1 The Beatson Institute for Cancer Research, CRC Beatson Laboratories, Glasgow,
G61 1BD, UK
2 Institute for Biomedical and Life Sciences, University of Glasgow, Glasgow,
G12 8QQ, UK
* Author for correspondence (e-mail: wkolch{at}beatson.gla.ac.uk )
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Summary |
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Key words: Raf, Apoptosis, ERK/MAPK, Knockout mice, Signal transduction
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Introduction |
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Since the biological outcome is determined by the strength and duration of
the activation of this pathway (Marshall,
1995), it is tightly regulated with the most intricate controls
operating at the level of Raf (Avruch et
al., 2001
; Houslay and Kolch,
2000
; Kolch,
2000
). Briefly, Raf activation is a consequence of its binding to
Ras and subsequent complex changes in phosphorylation and interaction with
lipids. Although all three Raf isoforms can interact with Ras, there are
important differences. For instance, Ras binding alone is sufficient to
activate B-Raf, but not Raf-1 or A-Raf, which require secondary signals
(Marais et al., 1997
). Rap1, a
Ras-related G protein, has been reported to activate B-Raf but to inhibit
Raf-1 (Vossler et al., 1997
).
Rap1 and Ras are activated by an overlapping, but distinct set of stimuli
(Bos et al., 2001
), whose
specific biological effects may be due to the selective activation of Raf
isoforms. Therefore, it would be surprising if all three Raf isozymes were to
rely on MEK alone as their only commonly accepted substrate. However, recent
reports have produced indirect, but strong, evidence for a branching of
signals at the level of Raf, in particular Raf-1, which is the focus of the
majority of Raf research.
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New Raf signalling pathways |
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Another candidate for a Raf effector is NF-B, a transcription factor
central to cell transformation and survival
(Mayo and Baldwin, 2000
).
NF-
B is sequestered in the cytosol by its inhibitor protein I
B,
which upon activation is phosphorylated and targeted for degradation
(Karin and Delhase, 2000
). It
is unclear how exactly these pathways interact, and both ERK-dependent and
ERK-independent mechanisms may be involved. The ERK substrate p90-RSK can
phosphorylate I
B and induce its degradation
(Ghoda et al., 1997
). In
addition, ERK can downregulate the expression of PAR-4, an inhibitor of
NF-
B activation (Barradas et al.,
1999
; Qiu et al.,
1999
). An emerging theme is that Raf-1 induces I
B
degradation via MEKK-1 (MEK kinase 1) independently of MEK and ERK and that
this is essential for Raf-1-mediated transformation
(Baumann et al., 2000
). MEKK-1
was originally described as a kinase capable of activating MEK, but this seems
to be an overexpression artefact; the physiological function of MEKK-1 is
instead in the NF-
B and apoptosis pathways
(Schlesinger et al., 1998
).
Regardless of the mechanism by which Raf-1 activates NF-
B, it does not
seem to be essential, since NF-
B activation proceeds unimpaired in
Raf-1-/- cells
(Jesenberger et al., 2001
;
Mikula et al., 2001
).
Other studies suggest that Raf-1 and ERK signalling can be distinct, or
even oppose each other. For example, the transcription of atrial natriuretic
factor (ANF) in cardiac myocytes is suppressed by ERK but activated by Raf
(Jette and Thorburn, 2000).
Likewise, in 3T3-L1 cells ERK impedes insulin-induced differentiation into
adipocytes (Font de Mora et al.,
1997
), whereas v-Raf promotes differentiation. However, in
contrast to insulin, v-Raf does not activate ERK, which suggests that v-Raf
promotes differentiation independently of ERK. Further, a dominant negative
Raf-1 mutant prevents insulin from stimulating differentiation but not from
activating ERK (Porras and Santos,
1996
). These findings suggest that during adipocytic
differentiation ERK is activated independently of Raf and that Raf supports
differentiation independently of ERK. Similarly, the neuronal differentiation
of rat hippocampal cells can be driven by activated Raf but not by activated
MEK (Kuo et al., 1996
), which
again suggests a bifurcation of Raf signalling and engagement of
MEK-independent Raf effector(s). This hypothesis was borne out by an elegant
study that generated a constitutively active Raf-1 point mutant that could not
interact with MEK (Pearson et al.,
2000
). This mutant failed to transform fibroblasts and activate
serum-response element (SRE)-mediated reporter gene expression. However, the
activation of NF-
B-dependent gene expression, neuronal differentiation
of PC12 cells and the activation of p90-RSK remained intact. Despite the
caveat that this Raf-1 mutant still may produce a small but sufficient level
of MEK activation, to date this is the clearest biochemical evidence for novel
Raf-1 signalling targets.
