Second nature: Biological functions of the Raf-1 ‘‘kinase’’
Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, The University of Vienna, Vienna Biocenter,
Dr. Bohr Gasse 9, 1030 Vienna, Austria
Accepted 16 March 2005
Available online 23 March 2005
Edited by Ga ´spa ´r Je ´kely
lular oncogene transduced by transforming retroviruses. Since
then, the three Raf isoforms have been intensively studied,
mainly as the kinases linking Ras to the MEK/ERK signaling
module. As this pathway is activated in human cancer, the Raf
kinases are considered promising therapeutic targets, and we
have learned a lot about their regulation, targets, and functions.
Do they still hold surprises? Recent gene targeting studies indi-
cate that they do. This review focuses on the regulation and biol-
ogy of the best-studied Raf isoform, Raf-1, in the context of its
? ? 2005 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
More than 20 years ago, Raf was discovered as a cel-
Keywords: Raf; Kinase; Knock-out; Survival; Motility
1. History of Raf
In 1983, Ulf Rapp reported the cloning of an acutely trans-
forming murine sarcoma virus (3611-MSV) and the character-
ization of its acquired oncogene, named v-raf (for rapidly
growing fibrosarcoma) . A similar sequence, named v-mil,
was identified by Klaus Bister and coworkers as the transform-
ing principle of a naturally occurring avian retrovirus MH2 .
Bister and Rapp then went on to show that 3611-MSV and
MH2 had incorporated orthologues of the same cellular pro-
tein , which Karin Moelling and Ulf Rapp proved to be
the first oncoprotein with serine/threonine kinase activity .
These seminal papers set the scene for the next 20 years of re-
search on the cellular counterparts of v-raf and v-mil. The Raf
kinase family comprises three isoforms, which differ in their
expression profile, regulation, and ability to function in the
context of the Ras–MEK–extracellularly regulated kinase
(ERK) cascade. Although A-Raf and B-Raf transcripts can
be detected, at different levels, in most embryonic and adult
mouse tissues , Raf-1 was first reported to be the only iso-
form expressed ubiquitously . Because of this and of reagent
availability, Raf-1 has been the most intensively studied mem-
ber of the family, and most of the groundbreaking contribu-
tions describing the role of Raf in signal transduction
actually deal with Raf-1. Thus, it was Raf-1 which was first re-
ported, in the late 1980s–early 1990s, to be phosphorylated and
activated in response to growth factor stimulation [7–12]. Acti-
vated Raf-1 was then linked to one of the rising stars in the sig-
nal transduction sky of those years, the ERK/MAP kinase
cascade. ERKs had just been discovered as proteins phosphor-
ylated on tyrosine and threonine upon stimulation of receptor
tyrosine kinases [13–16]. Soon after the description of the dual
specificity kinase MEK as the upstream activator of ERK [17–
19], Raf-1 was shown to activate MEK [20–22] and to physi-
cally associate with it . Finally, in 1993, a number of groups
demonstrated the recruitment of Raf-1 by activated Ras [24–
27], instating Raf-1 as the link between Ras and ERK and
completing the picture of the first MAPK pathway we all know
from the textbooks (Fig. 1). The MEK/ERK module, with its
impressive array of membrane, cytosolic, and nuclear sub-
strates , is an excellent candidate as the downstream effec-
tor carrying out all the functions attributed to activated Raf in
proliferation, differentiation, and survival by a variety of over-
expression studies. Indeed, for almost a decade, MEK was the
only commonly recognized substrate of the three Raf isoforms.
As we will see below, recent gene ablation studies are changing
this view radically, particularly in the case of Raf-1.
