Báo cáo khoa học: Compartmentalized signalling: Ras proteins and signalling nanoclusters pdf

MINIREVIEW
Compartmentalized signalling: Ras proteins and
signalling nanoclusters
Jasminka Omerovic and Ian A. Prior
Physiological Laboratory, University of Liverpool, UK
Ras proteins are small GTPases that operate as molec-
ular switches controlling the relay of signals from
cell-surface receptors to a diverse array of intracellular
effector cascades responsible for regulating cell prolif-
eration, differentiation and apoptosis [1]. Hyperactivat-
ing mutations of Ras promote cell transformation and
contribute to oncogenesis in 15% of human cancer
patients (data obtained from the Wellcome Trust
Sanger Institute Cancer Genome Project http://www.
sanger.ac.uk/genetics/CGP). Although clearly impor-
tant from a human health perspective, Ras has also
recently emerged as a key model system for investigat-
ing how the outputs of signalling networks are
spatially regulated. This is because the three Ras
proto-oncogenes encoding four isoforms (H-Ras,
K-Ras4A, K-Ras4B and N-Ras) are almost identical
and all (except K-Ras4A) are ubiquitously expressed;
yet do not exhibit complete functional redundancy [2].
It is proposed that this is because of their differential
localization to the plasma membrane and intracellular
organelles which potentially allows each isoform to
come into contact with different pools of activators
and effectors.
Guanine nucleotide-exchange factors (GEFs) are
Ras activators that promote the exchange of GDP for
GTP; this results in a conformational switch in the

Ras tertiary structure revealing an effector interaction
site. This is reversed by the intrinsic GTPase activity
of Ras proteins which is stimulated by GTPase-activat-
ing proteins (GAPs; Fig. 1). In human cells, there are
at least nine GEFs and eight GAPs with varied modes
of recruitment and regulation [3]; potentially allowing
considerable fine control of the location and dura-
tion of Ras signalling. Raf and phosphatidylinositol
Keywords
compartmentalization; GTPase; isoforms;
MAP kinase; microdomains; nanoclusters;
Raf; Ras; receptor; scaffold
Correspondence
I. A. Prior, Physiological Laboratory, School
of Biomedical Sciences, University of
Liverpool, Crown St, Liverpool L69 3BX, UK
Fax: +44 151 794 4434
Tel: +44 151 794 5332
E-mail:
(Received 1 September 2008, revised 16
October 2008, accepted 24 November
2008)
doi:10.1111/j.1742-4658.2009.06928.x
Differential subcellular compartmentalization of the three main Ras
isoforms (H-Ras, N-Ras and K-Ras) is believed to underlie their biological
differences. Modulatable interactions between cellular membranes and Ras
C-terminal hypervariable region motifs determine differences in trafficking
and the relative proportions of each isoform in cell-surface signalling
nanoclusters and intracellular endoplasmic reticulum ⁄ Golgi, endosomal
and mitochondrial compartments. Ras regulators, effectors and scaffolds

are also differentially distributed, potentially enabling preferential coupling
to specific signalling pathways in each subcellular location. Here we sum-
marize the mechanisms underlying compartment-specific Ras signalling and
the outputs generated.
Abbreviations
ER, endoplasmic reticulum; GAP, GTPase activating protein; GEF, guanine nucleotide exchange factors; HVR, hypervariable region; PtdIns3K,
phosphatidylinositol 3-kinase; RTK, receptor tyrosine kinase.
FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS 1817
3-kinase (PtdIns3K) are the archetypal Ras effectors
although over 20 others have since been identified,
many of which are GEFs and GAPs for other
GTPases [4,5]. After 20 years of intensive investigation,
most of the potential regulators and effectors of Ras
have been identified and characterized; the focus of
many laboratories is now switching towards under-
standing context-dependent regulation of interactions
and outputs within this large signalling web.
Ras trafficking and localization
H-, K-, and N-Ras share almost complete sequence
homology between residues 1–165 encompassing all of
the effector and nucleotide-binding motifs (Fig. 1). The
isoforms are principally distinguished from each other
by the final 23–24 amino acid stretch, the hypervari-
able region (HVR), where there is < 15% sequence
similarity between Ras proteins. The HVR contains all
of the motifs responsible for membrane binding and
trafficking of each isoform. After synthesis on cytosolic
polysomes, Ras isoforms undergo a series of post-
translational modifications to increase their membrane
affinity. The cysteine in the C-terminal CAAX motif is

