1. Biosci. Rep. (2011) / 31 / 231–244 (Printed in Great Britain) / doi 10.1042/BSR20100094
Signalling to actin: role of C3G, a multitasking
guanine-nucleotide-exchange factor
Vegesna RADHA1
, Aninda MITRA, Kunal DAYMA and Kotagiri SASIKUMAR
Centre for Cellular and Molecular Biology (CSIR), Uppal Road, Hyderabad 500 007, India
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Synopsis
C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of
molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways
triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions
in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion,
migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated
to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels
are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation.
In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can
therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes
our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin
cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a
potential therapeutic target.
Key words: actin cytoskeleton, C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor), differentiation,
embryonic development, GTPase, signalling
INTRODUCTION
The ability of small GTPases to switch between active GTP and
inactive GDP-bound states enables them to function as hubs in
signalling pathways. A small number of GTPases can respond
to a multitude of signals and also activate multiple downstream
effectors, resulting in diverse and specific responses. GTPases
are activated by GEFs (guanine-nucleotide-exchange factors) and
inhibited by GTPase-activating proteins that accelarate GTP hy-
drolysis [1]. Most upstream signals target GEFs to act on specific
GTPases and therefore GEFs serve to link activated receptors
to downstream signalling cascades and provide signalling spe-
cificity [2]. GEFs are classified on the basis of which family of
GTPases they act on, namely Ras GEFs, Rho GEFs etc. A com-
mon feature of Ras GEFs is the presence of a CDC25 homology
domain that helps in catalysis along with an REM (Ras exchanger
motif). In addition, Rap GEFs have multiple modular domains
that aid in protein and lipid interactions, and in their regulation
[3]. C3G (Crk SH3-domain-binding guanine nucleotide-releasing
factor) was the first Rap GEF identified with a domain showing
............................................................................................................................................................................................................................................................................................................
Abbreviations used: 3D, three-dimensional: AJ, adherence junction; C3G, Crk SH3-domain-binding guanine nucleotide releasing factor; CML, chronic myelogenous leukaemia; DC3G,
Drosophila C3G; EGF, epidermal growth factor; ERK, extracellular-signal-regulated kinase; GEF, guanine-nucleotide-exchange factor; JNK, c-Jun N-terminal kinase; MAPK,
mitogen-activated protein kinase; NB, neuroblastoma; NGF, nerve growth factor; pC3G, Y504 (Tyr504)-phosphorylated C3G; PDGF, platelet-derived growth factor; PV, pervanadate; REM,
Ras exchanger motif; SFK, Src family kinase; siRNA, small interfering RNA; T2D, Type 2 diabetes.
1 To whom correspondence should be addressed (e-mail: vradha@ccmb.res.in)
homology with the yeast CDC25, and was originally isolated as
an interacting partner of CRK (cellular homologue of the v-Crk
oncoprotein) [4,5]. Alternate names of C3G are Rap GEF1, GRF2
and DKFZ p781P1719. C3G has the catalytic domain along with
REM at the extreme C-terminus and lacks modular protein in-
teraction domains found in most other Rap GEFs (Figure 1A).
Domains found in Rap GEFs generally are DEP (disheveled-
EGL-10-pleckstrin domain), cNB-L (cyclic nucleotide-binding
domain-like) and PDZ (PSD-95/Dig/ZO-1). These are shown in
Figure 1(B) along with the primary structure of C3G. In humans,
the two primary protein products of about 140 kDa are over 1000
residues in length and have multiple proline-rich sequences in
the central region through which they interact with proteins con-
taining SH3 domains. Crk, Hck, c-Abl and Cas are molecules
known to interact directly with the central domain of C3G [4–8].
Residues in the N-terminus are responsible for interaction with
E-cadherin, indicating that the N-terminal sequences may also
aid in protein interaction [9]. The two isoforms arise due to al-
ternate splicing, and primarily differ in the N-terminus where
three amino acids of isoform a are replaced by 21 amino acids in
isoform b (Figures 2A and 2B).
