other pathways has not been reported so far, but it is conceivable that, by binding to C-Raf, B-Raf may inuence its interaction
with other proteins. Indeed, C-Raf:Rok-a complexes are more
abundant in B-Raf ablated keratinocytes [92], a fact that could possibly affect the intensity of the cross-talks mentioned above.

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6. Raf and Mek inhibitors success stories with pitfalls

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Activating B-RafV600E kinase mutations occur in up to 60% of

melanomas (http://www.cbioportal.org), making B-Raf an attractive drug target for this malignancy. In the last years, signicant
efforts have been made to nd compounds that inhibit B-RafV600E
and, ideally, cause melanoma regression.
New generation ATP-competitive Raf inhibitors, such as the
clinically approved vemurafenib (PLX4032) [9397] and dabrafenib (GSK2118436) [96,98], have improved selectivity for mutant
B-RafV600E (Table 1), resulting in high response rates and
increased progression-free and overall survival in patients with
BRAF mutant melanoma. However, cutaneous toxicities, such as
the onset of squamous cell carcinomas and keratoacanthomas,

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Table 1
FDA-approved Raf and Mek inhibitors.
Target IC50 (nM)
B-Raf

B-RafV600E

C-Raf

Mek1

Mek2

Raf inhibitors
Vemurafenib* (PLX4032 [100])
Dabrafenib* (GSK2118436 [101])
GDC-0879 [102]

100
3.2
n.a.

31
0.80
0.13

48
5
n.a.

n.a.
n.a.
n.a.

n.a.
n.a.
n.a.

Mek inhibitors
Trametinib* (GSK1120212 [103])
Selumetinib (AZD6244 [104])
PD0325901 [105]
CH5126766 [106])

n.a.
n.a.
n.a.
19.0

n.a.
n.a.
n.a.
8.20

n.a.
n.a.
n.a.
56

0.92
14
0.33
160

1.80
n.a.
0.33
n.a.

*
Vemurafenib, dabrafenib and trametinib have been approved by the FDA as single agent therapy for the treatment of unresectable metastatic melanomas harboring the
B-RafV600E mutation; dabrafenib and trametinib have also been approved by the FDA as combination therapy for the same disease. n.a., not applicable.

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occur in melanoma patients treated with vemurafenib (30%) or

dabrafenib (7%) [93,99]. These lesions contain activated Erk, a paradoxical nding considering that the patients are treated with an
inhibitor of the pathway.
Several explanations can be put forward to rationalize the Raf
inhibitor paradox. The rst involves Raf heterodimerization. In
the presence of active Ras and of limited amounts of the inhibitor,
Raf kinases adopt a conformation promoting hetero- and homodimerization. In the context of these dimers, the drug-bound protomer transactivates the inhibitor-free Raf kinase [107,108]. A more
complex mechanism, suggesting relocalization of B-Raf from an
inhibitory cytosolic complex and allowing RAS:B-Raf:C-Raf complex formation in Ras-transformed cells, has also been proposed
[24].
What is common to these models is that they both rely on Ras
activation as an initial event for inhibitor-induced Erk activation
and tumorigenesis. Ras mutations were found in secondary skin
lesions of melanoma patients treated with Raf inhibitors
[109,110], and the signicance of Ras activation for Raf inhibitorinduced tumor development was veried in mouse models in
which Ras activation was induced either by chemical carcinogenesis [110] or by targeting the GEF SOS to the plasma membrane
of basal keratinocytes [92]. The latter model conrmed that Ras
activation, in the absence of other mutations or of inammation,
is enough to trigger Raf inhibitor-driven tumorigenesis in both skin
and gastric epithelia [92]. Thus, Ras + inhibitor-induced Erk activation accelerates the growth of tumors originating from cells containing activated Ras [92,109,110]. Further common to both
models is the concept that paradoxical activation of the pathway
can only happen at low inhibitor concentration [92,107,108,110].
Unfortunately, the use of saturating concentrations of the inhibitor
is precluded by high cytotoxicity [100].
In mouse models, B-Raf and C-Raf are required for Ras + vemurafenib driven tumorigenesis. However, only C-Raf is necessary for
tumor development induced by GDC-0879, another ATP-competitive Raf inhibitor [111]. This specic role of C-Raf is not mediated
through Erk phosphorylation, which is similar in B- or C-Raf-decient epidermis and is necessary, but not sufcient, for the
development of inhibitor + Ras induced tumors. Concomitant dedifferentiation, induced by C-Raf as endogenous inhibitor of the
Rok-a signaling pathway [85,86,112], is indispensable for tumor
development [92]. Rok-a inhibition by C-Raf-Rok-a interaction is
not affected by Raf-inhibitor-induced Raf hetero- or homodimerization, but in general more C-Raf binds to Rok-a in B-Raf-decient
K5-SOS-F epidermis. The basis of this nding is currently unknown,
but it is possible that B-Raf ablation frees up an additional pool of
C-Raf for Rok-a interaction. The fact that Ras + Raf-inhibitorinduced tumorigenesis requires both Raf-mediated Erk activation
and kinase-independent Rok-a inhibition by C-Raf suggests that