farnesylated before the -AAX is proteolytically cleaved
and the farnesyl–cysteine is carboxymethylated [5]. The
farnesyl group promotes weak interaction with the
endoplasmic reticulum (ER) which is stabilized by an
adjacent set of motifs that vary among Ras isoforms.
This consists of mono- [N-, K(A)-Ras] or di-palmitoy-
lation (H-Ras) of cysteines or a hexalysine polybasic
sequence [K(B)-Ras].
The second signal motif and farnesylated cysteine
shared by all Ras isoforms comprise the targeting
domain (Fig. 2); a minimal motif that when fused to
GFP displays a superficially equivalent localization as
the cognate full length H- and K(B)-Ras proteins [6,7].
Recent data revealed that a third signal motif is neces-
sary for the correct localization of mono-palmitoylated
N- and K(A)-Ras isoforms. GFP conjugated to the
minimal targeting domain of N-Ras is restricted to the
Golgi, whereas when the adjacent linker region of the
HVR is included the construct localizes to the cell sur-
face [7]. When the HVR of the palmitoylated Ras
isoforms is compared, there is  70% sequence homol-
ogy including a six-residue basic ⁄ hydrophobic patch at
the N-terminus of the HVR (Fig. 2). Mutating this
sequence increases the amount of endomembranous
localization observed, indicating that this motif
contributes to cell-surface localization. Similarly, for
K(A)-Ras, the basic patch adjacent to the palmitoyl
group is sufficient to ensure cell-surface localization [7].
The second signal motif also determines the traffick-
ing routes taken by H-, N- and K(B)-Ras to the

plasma membrane. K(B)-Ras traffics via a poorly char-
acterized Golgi-independent route that in yeast
requires class C vps proteins which are normally
required to regulate endosome fusion [8–10]. Unlike
other small G proteins such as Rabs and Rho proteins,
there has been no chaperone such as GDI character-
ized for cytosolic Ras trafficking. Although palmitoy-
lated Ras isoforms have also been characterized to
traffic via Golgi-independent routes in yeast and
adipocytes [11,12], in fibroblasts they traffic through
the conventional secretory pathway [8,9]. Interestingly,
the two palmitoyl groups of H-Ras are not equally
necessary for trafficking to the plasma membrane,
palmitoylated Cys181 supports cell-surface localization,
whereas mono-palmitoylation on Cys184 confines
H-Ras to the Golgi [13]. Although based on mutagene-
sis studies, these observations are highly relevant to
in vivo trafficking because Ras palmitoylation is labile.
The measured half life is 10–20% of that of the 21 h
half-life of N- and H-Ras, and these cycles of acylation
and deacylation are important regulators of global Ras
compartmentalization by allowing recycling back to
the Golgi complex for re-palmitoylation [14,15]. When
de-palmitoylation is inhibited, H-Ras is non-specifi-
cally localizes to all endomembranes [16].
Upon reaching the cell surface, each Ras isoform
occupies distinct signalling nanoclusters, as discussed
in detail below. Activation of upstream receptor tyro-
Fig. 1. Ras activation and effectors. Ras is
a molecular switch cycling between inactive

GDP- and active GTP-bound conformations
controlled by GEFs and GAPs. Active Ras
stimulates many pathways, notably kinases
such as the Raf–MAP kinase cascade and
the PtdIns3K–Akt pathway and GEFs for
other GTPases. These pathways regulate
many cell functions including cell prolifera-
tion, differentiation migration and apoptosis.
Compartmentalized Ras signalling J. Omerovic and I. A. Prior
1818 FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS
sine kinases (RTKs) results in the internalization of up
to 60% of the surface receptor population that may be
targeted for lysosomal degradation by c-Cbl-dependent
ubiquitination [17]. An open question is the extent to
which Ras isoforms co-traffic with the activated recep-
tor complexes to facilitate signalling from the surface
of early ⁄ recycling endosomes. A tiny fraction of
H- and N-Ras is ubiquitinated, and ubiquitin–Ras
chimeras showed enhanced endosomal localization [18].
However, Ras ubiquitination appears to be uncoupled
from receptor activation because it is not dependent on
the Ras activation state. Further work is needed to
identify the enzymes responsible for ubiquitin turnover
on Ras to provide tools for analysing the role of this
post-translational modification in Ras biology.
In summary, whereas the palmitoylated Ras
isoforms traffic from the ER–Golgi to the plasma
membrane and endosomes on membranes, the lability
of palmitoylation provides a mechanism for cytosolic
recycling back to the Golgi complex. The lack of

hydrophobic acyl groups on K(B)-Ras facilitates cyto-
solic shuttling between the cell surface and intracellular
organelles. Although all isoforms have been observed
on a variety of organelles, the key difference is the
relative proportions of each isoform in each location.
Although the cell surface represents the prime location
for all isoforms, in many cell types the relative contri-
bution to the endomembranous component is typically
N ‡ H, K(A) > K(B)-Ras. When combined with
observed differences in relative abundance N- and
K(B)-Ras are the most abundant isoforms in most cell
lines [2], a broad conclusion is that the plasma mem-
brane is likely to be more coupled to K-Ras signalling
whereas the endomembrane is more coupled to N-Ras
signalling pathways.
Plasma membrane signalling
nanoclusters
Although the plasma membrane has traditionally been
viewed as a homogeneous organelle, in recent years
this model has been updated to incorporate a dense
mosaic of signalling domains [19]. These incorporate
nanoclusters of proteins and lipids that potentially
facilitate signalling by selectively concentrating the
components of effector cascades. Several laboratories,
using a combination of fractionation, chemical pertur-
bation and advanced microscopy techniques, have
A
B
Fig. 2. The Ras HVR and subcellular Ras
trafficking. The C-terminal HVR (A) contains