All these experiments involved the forced expression of activated or
dominant negative Raf-1 mutants, and hence are affected by the general
problems of overexpression artefacts. Two issues are of particular concern in
this context. First, constitutively active kinases may lose substrate
specificity. The activated Raf-1 mutants used in these studies were generated
by deletion of the regulatory domain, which not only suppresses kinase
activity (Cutler et al., 1998)
but also regulates the interaction with the activator Ras
(Vojtek et al., 1993
) and the
substrate MEK (Dhillon et al.,
2002
). The removal of such regulatory motifs may compromise
substrate specificity as well as the dynamic regulation of activity. Second,
the dominant negative Raf-1 mutants used were made either by removing the
kinase domain or by disabling the kinase activity through a point mutation in
the ATP-binding site. Both types of mutant can interact with Ras and hence can
block the access of other effectors to Ras. In addition, the latter mutant is
expected to compete for substrate(s) too. Therefore, as in the case of any
other results obtained through overexpression, these findings need to be
interpreted with caution. A definitive answer can be provided only by the
identification of a bona fide Raf substrate other than MEK.
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New substrates for Raf-1? |
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One range of substrates pertains to cell cycle regulation. pRb plays a
central role in the cell cycle, being the main repressive factor that holds
cells in G1. In G1, pRb acts as a transcriptional repressor by binding to the
transcription factor E2F and retaining it in the cytosol. To release E2F, pRb
must be phosphorylated on multiple sites by cyclin-dependent kinases (CDKs)
(Zheng and Lee, 2001). Raf-1
binds to pRb in growth-factor-stimulated cells, interacting only with the
active (hypophosphorylated) from of pRb
(Wang et al., 1998
). Raf-1
kinase activity is required for the interaction, but direct phosphorylation of
pRb by Raf-1 has been seen only in vitro. The authors propose that Raf-1 could
be part of the cell cycle machinery that together with CDKs inactivates pRb by
phosphorylation. This would link Raf-1 directly to cell cycle regulation.
However, these observations are at odds with the results of timed
microinjection studies. These show that, at least for cell cycle progression
in response to insulin, Raf-1 activity is required only in the first 20
minutes after stimulation (Rose et al.,
1998
), whereas pRb phosphorylation occurs much later in G1.
Another cell cycle protein postulated to be a Raf-1 substrate is Cdc25, a
phosphatase that triggers CDK activation by removing inhibitory phosphates
(Nilsson and Hoffmann, 2000
).
Active forms of Raf-1 interact with Cdc25, predominately the Cdc25A isoform,
which is involved in the G1/S phase transition. At least in vitro, Raf-1 can
activate Cdc25A by phosphorylation
(Galaktionov et al., 1995
).
Another report that was never followed up found that Raf-1 can phosphorylate
p53 in vitro and enhance its transcriptional activity
(Jamal and Ziff, 1995
).
The other group of proposed Raf-1 targets are proteins involved in
apoptosis regulation, most notably BAD and ASK-1 (for discussion, see below).