combination therapies targeting kinase and non-kinase functions

of Raf may be more efcient and safer for the treatment of skin
tumors.
More recently, a Ras-independent mechanism by which Raf
inhibitors can activate the Erk pathway has been described. Raf
inhibitors have been shown to block the autoinhibitory P-loop
phosphorylation that regulates wild-type Raf, but not BRafV600E.
This mechanism brings about the activation of wild-type Raf
monomers, is independent of Ras binding and of inhibitor-induced
dimerization, and may potentially have more widespread consequences than the mechanisms relying on activated Ras [113].
In addition to the onset of side effects due to paradox pathway
activation, the therapeutic success of Raf inhibitors is compromised by acquired drug resistance. Several mechanisms have been
described by which cells can evade Raf inhibition. A prominent one
is pathway reactivation by upstream components, such as receptor
tyrosine kinases (RTKs; EGFR, PDGFRb, IGF-1R, MET through HGF
secretion by the tumor microenvironment) [114118]. As a specic
example, in BRafV600E-expressing melanoma cells Raf inhibitors
disable the Erk dependent feedback which suppresses RTK-Ras signaling, reactivating mitogenic signaling [119]. Resistance caused
by RTK reactivation is not restricted to melanoma, being observed
in B-RafV600E expressing colon carcinoma cells [120,121]. This
mechanism has the additional advantage of enlisting parallel
survival pathways, such as the PI3K/AKT cascade [116118,122],
whose activation strongly reduces the sensitivity of K-Ras mutant
cancer to Erk inhibition [123].
Drug resistance can also be engendered in B-Raf-mutant tumors
by direct pathway reactivation, caused by secondary NRAS [115]
and Mek1 mutations [124,125] or by target amplication/diversication. B-RafV600E amplication [126], expression of B-RafV600E
splice variants promoting Ras-independent dimerization (p61BRafV600E) [30], C-Raf overexpression [116,122,127], Raf isoform
signal switching [116] and increased expression of the alternative
Mek kinase COT (Tpl2) [128] have all been reported and connected
to inhibitor resistance in melanoma cells. Finally, a gain of function
resistance study revealed that a melanocyte-specic signaling circuit involving the transcription factors CREB and MITF is also able
to mediate drug resistance [129].
Unlike Raf inhibitors, Mek inhibitors are unfortunately rather
toxic for normal tissues, which currently limits their clinical use
[130]. However, since the spectrum of activity of Mek inhibitors
is predicted to be broader, many efforts are being carried out to
develop more efcacious, less toxic substances, and to better
understand their mechanism of action.
In this context, one puzzling nding has been that some allosteric Mek inhibitors suppress Erk signaling and proliferation less
effectively in K-Ras-driven than B-RafV600E-driven tumors
[131,132]. This conundrum has been resolved recently by the

Please cite this article in press as: Cseh, B., et al. RAF neighborhood: Proteinprotein interaction in the Raf/Mek/Erk pathway. FEBS Lett. (2014), http://
dx.doi.org/10.1016/j.febslet.2014.06.025

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B. Cseh et al. / FEBS Letters xxx (2014) xxxxxx