membrane-trafficking motifs that regulate
the trafficking and nanocluster association
of Ras isoforms (B). Membrane interactions
can be modulated to allow recycling to the
ER ⁄ Golgi or targeting to alternative endo-
membranous locations.
J. Omerovic and I. A. Prior Compartmentalized Ras signalling
FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS 1819
characterized the association of Ras isoforms with dif-
ferent cell-surface nanoclusters that is driven by inter-
actions of their HVRs with membrane proteins and
lipids [20–22]. These nanoclusters are small (< 15 nm
diameter) and short-lived (t
1 ⁄ 2
£ 0.4 s) [23].
K(B)-Ras operates from actin-dependent, choles-
terol-independent nanoclusters that are stabilized by
galectin-3 and distinct from the H- and N-Ras signal-
ling nanoclusters [24,25]. A beneficial feature of the
K-Ras polybasic domain is its potential capacity to
aggregate the highly charged anionic lipid phosphati-
dylinositol-4,5-bisphosphate [26]. Phosphatidylinositol-
4,5-bisphosphate is the substrate for PtdIns3K, a key
Ras effector. In addition to 2D lateral diffusion, K-Ras
exhibits significant cytosolic exchange due to a mem-
brane residency half-life of only a few minutes [27].
This is modulated in vivo by disruption of K(B)-Ras
electrostatic interactions with the plasma membrane via
protein kinase C-dependent phosphorylation of Ser181
resulting in translocation to mitochondria [28].

The palmitoyl groups of H-Ras specify localization
in cholesterol-dependent nanoclusters, however upon
activation, H-Ras translocates into new cholesterol-
independent nanoclusters [13,22,29]. The activated
H-Ras nanoclusters are stabilized by galectin-1 interac-
tions with the C-terminal farnesyl group of the HVR
[22,30,31]. By contrast, N-Ras moves in the opposite
direction when activated [13]; although at present it is
unclear whether the H- and N-Ras nanoclusters are
actually identical or share a limited number of diag-
nostic markers.
Recent structural analysis of the interaction of the
H-Ras HVR with membranes gave an insight into how
Ras activation might translate into differential nano-
cluster association. Conformational changes in the
N-terminal switch regions of Ras re-orientate the
protein’s interaction with the plasma membrane by
regulating the membrane interactions of basic residues
located in the HVR (GDP-bound Ras) and a4 helix
(GTP-bound Ras) [32]. In effect, the changed mem-
brane interactions ‘tip over’ the main body of active
Ras, potentially facilitating effector and galectin-1
interactions [33]. Although this will promote interac-
tions with the active-Ras nanocluster components, the
GTP-dependent loss of affinity with the cholesterol-
dependent nanoclusters is likely to be due to modulat-
ing palmitoyl–membrane interactions [12,33,34].
In addition to characterizing the dynamic associa-
tion of Ras with different nanoclusters, the functional
consequences of these interactions have been analysed

in silico and in vivo. Importantly, if nanocluster forma-
tion is inhibited, output is reduced to just 3% of
maximal signalling [35]. Nanoclusters are highly
sensitive to low signalling inputs, giving them a switch-
like behaviour where, apart from a very narrow win-
dow, a maximal output is generated over a wide input
range [36]. The high abundance but very short life
span of Ras signalling domains means that although
each nanocluster effectively behaves digitally, the pop-
ulation as a whole generates a graded response in
which output is proportional to input [35,36].
Given that differential localization of Ras isoforms
to distinct signalling nanoclusters is believed to underlie
the lack of functional redundancy between Ras
isoforms, a key prediction is that effectors will have
different affinities for each type of nanocluster. Recent
high-resolution microscopy analysis revealed that
although both H- and K-Ras can recruit Raf to the
plasma membrane, the activated K-Ras nanoclusters
retain Raf, whereas activated H-Ras–Raf interactions
are transient [24]. These data fit comparative signalling
analysis that showed K-Ras to be a proportionally
better activator of the Raf-MAPK cascade than H-Ras
[37].
In summary, Ras activation-dependent conforma-
tional changes in HVR–membrane association result in
rapid redistribution to specialized signalling nanoclus-
ters stabilized by galectin proteins. The biophysical
and biochemical properties of different types of nano-
cluster are believed to regulate the variety and time

course of effector interactions allowing different
outputs.
Compartmentalization of accessory
proteins and organellar signalling
There are few organelles that Ras cannot access; an
important question is whether activators, regulators
and effectors show similarly compartmentalized
distributions. Figure 3 summarizes the locations occu-
pied by regulators of Ras signalling. Although all
organelles have at least one Ras regulator in residence,
the ER ⁄ Golgi and endosomes appear to be hotspots
for controlling intracellular Ras signalling. The
evidence for this organellar signalling and its func-
tional relevance are discussed in later paragraphs, but
at this point it is worth highlighting the compartmen-
talization of scaffold proteins that are key regulators
of Ras–Raf–MAP kinase and Ras–PtdIns3K–Akt
signalling.
Compartmentalized scaffolds
Scaffold proteins contribute to the control of signalling
kinetics, cross-talk between pathways and prevention
of activation of physiologically irrelevant signals. They
Compartmentalized Ras signalling J. Omerovic and I. A. Prior
1820 FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS
function by pre-assembling components of signalling
cascades in readiness for recruitment to the site of
receptor activation or on intracellular organelles.
Several Ras pathway scaffolds have been identified,
most of which coordinate Raf–MEK–ERK (Raf–MAP
kinase) signalling from different locations. These