2. Regulation of Raf kinase activity
Despite intensive efforts, Raf regulation is far from com-
pletely understood. Again, most of the work in this area fo-
cused on Raf-1, and has revealed a complex process
involving membrane recruitment, intra- and intermolecular
interactions, and phosphorylation/dephosphorylation events
resulting in kinase activation/release from repression. Raf-1
regulation has been the subject of recent reviews [29–31], and
we will limit ourselves to an outline with an angle on repres-
All three Raf kinases share a common structure comprising
three conserved regions (CR; see Fig. 2): CR1, containing the
two Ras-binding sites Ras-binding domain (RBD), and cys-
teine-rich domain (CRD); CR2, rich in Ser/Thr residues; and
CR3, representing the business end of the molecule, the kinase
domain. The carboxy-terminal half of Raf-1 contains all the
phosphorylation sites which stimulate activity, including the
conserved Thr and a Ser residue in the so-called ‘‘activation
segment’’ necessary for the activation of Raf-1 and B-Raf,
and in all likelihood, of A-Raf ; and Ser and Tyr residues
relevant for the activation of Raf-1 and A-Raf (S338 and
Tyr341 in Raf-1). The kinases phosphorylating these residues
in a physiological growth factor response have been searched
*Fax: +43 1 4277 9546.
E-mail address: email@example.com.
0014-5793/$30.00 ? 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS 29449 FEBS Letters 579 (2005) 3271–3277
for by a number of different approaches, but their identity is
still controversial (for review, see ). In addition, the car-
boxy-terminus contains a conserved constitutively phosphory-
lated Ser residue (S621 in Raf-1), which mediates the binding
of Raf to the 14-3-3 and has been shown by mutational anal-
ysis to be necessary for Raf activity in vivo. Phosphorylation
of the amino-terminal half of Raf-1, in contrast, mediates
repression of kinase activity. Indeed, this region contains 7
out the 8 negative phosphorylation sites present in Raf-1: the
inhibitory sites phosphorylated by PKA (S43, S233, and 259)
or PKB (S259) which create additional binding sites for the
14-3-3 proteins; and 5 out of 6 newly discovered ERK phos-
phorylation sites (S29, S43, S289, and 296, and 301) . A
(S624) is found in the carboxy-terminus. The discovery of these
sites suggests the existence of a negative regulatory feedback
Fig. 2. Schematic view of Raf-1 (A) and its activation mechanism (B). Aminoacid residues and protein regions that participate in the positive
regulation of Raf-1 MEK Kinase activity are indicated in different shades of green, whereas aminoacid residues and protein domains participating in
the negative regulation of Raf-1 kinase activity are indicated in red. The CR1 region contains the RBD and the CRD involved in the relief from
autoinhibition by Ras. The CR2 region is rich in serine residues which participate in the negative regulation of kinase activity. The CR3 region
contains the kinase domain and the serine, threonine, and tyrosine residues involved in kinase activation (see text for details). (B) Raf-1 is regulated
by intramolecular autoinhibition. In the quiescent state, phosphorylation of the S259 and S621 residues creates binding sites for the 14-3-3 proteins,
which stabilizes the inactive conformation. Upon mitogenic stimulation, Ras and PP2A cooperate to release autoinhibition; the subsequent
phosphorylation of activating sites by as yet incompletely identified upstream kinases yields a fully active MEK1 kinase. Following ERK activation,
Raf-1 is phosphorylated on 6 negative regulatory sites, yielding a desensitized kinase. The prolyl isomerase Pin1 co-operates with PP2A to
dephosphorylate the ERK-dependent negative regulatory residues. Finally, S259 becomes phosphorylated again, and Raf-1 reassumes its quiescent
conformation, stabilized by a 14-3-3 dimer.
Fig. 1. General scheme of the ERK pathway. An activated receptor tyrosine kinase (RTK) causes the relocalization of the GRB2–SOS complex. SOS
is a guanine nucleotide exchange factor (GEF) for Ras and causes the conversion of Ras-GDP into Ras-GTP. Ras-GTP recruits Raf to the
membrane, thereby promoting its activation. Activated Raf phosphorylates and activates MEK, which, in turn, stimulates ERK activity. ERK has
an impressive roster of substrates which have been implicated in the regulation of proliferation, survival, and differentiation.
M. Baccarini / FEBS Letters 579 (2005) 3271–3277
loop linking ERK stimulation to Raf-1 deactivation and there-
fore possibly limiting ERK activation.