Rosen and the Hatzivassiliou groups. Using inhibitors that preferentially bind to active or inactive Mek, they have shown that the
rst class of substances is more effective in cells harboring BRafV600E, which have high concentrations of phosphorylated
Mek, while the second is more effective in K-Ras transformed cells,
where the concentration of phosphorylated Mek is lower [133].
This is due to the fact that in Ras transformed cells, signaling
through the Erk pathway is reduced by the phosphorylation of
negative regulatory residues of C-Raf by Erk, which inhibits both
C-Raf kinase activity and its interaction with Ras. Mek inhibitors
interfere with this negative feedback loop, and the resulting CRaf reactivation limits their efcacy in this system [132]. B-Raf
mutant tumors are insensitive to this negative feedback [134],
which explains why Mek inhibitors can more effectively downregulate the Erk pathway in these cells.
The second class of inhibitors interferes with the binding of Raf
to Mek, improving efcacy in K-Ras transformed cells. Within this
class, inhibitors such as Selumetinib, PD0325901, and CH5126766
(Table 1), stabilize a complex in which Mek cannot be phosphorylated by Raf, essentially generating a dominant-negative inhibitor
of Raf [106,132]. Stabilization of the Raf:Mek complex also has
negative consequences for the formation of Raf heterodimers,
and therefore for Ras-induced Raf activation [133].
In contrast, the allosteric inhibitor Trametinib reduces Raf binding to Mek. Trametinib inhibits the proliferation of both RAS and BRafV600E mutant cell lines and xenografts [132,135] and decreases
both Ras and Ras + Raf inhibitor-induced tumor formation in a
transgenic model of Ras-driven epidermal tumorigenesis [92]. Trametinib is also the only Mek inhibitor that has been approved as a
single agent for treatment of unresectable or metastatic melanoma
harboring the B-Raf V600E or V600K mutation.
From all of the above, it is clear that alternative therapeutic
strategies are needed to overcome drug resistance. Two main avenues are being explored. The rst, approved for clinical use by the
FDA on January 2014 for the treatment of metastatic B-Raf-driven
melanoma, is a combination of Raf and Mek inhibitors (dabrafenib
plus trametinib) [136]. This double hit is expected to circumvent
and/or delay acquired resistance originating from pathway reactivation, and has been recently shown to prevent melanoma metastasis in a preclinical model [137]. It is likely that in the future, Raf
inhibitor monotherapy will be replaced by Raf and Mek inhibitor
combination therapy as the rst-line treatment for B-Raf-driven
melanoma.
In addition to this vertical inhibitor combination, preclinical
studies have also shown the benets of co-targeting PI3K, mTOR,
Hsp90 CDK 4/6, FGFR, c-Met (parallel inhibitor combination) or
using immunotherapy to overcome drug resistance (reviewed in
[138,139]).
Drug holidays, the temporary cessation of drug treatment,
may prove effective in reverting drug resistance. Raf inhibitorresistant melanomas revert to drug-sensitivity when the treatment
is interrupted in preclinical models [140] and, most importantly, in
the clinic [141]. This indicates that drug resistance is adaptive and,
most importantly, reversible. The preclinical model has shown that
B-Raf-driven tumors become not only resistant, but addicted to Raf
inhibitors in vivo, resulting in lethal, drug-resistant disease the
onset of which can be delayed by intermittent inhibitor treatment
[140]. Along the same lines, a recent report from the Bernards lab
has shown that melanoma cells that survive Raf inhibitor treatment through the reversible upregulation of RTKs enter senescence
due to supraphysiological Erk pathway stimulation when the drug
is removed. These cells, which acquire inhibitor resistance at the
cost of their general tness, are negatively selected during drug
holidays, restoring the sensitivity of the tumor population to the
Raf inhibitor axis [114].

The last 5 years have seen tremendous progress in the area of

clinical Erk pathway inhibition, with exceptional fast rates of
bench-to-bedside translation. In addition, however, research on
the inhibitors mode of action and on the mechanisms underlying
resistance has advanced our understanding of how the Erk pathway is wired not only in tumors, but also in normal cells. Thus, this
area of research represents a prime example of what can be
achieved by the concerted efforts of academia, companies and clinicians investigating the same problems from different angles.

473

7. Conclusions

482

The investigation of the Ras/Raf/Mek/Erk pathway has provided

us with a wealth of insight into the regulation of complex signaling
networks, some of which are likely applicable to other MAPK cascades. Proteinprotein interaction has emerged as one recurrent
theme, with major consequences for pathway activation, regulation and cross-talk; it is becoming clear that using inhibitors that
target complex formation in addition to catalytic activity may yield
superior specicity and broaden the range of susceptible tumors.
The dynamics of complex formation strongly depend on the stoichiometry of the proteins involved in the interaction(s), which in
turn varies in different healthy tissues and is individually modulated in transformed cells. The resulting changes in the assembly
and localization of signaling complexes are likely to specify individual biochemical and biological outcomes. The big challenge now
will be to obtain a complete map of the interaction partners in different tissues, to determine which of the interactions are essential
in development and disease, and identify those that can be
exploited for the purpose of molecule-targeted therapy.

483

Acknowledgments

501

We thank all the members of the Baccarini laboratory for helpful discussions and apologize to all the colleagues whose work, for
reason of space, could not be cited in this review. Work in the Baccarini lab is/was supported by grants of the Austrian Research Fund
(FWF W-1220, SFB-021, P-19530), EU-project INFLACARE, Bridge
project 827500 (Austrian Research Promotion Agency, FFG), and
the Johanna Mahlke geb. Obermann-Stiftung of the University of
Vienna.

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Please cite this article in press as: Cseh, B., et al. RAF neighborhood: Proteinprotein interaction in the Raf/Mek/Erk pathway. FEBS Lett. (2014), http://
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Please cite this article in press as: Cseh, B., et al. RAF neighborhood: Proteinprotein interaction in the Raf/Mek/Erk pathway. FEBS Lett. (2014), http://
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