include KSR, AF6 and IQGAP-1 (plasma membrane),
b-arrestin (plasma membrane, endosomes, nucleus),
p14-MP1 (late endosomes) and Sef (Golgi) that typi-
cally undergo regulated redistribution from the cytosol
to these locations [38–43].
A key function of scaffolds is to regulate the kinetics
of signalling, an example of this can been seen when the
late endosomal MP1 scaffold adaptor protein p14 is
knocked-down using RNAi resulting in mislocalization
of MP1 to the cytosol [44]. Acute MAP kinase activa-
tion that occurs at the plasma membrane was unper-
turbed; however, MAP kinase activation following
prolonged (10–30 min) epidermal growth factor stimula-
tion was significantly reduced. This fits with the model
in which prolonged growth factor stimulation results in
receptor endocytosis and signalling from endosomes
and suggests that activated receptors engage new signal-
ling complexes resident within the endocytic system.
A final scaffold, Appl1, modulates the Akt signalling
pathway and operates from a subpopulation of signal-
ling early endosomes [45,46]. Akt has many substrates,
however endosomal interaction of Appl1 with Akt speci-
fies activation of the GSK3 cell survival pathway in
zebrafish [46]. Importantly, when Appl1 was mis-tar-
geted to other cellular locations this pathway could not
be engaged indicating highly context-dependent signal-
ling outputs. Although identified as an effector for the
small GTPase Rab5, it will be interesting to see whether
endosomal Ras can also activate this signalling complex.
Endosomal signalling

The initiation points of most signalling cascades are
the cell-surface localized RTKs and G-protein-coupled
Fig. 3. Compartmentalization of Ras GEFs,
GAPs and scaffolds. Ras has been localized
to and signals from both cell surface and
endomembrane platforms. Ras regulators
are also differentially localized, many of the
proteins illustrated exhibit regulated
recruitment from the cytosol. See text and
Omerovic et al. [5] for primary references.
J. Omerovic and I. A. Prior Compartmentalized Ras signalling
FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS 1821
receptors. A proportion of activated receptors are
internalized and sorted through the endosome for
recycling back to the surface or delivery to lyso-
somes for degradation. Because the tails of early
endosomal receptors are still exposed to the cytosol
they are also potentially able to initiate signalling
cascades if still active. This observation, together with
the localization of the Ras GEF, mSos, the
activated Raf-MAP kinase cascade and scaffold
proteins MP1 and APPL on endosomes, suggests that
they are a viable platform for RTK–Ras signalling
[47,48].
Several lines of evidence support this idea; first,
selective activation of endosomal epidermal growth
factor receptor or platelet-derived growth factor
receptor stimulated cell proliferation and cell survival,
indicating that the cell-signalling machinery on
endosomes is functional and capable of generating a

biologically relevant output [49,50]. Activation of the
full complement of epidermal growth factor receptor
effectors requires receptor endocytosis [51]; and
inhibition of endocytosis attenuates growth factor
stimulated H- and N-Ras but not K-Ras activation of
the Raf–MAP kinase pathway [2].
Importantly, whereas acute growth factor stimu-
lated MAP kinase activation is primarily believed to
emanate from the plasma membrane, endosomes
support sustained MAP kinase activation [52,53].
The ability to modulate the kinetics of MAP kinase
activation has cell phenotypic consequences; for
example, in PC12 cells this results in a switch from
proliferation to differentiation [54]. In addition to
facilitating endosomal Ras–MAP kinase signalling,
the scaffold protein MP-1 also regulates coupling to
specific upstream stimuli. This is controlled by the
MP-1 interaction partner MORG1 that facilitates
lysophosphatidic acid, phorbol ester or serum-depen-
dent but not growth factor-dependent MAP kinase
activation [55].
The evidence for endosomal Ras pathway signalling
conferring signal specificity distinct from that gener-
ated from other subcellular signalling platforms is
currently limited. However, a clear example is provided
by studies of the endosomal Akt scaffold Appl1.
During zebrafish development, loss of Appl1 results in
apoptosis due to the loss of the Akt–GSK–3b survival
signal [46]. In this case, endosomal Akt signalling is an
important driver of cell survival in tissues where Appl1

is highly expressed. Presumably the abundance and
localization of other co-factors will also be modulated
in these cells to ensure that the endosomal microenvi-
ronment is favoured for regulating anti-apoptotic
signalling.
ER
⁄
Golgi signalling
Studies of ER ⁄ Golgi signalling provide some of the
best evidence for isoform- and compartment-specific
Ras signalling having phenotypic consequences. Ras
GEFs and GAPs, the Ras effector Rain1 and the scaf-
fold Sef have all been localized to the Golgi (Fig. 3)
[5,43,56]. Although endogenous Golgi Ras activation
has largely proved difficult to visualize [57], an elegant
bystander fluorescence technique revealed that delayed
and sustained endogenous Golgi Ras activation could
be stimulated by growth factors [58].
Ras activation on the Golgi is mediated by calcium ⁄
diacylglycerol (Ca
2+
⁄ DAG)-dependent stimulation of
RasGRP1 [59], and on the ER by lysophosphatidic
acid-dependent stimulation of RasGRF1 and Ras-
GRF2 [60]. Although most studies have relied on
overexpression of Ras or Ras modulators to study
compartmentalized Ras activation and signalling,
recent work by Philips and colleagues revealed the
different wiring involved in activating Ras on the
cell surface and Golgi in T cells [61]. T-cell receptor