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2. V. Radha and others
Figure 1 Domain organization of C3G and comparison with other Rap GEFs
(A) Schematic diagram showing the domain organization of C3G protein. The C-terminal catalytic domain of C3G is
homologous with CDC25 and is responsible for target G protein activation. The N-terminal region has a domain that
interacts with E-cadherin. The central protein interaction domain (also known as Crk-binding region, CBR) contains multiple
proline-rich sequences that bind SH3 domains of Crk, Cas, c-Abl and Hck. The non-catalytic sequences negatively regulate
the catalytic activity of C3G. (B) Domain organization of different Rap GEFs. C3G is a unique Rap GEF member that
lacks modular protein interaction domains found in other Rap GEFs. cAMP: cAMP-binding site; EF, EF-hand calcium-binding
domain; Ras GEF or CDC25 homology domain; RA, Ras-association domain; Y504, Tyr504
.
A single-copy gene at chromosomal location 9q34.3 en-
codes C3G [10]. Other proteins encoded from the same region
are c-Abl, nucleoporin 214, laminin γ 3, NET39, protein-O-
mannosyltransferase, uridine-cytidine kinase 1, mediator com-
plex subunit 27 and sarcosine dehydrogenase. The human C3G
gene comprises 24 exons spanning 163 kb (Figure 2A). Tran-
script size of isoform a is 6085 bp and that of isoform b, 6256 bp.
Its homologues have been cloned from several organisms and
show a high degree of conservation in the catalytic domain
(Figure 3). The proline-rich stretches and E-cadherin-binding
domain are conserved among all the vertebrates from which
C3G has been cloned. In invertebrates, some putative proline-
rich SH3-binding stretches could be identified in the primary
sequence, but the E-cadherin-binding domain shows poor con-
servation. C3G may therefore have evolved to perform a broader
range of functions in the vertebrates. A variety of stimuli such
as growth factors, cytokines, integrins, neurotrophins, hormones
and mechanical stress have been shown to engage C3G-mediated
signalling (Table 1).
The GTPases known to be regulated by C3G are Ras
family members Rap1, Rap2, R-Ras, TC-21 and the Rho
family member TC10 leading to the activation of MAPK
(mitogen-activated protein kinase) and other effector pathways
[11–19]. Generally, GEFs do not show promiscuity in targeting
members of the various G-protein subfamilies and act within
their specific family of G-proteins. C3G is an example of
a GEF that targets a Rho family member in addition to the
Ras family GTPases. Over the past 15 years, several studies
have thrown light on the involvement of C3G in multiple
signalling pathways and its role in regulating diverse cellular
functions. Through the activation of ERK (extracellular-signal-
regulated kinase), JNK (c-Jun N-terminal kinase) and Rac
signalling, C3G plays a crucial role in integrin-mediated cell
adhesion and migration and also regulates cell proliferation,
differentiation and apoptosis. These cellular properties are often
associated with, or are a consequence of, cytoskeletal reorganiz-
ation. In the present review, we highlight these properties of C3G
and describe the consequence of its deregulation in cells as well
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3. C3G signals to actin remodelling
Figure 2 Isoforms of C3G
(A) Schematic diagram showing the genome organization of C3G gene. The human C3G gene comprises 24 exons, spanning
163 kb on chromosome 9q34.3. Human C3G has two predominant isoforms, a and b, which arise due to alternate splicing
and differ in their N-termini. Isoform a has 6085 bp of transcript length, whereas isoform b has 6256 bp of transcript
length. (B) Characterized mammalian isoforms of C3G. The two isoforms of C3G protein differ such that 3 aa (amino acids)
of Isoform a are replaced by 21 aa in isoform b. A truncated isoform is expressed in CML cells (K562), p87C3G which
arises from a 4.5 kb transcript. An alternate isoform in rat, which is expressed only in testis and brain, has a 51 aa (153
bp) insertion, just after the proline-rich domain. In mouse an additional isoform is found which has a deletion of 38 aa at
the N-terminal.
as during embryonic development. The examples of defective
signalling due to C3G leading to pathological states are also
presented.