The ankyrin-repeat protein Tvl-1 is one such potential target. Tvl-1 has been
reported to promote the assembly of pro-apoptotic Apaf-1 complexes, caspase
activation and apoptosis (Patriotis et
al., 2001), and bound to Raf-1 in a yeast two-hybrid screen with a
murine T-cell cDNA library (Lin et al.,
1999
). Tvl-1 is both an in vitro substrate for Raf-1 and a novel
regulator that enhances Raf-1 activation by EGF and Src plus Ras. At present,
the functional relationships between these diverse Tvl-1 functions are
unclear. Raf-1 may inhibit the pro-apoptotic function of Tvl-1 given that
Raf-1-/- macrophages are hypersensitive to caspase-1
activation and apoptosis (Jesenberger et
al., 2001
). Another suggested Raf-1 substrate, RIP2 (also called
CARDIAK or RICK) was originally described as a pro-apoptotic serine/threonine
kinase involved in caspase activation and tumour necrosis factor (TNF)
receptor and Fas signalling (Inohara et
al., 1998
; McCarthy et al.,
1998
; Thome et al.,
1998
). Recently, RIP2 was reported to activate ERK and be
activated by Raf-1 in response to TNF stimulation
(Navas et al., 1999
). Thus, in
the TNF signalling pathway, RIP2 would take the place of MEK to couple Raf-1
to ERK. The physiological purpose of such a change in ERK activation is
obscure but intriguing, because RIP2 can activate NF-
B
(McCarthy et al., 1998
;
Thome et al., 1998
). It thus
may provide another link between Raf-1 and NF-
B.
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Raf-knockout mice |
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Last year saw the back-to-back publication of two Raf-1-knockout
mice studies (Huser et al.,
2001; Mikula et al.,
2001
) in which Raf-1 protein expression was abolished completely.
Both groups observed embryonic lethality and poor development of the placenta
and embryonic organs, in particular of the liver and the haematopoietic
compartment. As in most knockout mice, the phenotypes were modified by genetic
background and ameliorated in outbred strains. However, two common themes
emerged: a normal proliferative capacity and an increased propensity towards
apoptosis. Surprisingly, both studies found ERK activation by mitogens
unimpaired in Raf-1-/- fibroblasts. This could reflect
compensation by the other Raf isozymes, probably B-Raf, which was previously
reported to be the main MEK activator in fibroblasts
(Reuter et al., 1995
). B-Raf
has been shown to protect established fibroblasts against apoptosis through
ERK-dependent interference with caspase activation
(Erhardt et al., 1999
).
However, this study employed overexpressed B-Raf, which activated ERK
constitutively. Thus, it is difficult to compare these results with those
obtained from the Raf-1-/- fibroblasts, in which ERK was
activated transiently by growth factors, but they indicate a significant
antiapoptotic role for Raf-1 that is independent of ERK.
All Raf-1 gene-knockout studies observed similar defects in the
placenta: hypovascularisation and a severe reduction of the spongiotrophoblast
and labyrinth layers (Murakami and
Morrison, 2001). This placenta phenotype also occurs in
MEK-1-/- mice (Giroux
et al., 1999
), which suggests that the Raf-1-MEK axis is required
for proper placenta development. However, despite MEK-1-/-
embryos dying in midgestation, no increase in apoptosis was noted.
MEK-1-/- fibroblasts fail to migrate on fibronectin
(Giroux et al., 1999
), a
feature that unfortunately was not tested in the Raf-1-knockout
studies. Thus, a comparison of all the knockout data argues for MEK-dependent
as well as MEK-independent roles of Raf-1.
Huser and colleagues also made Raf-1FF-knock-in mice
(Huser et al., 2001). Raf-1FF
is a point mutant which, at least in cultured cells, cannot be efficiently
activated by mitogens owing to the mutation of tyrosine residues 340 and 341
to phenylalanine. Astonishingly, the phenotype of these mice is normal. They
survive to adulthood, are fertile and display none of the abnormalities
associated with any of the Raf knockouts. The Raf-1FF mutant
retains a low level of kinase activity, and it is unknown whether and how this
activity is regulated in tissues. Thus, this mutant still could signal through
MEK, and a conclusive answer has to await the knock-in of a true
kinase-negative Raf-1 mutant.