activation stimulates Golgi-Ras, however, co-stimula-
tion with the integrin LFA-1 also activated Ras
on the plasma membrane by raising the local
concentration of DAG and phosphatidic acid to also
activate cell surface localized RasGRP1 [61].
Therefore, whereas Ras and the GEF RasGRP1 are
found in two compartments, their organelle-specific
activation can be precisely regulated by different
extracellular ligands inducing localized concentrations
of second messengers.
The difficulty in observing Golgi Ras activation
compared with plasma membrane Ras activation
could lead to the conclusion that it represents a minor
inconsequential component of global Ras signalling.
However, the cell is unlikely to waste resources on
redundant signalling pathways. Moreover, strong
evidence for physiologically relevant Golgi Ras signal-
ling recently emerged in studies of thymocyte selection
which revealed that the endogenous Golgi Ras pool is
necessary for positive thymocyte selection whereas the
plasma membrane specifies negative selection [62].
Finally, the Golgi also interestingly provides a site
for negative regulation of Ras signalling and stimula-
tion of cell proliferation and differentiation via the
action of RKTG. This is a seven transmembrane
protein that sequesters cytosolic Raf to the Golgi,
competitively inhibiting interaction with activated Ras
and the MAP kinase cascade [63]. On a more general
point, it is also tempting to speculate that negative
regulation of Ras pathways via phosphatases may also

be highly compartmentalized.
Compartmentalized Ras signalling J. Omerovic and I. A. Prior
1822 FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS
Other compartmentalized Ras signalling
Although the ER ⁄ Golgi and endosomes have been
strongly identified as sites of Ras signalling, both the
mitochondria and nucleus have also been shown to
contain specific Ras isoforms. An H-Ras splice variant
lacking the C-terminal HVR, p19
ras
localizes to the
nucleus and cytosol where it regulates the activity of
the tumour suppressor p73 [64]. Mitochondrial Ras
signalling by N-Ras and K(B)-Ras has also been
characterized. Phosphorylation of Ser181 within the
K(B)-Ras HVR destabilizes the electrostatic interac-
tions of the polybasic domain with the plasma
membrane and promotes redistribution to mitochon-
dria where it induces Bcl-X
L
-dependent apoptosis [28].
N-Ras and K-Ras also play a role in maintaining
normal mitochondrial morphology and function [65].
Conclusion
Compartmentalized Ras signalling enables a spectrum
of outputs to be tuned from a minimal core-signalling
machinery by modulating access to a variety of activat-
ing and effector proteins. These are evolutionarily
conserved mechanisms because in yeast one Ras
protein controls two outputs related to mating or

morphology via signalling from the plasma membrane
or endomembrane respectively [66]. In mammalian
systems we are still at an early stage in the identifica-
tion and validation of phenotypes regulated by local-
ized Ras signalling. In future the major challenge
remains to understand what output each compartment
is capable of controlling and how this is achieved. A
key problem lies in the tools available to investigate
this that typically utilize overexpression and therefore
potential distortion of information flow through
signalling pathways. Also, differences in compartmen-
talized signalling are likely to mostly involve the same
pathways stimulated to different extents in different
locations; i.e. only by looking across a wide range of
potential pathways are we likely to understand what is
really happening. Novel large-scale quantitative screen-
ing approaches and new tools that locally stimulate or
inhibit the endogenous Ras pathway represent ideals
that many groups are working towards combining to
try to understand this important phenomenon.
Acknowledgements
IAP is a Royal Society University Research Fellow;
work in our laboratory is funded by Cancer Research
UK, the Wellcome Trust and the North West Cancer
Research Fund.
References
1 Karnoub AE & Weinberg RA (2008) Ras oncogenes:
split personalities. Nat Rev Mol Cell Biol 9, 517–531.
2 Omerovic J, Hammond DE, Clague MJ & Prior IA
(2008) Ras isoform abundance and signalling in human