The current model of Raf-1 regulation postulates that the
N-terminal domain of Raf-1 binds to the kinase domain and
suppresses the catalytic activity of the enzyme. This intramo-
lecular autoinhibition is favored by 14-3-3 proteins binding
to phosphorylation sites at the N- and C-terminus of Raf-1,
and must be disrupted to permit Raf-1 activation. We and oth-
ers have shown that this is accomplished via dephosphoryla-
phosphatase PP2A, which interacts with Raf-1 in mitogen-
stimulated cells. Upon S259 dephosphorylation, Raf-1 is re-
cruited to the membrane and binds to Ras [33–35] via the
RBD and the CRD contained in the CR1 region. Ras binding
is then followed by the phosphorylation of the activating resi-
dues in the CR3 region, which stabilizes an activated confor-
mation. Activation is terminated by a negative feedback loop
in which ERK and PKB phosphorylate Raf-1 on inhibitory
sites. The activation-competent conformation of Raf-1 is finally
re-established by the co-ordinated action of Pin1, a prolyl
isomerase that converts pSer and pThr residues from the cis
to the trans conformation, which is preferentially recognized
and dephosphorylated by PP2A. Thus, at least two distinct
Ras effectors, PKB and ERK, contribute to the negative regu-
lation of Raf-1, and dephosphorylation of inhibitory sites is as
important as activating phosphorylation for the stimulation of
Raf-1 kinase activity.
In contrast to this complicated process, B-Raf activation
seems to be much more direct, requiring basically only Ras
binding  and phosphorylation of the activation segment
 to disrupt intramolecular autoinhibition. In B-Raf, the
aminoacids involved in Raf-1 activation are either constitu-
tively phosphorylated (Ser 445, corresponding to Raf-1
Ser338) or negatively charged (Asp448, corresponding to
Raf-1 Tyr341). The constitutive presence of negative charges
in this region of B-Raf likely reduces the threshold for mito-
gen-induced kinase stimulation. In addition, it is unclear
whether a 14-3-3 dimer stabilizes intramolecular autoinhibi-
tion in the case of B-Raf. 14-3-3 bind to B-Raf pS728 and
a further potential 14-3-3 binding site in the CR2 (S364)
can be generated by PKB . Although phosphorylation
of this site inhibits B-Raf activity, it has not been tested
whether endogenous B-Raf is phosphorylated on pS364 in
quiescent cells, and whether dephosphorylation of this site
is necessary for B-Raf activation. Finally, only two of the
six residues phosphorylated by ERK in Raf-1 are conserved
in the other Raf proteins, and it is not know whether phos-
phorylation of these two residues only is sufficient for deacti-
vation. Thus, comparison of Raf-1 and B-Raf regulation
reveals that the latter kinase is ‘‘primed’’ for Ras-induced
3. Raf and the MEK/ERK module
Although most of the work published on the activation of
the MEK/ERK module was performed with Raf-1, evidence
has been accumulating that B-Raf is the main MEK kinase
in vivo. Cell fractionation and immunodepletion studies have
shown that B-Raf is the main MEK kinase found in cell and
brain lysates [38–40]. Furthermore, comparison of the three
Raf kinases has shown that B-Raf binds best to MEK 
and has the highest basal MEK kinase activity both in vitro
 and in fibroblasts, when expressed as a conditionally
oncogenic form . Finally, growth factor-stimulated ERK
activation is reduced in B-Raf-deficient, but not in A-Raf- or
Raf-1-deficient cells [5,44–47]. These experimental facts corre-
late well with the observation that the Raf kinases from lower
organisms, like C. elegans? lin-45 or Drosophila?s D-Raf, are
more similar to B-Raf than to the other two mammalian Raf
kinases. Thus, B-Raf is likely to be the archetypal MEK
kinase, whereas Raf-1 and A-Raf have likely diverged to per-
form other functions. Although at present the only confirmed
substrate of B-Raf is MEK, recent work on B-Raf mutations
found in human tumors has revealed an unexpected twist in
the story: B-Raf mutants unable to phosphorylated MEK in
vitro can still activate the MEK/ERK cascade in vivo, and they
do so by binding to, and activating, Raf-1 . It is yet com-
pletely unclear whether the mutations abrogate B-Raf kinase
activity completely or whether they shift substrate specificity,
whether kinase activity is required for the effect on Raf-1, or
whether heterodimerization between Raf-1 and mutant B-
Raf causes a conformational change promoting Raf-1 MEK
kinase activity. In this context, it is noteworthy that wild-type
Raf-1 and B-Raf can heterodimerize  and, more specifi-
cally, that the isolated autoinhibitory domain of Raf-1 can
interact with, and inhibit, the catalytic domain of B-Raf .