At present it is unclear whether the strikingly different phenotypes of the
Raf-knockout mice reflect the existence of isoform-specific
substrates, tissue-specific expression or different potencies of activation of
the ERK pathway. A-Raf and B-Raf expression appears more restricted, whereas
Raf-1 is ubiquitously expressed (Storm et
al., 1990). However, the knockout mice clearly show that Raf-1
cannot compensate for the loss of another Raf isoform. Indeed, Wiese and
colleagues have shown that neurotrophic factors are unable to sustain the
viability of sensory and motor neurons explanted from
B-Raf-/- mice, whereas neurons from
Raf-1-/- mice survive with the same efficiency as
wild-type cells (Wiese et al.,
2001
). The basis for this difference is unknown at present. B-Raf
is a more potent activator of MEK than Raf-1, and Raf-1 is better than A-Raf
(Marais et al., 1997
). The
strength and duration of ERK activity can dictate diverse biological outcomes.
For instance, in PC12 cells the sustained activation of ERK promotes
differentiation, whereas a transient activation is associated with
proliferation (Marshall,
1995
). However, the observation that the regulation of ERK by
mitogens is indistinguishable between Raf-1-/- and
wild-type cells (Huser et al.,
2001
; Mikula et al.,
2001
) strongly suggests that the phenotype of the Raf-1
knockout cells is due to a deficiency in another as yet unknown
Raf-1-dependent signalling pathway.
In summary, the knockout studies yield three important and provocative
conclusions. First, Raf isozymes are not functionally redundant. Second, the
phenotype of the Raf-1-knockout mice cannot be explained by
alterations in ERK regulation alone. Third, the kinase activity of Raf-1 may
not be required for its biological functions. This latter point is especially
intriguing in light of the fact that KSR (kinase suppressor of Ras), the
closest relative of Raf kinases, seems to function more as a scaffolding
protein than as a kinase (Morrison,
2001).
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Raf and apoptosis |
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Salomoni and colleagues showed that the transformation of 32D
haematopoietic cells by the Bcr-Abl oncogene is crucially dependent on Raf-1
to prevent apoptosis (Salomoni et al.,
1998). Bcr-Abl induces the translocation of a fraction of
endogenous Raf-1 to mitochondria, and a transformation-defective Bcr-Abl
mutant can be complemented by the MasRaf-1 kinase domain. Again MasRaf-1
induced phosphorylation of BAD and its sequestration in the cytosol,
confirming that Raf-1 can stimulate BAD phosphorylation. A more detailed
dissection of the mechanism suggested that the anti-apoptotic function of
Bcr-Abl is executed through BAD-independent and BAD-dependent pathways
(Neshat et al., 2000
). Apart
from a requirement for phosphinositide 3-kinase kinase (PI-3K), the
BAD-independent pathway remains elusive. The BAD-dependent pathway also needs
PI-3K activity, but depends on the mitochondrial targeting of Raf-1 and is ERK
independent. It can be completely blocked by a kinase-negative MasRaf-1
mutant. These findings imply that in the anti-apoptotic pathway involving
mitochondrial Raf-1, Raf-1 functions downstream of PI-3K. A main executor of
PI-3K in the survival pathway is Akt, a kinase that can phosphorylate and
inactivate several inducers of apoptosis, including BAD
(Datta et al., 1999
).
Curiously, the antiapoptotic effect of Akt requires mitochondrial localisation
and PKC-dependent activation of Raf-1, whereas ERK activation is dispensable
(Majewski et al., 1999
). Given
that BAD is a direct substrate for Akt, but not Raf-1, these findings hint at
an intricate relationship between the Raf and Akt pathways in apoptosis
regulation.
There is also evidence that mitochondrion-located Raf-1 interacts with
Grb10 (Nantel et al., 1999),
an adapter protein that interacts with a variety of signalling molecules
(including IGF-1, insulin and EGF), Eph family and growth hormone receptors,
and kinases (including Bcr-Abl and JAK2). In Cos-7 and HeLa cells, Grb10 is
located mostly at mitochondria. Nantel et al. have suggested that a
Raf-1-Grb10 interaction at mitochondria could modify the activity of Raf-1.