cancer cell lines. Oncogene 27 , 2754–2762.
3 Mitin N, Rossman KL & Der CJ (2005) Signaling inter-
play in Ras superfamily function. Curr Biol 15, 563–574.
4 Goldfinger LE, Ptak C, Jeffery ED, Shabanowitz J,
Han J, Haling JR, Sherman NE, Fox JW, Hunt DF &
Ginsberg MH (2007) An experimentally derived data-
base of candidate Ras-interacting proteins. J Proteome
Res 6, 1806–1811.
5 Omerovic J, Laude AJ & Prior IA (2007) Ras proteins:
paradigms for compartmentalised and isoform-specific
signalling. Cell Mol Life Sci 64, 2575–2589.
6 Hancock JF, Paterson H & Marshall CJ (1990) A poly-
basic domain or palmitoylation is required in addition
to the CAAX motif to localize p21ras to the plasma
membrane. Cell 63, 133–139.
7 Laude AJ & Prior IA (2008) Palmitoylation and locali-
sation of RAS isoforms are modulated by the hyper-
variable linker domain. J Cell Sci 121, 421–427.
8 Apolloni A, Prior IA, Lindsay M, Parton RG &
Hancock JF (2000) H-ras but not K-ras traffics to the
plasma membrane through the exocytic pathway. Mol
Cell Biol 20, 2475–2487.
9 Choy E, Chiu VK, Silletti J, Feoktisitov M, Morimoto
T, Michaelson D, Ivanov IE & Philips MR (1999)
Endomembrane trafficking of Ras: the CAAX motif
targets proteins to the ER and Golgi. Cell 98, 69–80.
10 Wang G & Deschenes RJ (2006) Plasma membrane
localization of Ras requires class C vps proteins and
functional mitochondria in Saccharomyces cerevisiae.
Mol Cell Biol 26, 3243–3255.

11 Dong X, Mitchell DA, Lobo S, Zhao L, Bartels DJ &
Deschenes RJ (2003) Palmitoylation and plasma mem-
brane localization of Ras2p by a nonclassical trafficking
pathway in Saccharomyces cerevisiae. Mol Cell Biol 23,
6574–6584.
12 Watson RT, Furukawa M, Chiang SH, Boeglin D,
Kanzaki M, Saltiel AR & Pessin JE (2003) The exocytic
trafficking of TC10 occurs through both classical and
nonclassical secretory transport pathways in 3T3L1
adipocytes. Mol Cell Biol 23, 961–974.
13 Roy S, Plowman S, Rotblat B, Prior IA, Muncke C,
Grainger S, Parton RG, Henis Y, Kloog Y & Hancock
JF (2005) Individual palmitoyl residues serve distinct
roles in H-ras trafficking, microlocalization and
signaling. Mol Cell Biol 25, 6722–6733.
14 Baker TL, Zheng H, Walker J, Coloff JL & Buss JE
(2003) Distinct rates of palmitate turnover on
membrane bound cellular and oncogenic H-ras. J Biol
Chem 278, 19292–19300.
J. Omerovic and I. A. Prior Compartmentalized Ras signalling
FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS 1823
15 Magee AI, Gutierrez L, McKay IA, Marshall CJ &
Hall A (1987) Dynamic fatty acylation of p21
N-ras
.
EMBO J 6, 3353–3357.
16 Rocks O, Peyker A, Kahms M, Verveer PJ, Koerner C,
Lumbierres M, Kuhlmann J, Waldmann H, Wittingho-
fer A & Bastiaens PI (2005) An acylation cycle regulates
localization and activity of palmitoylated Ras isoforms.

Science 307, 1746–1752.
17 Levkowitz G, Waterman H, Zamir E, Kam Z, Oved S,
Langdon WY, Beguinot L, Geiger B & Yarden Y
(1998) c-Cbl ⁄ Sli-1 regulates endocytic sorting and ubiq-
uitination of the epidermal growth factor receptor. Gene
Dev 12, 3663–3674.
18 Jura N, Scotto-Lavino E, Sobczyk A & Bar-Sagi D
(2006) Differential modification of Ras proteins by
ubiquitination. Mol Cell 21, 679–687.
19 Laude AJ & Prior IA (2004) Plasma membrane microd-
omains: organization, function and trafficking. Mol
Membr Biol 21, 193–205.
20 Murakoshi H, Iino R, Kobayashi T, Fujiwara T, Ohshi-
ma C, Yoshimura A & Kusumi A (2004) Single-mole-
cule imaging analysis of Ras activation in living cells.
Proc Natl Acad Sci USA 101, 7317–7322.
21 Niv H, Gutman O, Kloog Y & Henis Y (2002) Acti-
vated K-ras and H-ras display different interactions
with saturable nonraft sites at the surface of live cells.
J Cell Biol 157, 865–872.
22 Prior IA, Muncke C, Parton RG & Hancock JF (2003)
Direct visualization of Ras proteins in spatially distinct
cell surface microdomains. J Cell Biol 160, 165–170.
23 Nicolau DV, Burrage K, Parton RG & Hancock JF
(2006) Identifying optimal lipid raft characteristics
required to promote nanoscale protein–protein interac-
tions on the plasma membrane. Mol Cell Biol 26, 313–
323.
24 Plowman SJ, Ariotti N, Goodall A, Parton RG &
Hancock JF (2008) Electrostatic interactions positively