The relevance of these data for the regulation of the wild-type
enzymes during physiological responses has not yet been
tested; however, they raise the interesting possibility that
Raf-1 and B-Raf may cross-regulate each other in this context
Complex formation is a recurring theme in Ras-ERK sig-
naling, and a number of scaffold proteins have been described
that, by recruiting selected signaling components, help main-
taining signal fidelity and favor signal propagation through
the cascade. KSR, for instance, is a Raf-related pseudokinase
which binds to MEK, ERK, and Raf ; CNK interacts
both with Raf and with components of the Ral signaling
pathway ; and Sur-8 facilitates the interaction between
Ras and Raf . On the other hand, proteins have been
identified that disrupt interactions in the cascade: RKIP,
which decreases interaction between Raf and MEK and
may regulate Raf activation ; Sprouty and Spred, which
suppress Raf activation [55,56]; and IMP, which inactivates
KSR . Most of these proteins have been first identified
in Drosophila or C. elegans; therefore, the prediction would
be that they interact both with Raf-1 and B-Raf. Indeed,
whenever tested, this was the case. In several cases, interac-
tion of the scaffold with their target proteins or correct local-
ization of the scaffold are modulated by phosphorylation/
dephosphorylation events ; these multiple levels of regula-
tion provide a high degree of plasticity, allowing the cell to
redirect the signals towards, or away from, the ERK signaling
pathway, and thereby to fine-tune its output.
4. Lessons from knock-out mice – Novel targets and novel
functions for Raf-1
A-raf, B-raf and c-raf-1-deficient mice have been generated.
A-raf-deficient mice are born alive and show neurological and
intestinal defects, depending on the genetic background . In
contrast, B-raf and c-raf-1-deficient embryos both die around
M. Baccarini / FEBS Letters 579 (2005) 3271–3277
midgestation. The former succumb to vascular hemorrhage
due to apoptotic death of differentiated endothelial cells ,
whereas c-raf-1-deficient embryos show increased apoptosis
of embryonic tissues  or, more selectively, of the fetal liver
, depending on the genetic background. Ablation of the
common Raf kinase target, MEK-1, results in embryonic
lethality due to a placentation defect correlating with reduced
cell motility . These divergent phenotypes show that Raf-1,
B-Raf, and MEK-1 serve distinct essential functions in embry-
While little follow-up work has been done on the B-Raf and
MEK-1 knock-out, a number of papers have advanced our
understanding of the biological role of Raf-1. It has quickly
become clear that one of the main functions of this protein
is to restrict caspase activation in response to selected stimuli,
notably Fas stimulation [45,46], pathogen-mediated macro-
phage apoptosis , and erythroid differentiation . The
MEK/ERK module is in principle capable of antagonizing
apoptosis in a number of ways, including the expression of cas-
pase inhibitors and the neutralization of pro-apoptotic Bcl-2
family members; a further prominent prosurvival molecule,
the transcription factor NF-jB, has been proposed as a down-
stream target of Raf-1 (reviewed in ). However, neither
MEK/ERK nor NF-jB activation are altered in Raf-1-defi-
cient cells and embryos [45,46,62], indicating that the prosur-
vival role of Raf-1 does not depend on these functions.