Two recent studies provide a clue to the physiological significance of such an
interaction, showing that apoptosis protection by insulinlike growth factor 1
(IGF-1) is mediated by three pathways, all of which culminate in BAD
phosphorylation (Peruzzi et al.,
1999
). One pathway induces the mitochondrial translocation of
Raf-1 and Nedd4 (Peruzzi et al.,
2001
). Nedd4 is a target of caspases in apoptosis and also
interacts with Grb10. Nedd4 is also a ubiquitin ligase, and Peruzzi et al.
suggest that Raf-1-mediated translocation of BAD away from the mitochondria
may stimulate ubiquitination and consequent degradation of BAD by the
proteasome.
Another interesting anti-apoptotic function of Raf-1 was described by Chen
and colleagues (Chen et al.,
2001), who observed that Raf-1 can bind and inhibit apoptosis
signal-regulated kinase 1 (ASK-1). ASK-1 is an important mediator of apoptotic
signalling initiated by a variety of apoptotic stimuli; indeed, suppression of
ASK-1 signalling is likely to be a general mechanism of cell survival
(Tobiume et al., 2001
).
Curiously, a kinase-negative Raf-1 inhibits ASK-1-induced apoptosis with the
same efficiency as wild-type Raf-1. Such a kinase-independent mechanism to
counteract apoptosis could explain why the non-mitogen responsive Raf-1FF
mutant can rescue the Raf-1-knockout mice.
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Open questions and future prospects |
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The challenge will be to identify such substrates and prove that they are
phosphorylated by Raf-1 in cells. This endeavour is hampered by the low
specific activity of Raf-1 and the lack of a recognizable consensus motif for
Raf-1 phosphorylation (Force et al.,
1994). A combination of innovative genetics and biochemistry is
needed. For instance, src family kinases have been engineered by point
mutation to use substitute ATP analogues that are not accepted by other
kinases as phosphate donors (Shah et al.,
1997
). These ATP analogues do not change the substrate specificity
(Shah et al., 1997
), but allow
selective visualisation and identification of the direct substrates of the
engineered kinase. Clues are also expected from the proteomic analysis of the
composition of Raf-1 multiprotein signalling complexes. Because it has been
demonstrated that physical interaction between Raf-1 and MEK is essential for
MEK phosphorylation (Yeung et al.,
2000
), other substrates are likely to be found in association with
Raf-1. The availability of murine Raf-1-knockout cells in conjunction
with efficient gene transduction techniques allows us to employ cDNA libraries
in functional screens for suppressors of the apoptosis susceptibility of
Raf-1-/- cells. Such suppressors are strong candidates for
Raf-1 effectors in the apoptosis pathway. The Raf-1-/-
cells also provide an excellent system to validate potential effectors found
biochemically. The classical genetic model organism D. melanogaster
expresses a single Raf (D-Raf), which resembles B-Raf. In the widely studied
eye-development pathway, D-Raf is fully dependent on MEK. However, D-Raf has a
much broader function in embryonic pattern formation
(Radke et al., 2001
) and the
development of imaginal disks (Li et al.,
2000
). It will be interesting to find out whether all these
functions are mediated by MEK and ERK.
As an additional level of complexity, both genetic
(Huser et al., 2001) and
biochemical (Chen et al., 2001
)
studies raise the provocative possibility that Raf-1 may be able to signal
independently of its kinase activity. In this manner, Raf-1 could act as a
scaffold or regulatory subunit. Such a function is even more difficult to
prove. Genetic rescue studies in D. melanogaster indicate that kinase
activity is essential for D-Raf to function
(Radke et al., 2001
). This may
be different in mammalian cells, but clearly a kinase-negative
Raf-1-knock-in is needed to prove the point. The embryonic lethality
and the placenta defects of Raf-1-/- mice may obscure such
effects. Thus, meaningful answers will require conditional geneknockout
technology whereby the Raf-1 gene can be removed at will in selected
tissues and at a chosen point in time. In any case the Raf field, thought to
be mature, may be about to watch an old dog learn new tricks.
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