regulate K-ras nanocluster formation and function. Mol
Cell Biol 28, 4377–4385.
25 Prior IA, Parton RG & Hancock JF (2003) Observing
cell surface signalling domains using electron micros-
copy. Science’s STKE 2003, 19.
26 McLaughlin S, Hangyas-Mihalyne G, Zaitseva G &
Golebiewska U (2005) Reversible – through calmodulin
– interactions between basic residues on proteins and
acidic lipids in the plasma membrane. Biochem Soc
Symp 75, 189–198.
27 Silvius JR, Bhagatji P, Leventis R & Terrone D (2006)
K-ras4B and prenylated proteins lacking ‘second
signals’ associate dynamically with cellular membranes.
Mol Biol Cell 17, 192–202.
28 Bivona TG, Quatela SE, Bodemann BO, Ahearn IO,
Soskis MJ, Mor A, Miura J, Wiener HH, Wright L,
Saba SG et al. (2006) PKC regulates a farnesyl-electro-
static switch on K-ras that promotes its association with
Bcl-XL on mitochondria and induces apoptosis. Mol
Cell 21, 481–494.
29 Prior IA, Harding A, Yan J, Sluimer J, Parton RG &
Hancock JF (2001) GTP-dependent segregation of
H-ras from lipid rafts is required for biological activity.
Nat Cell Biol 3, 368–375.
30 Belanis L, Plowman SJ, Rotblat B, Hancock JF &
Kloog Y (2008) Galectin-1 is a novel structural
component and a major regulator of H-ras
nanoclusters. Mol Biol Cell 19, 1404–1414.
31 Paz A, Haklai R, Elad-Sfadia G, Ballan E & Kloog Y
(2001) Galectin-1 binds oncogenic H-ras to mediate Ras

membrane anchorage and cell transformation. Oncogene
20, 7486–7493.
32 Abankwa D, Hanzal-Bayer M, Ariotti N, Plowman SJ,
Gorfe AA, Parton RG, McCammon JA & Hancock JF
(2008) A novel switch region regulates H-ras membrane
orientation and signal output.
EMBO J 27, 727–735.
33 Gorfe AA, Hanzal-Bayer M, Abankwa D, Hancock JF
& McCammon JA (2007) Structure and dynamics of
the full-length lipid-modified H-ras protein in a 1,2-di-
myristoylglycero-3-phosphocholine bilayer. J Med Chem
50, 674–684.
34 Vogel A, Tan K-T, Waldmann H, Feller S, Brown M &
Huster D (2007) Flexibility of Ras lipid modifications
studied by 2H solid-state NMR and molecular
dynamics simulations. Biophys J 93, 2697–2712.
35 Tian T, Harding A, Inder K, Plowman S, Parton RG &
Hancock JF (2007) Plasma membrane nanoswitches
generate high-fidelity Ras signal transduction. Nat Cell
Biol 9, 905–914.
36 Harding A & Hancock JF (2008) Ras nanoclusters:
combining digital and analog signaling. Cell Cycle 7,
127–134.
37 Yan J, Roy S, Apolloni A, Lane A & Hancock JF
(1998) Ras isoforms vary in their ability to activate
Raf-1 and phosphoinositide 3-kinase. J Biol Chem 273,
24052–24056.
38 Therrien M, Michaud NR, Rubin GM & Morrison DK
(1996) KSR modulates signal propagation within the
MAPK cascade. Genes Dev 10 , 2684–2695.

39 Yamamoto T, Harada N, Kano K, Taya S, Canaani E,
Matsuura Y, Mizoguchi A, Ide C & Kaibuchi K (1997)
The Ras target AF-6 interacts with ZO-1 and serves as
a peripheral component of tight junctions in epithelial
cells. J Cell Biol 139, 785–795.
40 Hart MJ, Callow MG, Souza B & Polakis P (1996)
IQGAP1, a calmodulin-binding protein with a rasGAP-
related domain, is a potential effector for cdc42Hs.
EMBO J 15, 2997–3005.
41 Luttrell LM, Roudabush FL, Choy EW, Miller WE,
Field ME, Pierce KL & Lefkowitz RJ (2001) Activation
and targeting of extracellular signal-regulated kinases
by beta-arrestin scaffolds. Proc Natl Acad Sci USA 98,
2449–2454.
Compartmentalized Ras signalling J. Omerovic and I. A. Prior
1824 FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS
42 Wunderlich W, Fialka I, Teis D, Alpi A, Pfeifer A,
Parton RG, Lottspeich F & Huber LA (2001) A novel
14-kilodalton protein interacts with the mitogen-acti-
vated protein kinase scaffold mp1 on a late endoso-
mal ⁄ lysosomal compartment. J Cell Biol 152, 765–776.
43 Torii S, Kusakabe M, Yamamoto T, Maekawa M
& Nishida E (2004) Sef is a spatial regulator for
Ras ⁄ MAP kinase signaling. Dev Cell 7, 33–44.
44 Teis D, Wunderlich W & Huber LA (2002) Localization
of the MP1-MAPK scaffold complex to endosomes is
mediated by p14 and required for signal transduction.
Dev Cell 3, 803–814.
45 Miaczynska M, Christoforidis S, Giner A, Shevchenko
A, Uttenweiler-Joseph S, Habermann B, Wilm M, Par-