What, then, are the essential downstream targets of Raf-1 in
Recently, conditional mutagenesis has confirmed apoptosis
signal-regulated kinase 1 (ASK1) as a pro-apoptotic molecule
inhibited by Raf-1 in vivo . ASK1 is a protein kinase which
works upstream of JNK and p38 to promote apoptosis in-
duced by stress or by death receptors, like the TNF-aR or
Fas. A few years ago, Hanan Fu reported that Raf-1 forms
complexes with, and antagonizes, ASK1, and that Raf-1 does
so independently of its kinase activity . More recently,
ASK1 binding to Ha-Ras has been shown to inhibit the proa-
poptotic activity of the kinase . Last year, elegant work by
Kinyia Otsu has shown that cardiac-specific Raf-1 ablation in-
duces cardiomyocyte apoptosis in vivo. This defect is accom-
paniedby the transient activation
downstream targets p38 and JNK, and could be rescued by
inactivation of the ASK1 gene . This work has firmly estab-
lished that Raf-1 antagonizes ASK1 in vivo, at least in cardio-
myocyte survival. Whether Raf-1 modulates ASK1 activity by
direct binding or by competing for a common binding partner
responsible for the inhibition of ASK1-induced apoptosis is at
Hyperactivation of ASK1, however, does not explain the
selective hypersensitivity of Raf-1-deficient fibroblasts towards
FasL, but not TNF-a-induced apoptosis. In particular, con-
ventional ablation of ASK1 reveals that this kinase is essential
for TNF-a, but not Fas-induced apoptosis, making it unlikely
that ASK1 is the Raf-1 target in this context. A protein with all
the right credentials has been recently identified as a result of a
proteomic effort combined with the analysis of knock-out cells
and RNA interference. As in the case of ASK1, the protein in
question is a kinase, MST2, and it is hyperactive in Raf-1
knock-out/knock-down cells . Raf-1 binds to MST2 via
its N-terminus, and specifically via the CR2 region that is
not conserved in B-Raf; therefore, MST2 qualifies as
‘‘Raf-1-only’’ target. MST2 is activated selectively by Fas in
Raf-1-deficient cells, indicating that MST2 inhibition is an
essential function of Raf-1 in the context of Fas-induced apop-
tosis. Mechanistically, Raf-1 appears to prevent MST2 homo-
dimerization, which leads to activation of this kinase, and
additionally to recruit a phosphatase, (PP2A?), which dephos-
phorylates, and therefore inactivates, MST2 (Fig. 3). Kinase-
dead Raf-1 is as efficient as wild-type Raf-1 in binding to,
and antagonizing, MST2, proving that the kinase activity of
Raf-1 is dispensable for this prosurvival function. Although
the significance of MST2 inhibition in the context of the whole
organism has not been assessed, these data identify a novel, ki-
nase-independent target of Raf-1 in apoptosis.
of ASK1and its
Fig. 3. Novel targets and functions of Raf-1. (A) Schematic view of Raf-1 and Rok-a, highlighting the similarities between the two molecules. RB,
Ras-binding domain; PH, pleckstrin homology domain; K, kinase domain. (B) In wild-type cells, the presence of Raf-1 regulates the level of activity
of Rok-a (right side), possibly by cross-inhibition via its pleckstrin homology domain; and of MST2 (left side), by inhibiting the formation of MST2
dimers and by recruiting a phosphatase (PPase) that dephosphorylates and inactivates MST2. In Raf-1 knock-out cells, hyperactivation of Rok-a
leads to defects in actin remodeling and to impaired migration via the hyperphosphorylation of ezrin; phosphorylated ezrin further appears to
impinge on apoptosis by reducing Fas internalization and enhancing the Fas signal. in addition, in the absence of Raf-1 Fas stimulation causes the
dimerization and activation of MST2, which contributes to the increased apoptosis observed in Raf-1 knock-out cells. The possibility that ERK may
redirect Raf-1 towards kinase-independent targets by phosphorylating inhibitory residues on Raf-1 is indicated.