ton RG & Zerial M (2004) APPL proteins link Rab5 to
nuclear signal transduction via an endosomal compart-
ment. Cell 116, 445–456.
46 Schenck A, Goto-Silva L, Collinet C, Rhinn M, Giner A,
Habermann B, Brand M & Zerial M (2008) The endoso-
mal protein Appl1 mediates Akt substrate specificity and
cell survival in vertebrate development. Cell 133, 486–497.
47 Di Guglielmo GM, Baass PC, Ou WJ, Posner BI &
Bergeron JJ (1994) Compartmentalization of SHC,
GRB2 and mSOS, and hyperphosphorylation of Raf-1
by EGF but not insulin in liver parenchyma. EMBO J
13, 4269–4277.
48 Pol A, Calvo M & Enrich C (1998) Isolated endosomes
from quiescent rat liver contain the signal transduction
machinery. Differential distribution of activated Raf-1
and Mek in the endocytic compartment. FEBS Lett
441, 34–38.
49 Pennock S & Wang Z (2003) Stimulation of cell prolif-
eration by endosomal epidermal growth factor receptor
as revealed through two distinct phases of signaling.
Mol Cell Biol 23, 5803–5815.
50 Wang Y, Pennock SD, Chen X, Kazlauskas A & Wang
Z (2004) Platelet-derived growth factor receptor-medi-
ated signal transduction from endosomes. J Biol Chem
279, 8038–8046.
51 Vieira AV, Lamaze C & Schmid SL (1996) Control of
EGF receptor signaling by clathrin-mediated endocyto-
sis. Science 274, 2086–2089.
52 Oksvold MP, Skarpen E, Wierod L, Paulsen RE &
Huitfeldt HS (2001) Re-localization of activated EGF

receptor and its signal transducers to multivesicular
compartments downstream of early endosomes in
response to EGF. Eur J Cell Biol 80, 285–294.
53 Taub N, Teis D, Ebner HL, Hess MW & Huber LA
(2007) Late endosomal traffic of the epidermal growth
factor receptor ensures spatial and temporal fidelity of
mitogen-activated protein kinase signaling. Mol Biol
Cell 18, 4698–4710.
54 Marshall C (1995) Specificity of receptor kinase signal-
ing: transient versus sustained extracellular signal-regu-
lated kinase activation. Cell 80 , 179–185.
55 Vomastek T, Schaeffer HJ, Tarcsafalvi A, Smolkin ME,
Bissonette EA & Weber MJ (2004) Modular construc-
tion of a signaling scaffold: MORG1 interacts with
components of the ERK cascade and links ERK signal-
ing to specific agonists. Proc Natl Acad Sci USA 101,
6981–6986.
56 Mitin NY, Ramocki MB, Zullo AJ, Der CJ, Konieczny
SF & Taparowsky EJ (2004) Identification and charac-
terization of rain, a novel Ras-interacting protein with
unique subcellular localization. J Biol Chem 279,
22353–22361.
57 Augsten M, Pusch R, Biskup C, Rennert K, Wittig U,
Beyer K, Blume A, Wetzker R, Friedrich K & Rubio I
(2006) Live-cell imaging of endogenous Ras-GTP
illustrates predominant Ras activation at the plasma
membrane. EMBO Rep 7, 46–51.
58 Chiu VK, Bivona T, Hach A, Sajous JB, Silletti J,
Wiener H, Johnson RL, Cox AD & Philips MR (2002)
Ras signalling on the endoplasmic reticulum and the

Golgi. Nat Cell Biol 4, 343–350.
59 Bivona TG, Perez de Castro I, Ahearn IM, Grana TM,
Chiu V, Lockyer PJ, Cullen PJ, Pellicer A, Cox AD &
Philips MR (2003) Phospholipase Cc activates Ras on
the Golgi apparatus by means of Ras-GRP1. Nature
424
, 694–698.
60 Arozarena I, Matallanas D, Berciano MT, Sanz-
Moreno V, Calvo F, Munoz MT, Egea G, Lafarga M
& Crespo P (2004) Activation of H-ras in the endoplas-
mic reticulum by the rasGRF family of guanine nucleo-
tide exchange factors. Mol Cell Biol 24, 1516–1530.
61 Mor A, Campi G, Du G, Zheng Y, Foster DA, Dustin
ML & Philips MR (2007) The lymphocyte function-
associated antigen-1 receptor costimulates plasma mem-
brane Ras via phospholipase D2. Nat Cell Biol 9, 713–
719.
62 Daniels MA, Teixeiro E, Gill J, Hausmann B, Roubaty
D, Holmberg K, Werlen G, Hollander GA, Gascoigne
NR & Palmer E (2006) Thymic selection threshold
defined by compartmentalization of Ras ⁄ MAPK signal-
ling. Nature 444, 724–729.
63 Feng L, Xie X, Ding Q, Luo X, He J, Fan F, Liu W,
Wang Z & Chen Y (2007) Spatial regulation of Raf
kinase signaling by RKTG. Proc Natl Acad Sci USA
104, 14348–14353.
64 Jeong MH, Bae J, Kim WH, Yoo SM, Kim JW, Song
PI & Choi KH (2006) p19ras interacts with and acti-
vates p73 by involving the MDM2 protein. J Biol Chem
281, 8707–8715.

65 Wolfman JC, Planchon SM, Liao J & Wolfman A
(2006) Structural and functional consequences of c-N-
ras constitutively associated with intact mitochondria.
Biochim Biophys Acta 1763, 1108–1124.
66 Onken B, Wiener H, Philips MR & Chang EC (2006)
Compartmentalized signaling of Ras in fission yeast.
Proc Natl Acad Sci USA 103, 9045–9050.
J. Omerovic and I. A. Prior Compartmentalized Ras signalling
FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS 1825