M. Baccarini / FEBS Letters 579 (2005) 3271–3277
Protection from apoptosis is not the only physiologically
relevant function of Raf-1. Using conditional mutagenesis,
we have recently demonstrated that Raf-1 is required for
normal wound healing in vivo and for the migration of
keratinocytes and fibroblasts in vitro. Strikingly, this novel
function of Raf-1 can also be carried out by a kinase-dead
mutant, and, just like prosurvival, it involves the inhibition
of another kinase. The target of Raf-1 in motility is the
Rho effector Rok-a, which is hyperactive and mislocalizes
to the membrane of Raf-1-deficient cells. As a consequence
of Rok-a hyperactivation, Raf-1 knock-out fibroblast and
keratinocytes have a contracted appearance, a defective cyto-
skeleton characterized by tight cortical actin bundles, and fail
to migrate. Chemical inhibition of Rok-a or expression of a
dominant-negative Rok-a mutant rescue all defects of the
Raf-1-deficient cells, indicating that Rok-a is the only target
of Raf-1 in motility . But how does Raf-1 regulate Rok-
a? We know that inhibition is mediated by the Raf-1 aut-
oregulatory region, which contains a cystein-rich pleckstrin
homology (PH) domain (aa 100–144). Rok-a, like Raf, is
regulated by autoinhibition, and its carboxy-terminal aut-
oregulatory region features a PH highly homologous to the
one found in Raf-1 (Fig. 3A). This leads to the hypothesis
that Raf-1 may keep activated Rok-a in check by binding
to the Rok-a kinase domain and repressing its function
5. Conclusions and future perspectives
Two surprising lessons emerge from the data summarized
above: first, the MEK kinase activity of Raf-1 is not required
for the essential functions of this protein in survival and motil-
ity; and second, the autoinhibitory N-terminus of Raf-1, which
is deleted in the retroviral oncogene, is used by the cell as a
negative regulator of at least two other kinases, one promoting
apoptosis, the other controlling cell shape and motility. Natu-
rally, these insights raise a whole host of new questions. For
instance, if MEK kinase activity is not the main function of
Raf-1, why is it so tightly regulated? One possibility is that
the negative regulatory mechanisms targeting Raf-1 kinase
activity have evolved to separate Raf-1 from the MEK/ERK
module, or even to redirect it towards other targets, which
do not require kinase activity. Both MST2 and Rok-a bind
to the N-terminal region of Raf-1, which should not be acces-
sible in the ‘‘quiescent’’ state of the protein. Do they bind bet-
ter to the ‘‘desensitized’’ Raf-1 produced as a consequence of
ERK activation? We are currently performing structure-
function studies with the Raf-1/Rok-a pair to answer these
On a different note, are the functions of Raf-1 in apoptosis
and motility completely separated, or do they intersect? Recent
studies in the lab indicate that the latter may be the case. Rok-
a and its target ezrin, hyperphosphorylated in Raf-1-deficient
fibroblasts and responsible for the bundling of cortical actin
in these cells, appear to mediate Fas clustering and inhibit
Fas internalization, thereby rendering the cells selectively
hypersensitive to Fas-induced apoptosis.
Finally, are the kinase-independent function all there is to
Raf-1 physiology, or are there other, tissue-specific functions
of Raf-1 as a (MEK1) kinase? What is the relative significance
of the prosurvival function of Raf-1 and of its role in motility
for embryonic development? And, possibly the most burning
question, may one of these kinase-independent functions be
of importance in tumor development or maintenance? Condi-
tional mutagenesis coupled with the use of mouse tumor mod-
els will enable us to address these issue. And Raf biology will
keep us intrigued for the next 20 years.
Acknowledgments: We apologize to our colleagues whose work could
not be cited because of space constraints, and thank all the members
of the lab for helpful discussions. Work in the Baccarini lab is sup-
ported by Austrian Research Fund Grants P15784-B07 and P16398-
B07 and by EC Grant LSH-CT-2003-506803.
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