Isoforms and expression
Although C3G is ubiquitously expressed, some tissue-specific
differences in expression levels have been seen. C3G transcripts
are subject to alternate splicing and variant isoforms have been
cloned from different species (Figure 2B). Rat tissues have shown
the presence of a major ubiquitously expressed 7 kb transcript
and a 4 kb transcript in some tissues [20]. An isoform contain-
ing a 153 bp insert after the fifth proline-rich region has also
been cloned from rat testis [20]. This isoform is predominantly
expressed in the testis and to some extent in the brain unlike
all other tissues which show predominant expression of only the
isoform without this insert. Specific functions served by these
isoforms have not been studied yet.
In human tissues, too, C3G shows ubiquitous expression, but
levels of a 7.5-kb transcript were high in adult skeletal muscle
and placenta, fetal heart and brain and low in the liver [4]. A short
87-kDa isoform encoded by a 4.4-kb transcript is expressed in
myeloid leukaemic cells, and lacks N-terminal 305 amino acids
of the full-length C3G [21]. This lacks the first two polyproline
regions and interacts with Bcr-Abl through the third proline-rich
sequence. The p87 isoform showed differences in expression
levels depending on disease remission during treatment, suggest-
ing a role for C3G in CML (chronic myelogenous leukaemia)
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4. V. Radha and others
Figure 3 C3G protein from various species
A comparative analysis of C3G protein from various species is shown with human (H. sapiens) C3G. Human C3G (isoform
b) shows E-cadherin-binding domain (red), proline-rich regions (light blue), REM (dark blue) and Ras GEF domain (black).
Amino acid residues belonging to each domain are mentioned and the position of amino acid residue at the start of
each proline-rich region is also mentioned. The longest isoform of C3G from each species is depicted along with all the
domains and the regions showing significant homology with human C3G with percentage identity. Corresponding regions
are indicated (in red). Analysis reveals the presence of putative proline-rich SH3 binding sequences in C3G protein from
various species. The N-terminal region in C3G protein from rat (R. norvegicus), mouse (M. musculus), Xenopus (X. laevis)
and zebrafish (D. rerio) showing high identity with E-cadherin-binding domain of human C3G is also depicted.
pathogenesis. In mouse tissues, two transcripts with and without
a 114 bp insertion in the N-terminal were expressed in most tis-
sues. C3G expression was high in brain, heart, liver and muscle
and low in adipose tissue, kidney and spleen [22].
Regulation
Currently, very little information is available on regulation of
C3G expression. Difference in relative expression of the two
mouse isoforms was seen during adipocyte differentiation [22].
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5. C3G signals to actin remodelling
Table 1 Stimuli that engage C3G
JAK, Janus kinase; STAT, signal transducer and activator of transcription; TIMP2 tissue inhibitor of metallo-
proteinases 2.
Stimulus Molecules involved in the pathway Reference(s)
Integrin binding VLA-4/VLA-5/R-Ras [33,78,98]
T-cell receptor c-Cbl/CrkL [99]
B-cell receptor c-Cbl/CrkL [100]
Insulin Cbl/Crk/C3G/TC10 [17,36,47]
EGF EGF/Crk/C3G/Rap1/B-Raf [47,48]
NGF FRS2/Crk/C3G /Rap1/B-Raf [14,48]
Interferon γ c-Cbl/CrkL [101]
Erythropoetin, interleukin-3 and interleukin-5 CrkL/STAT5 [102,103]
Hepatocyte growth factor Gab1-CrkL [34]
Growth hormone JAK2 and c-Src [18]
Reelin stimulation via Dab1/CrkL [104]
Mechanical force CrkII/Cas [60]
Nectin c-Src/Crk [66]
Cadherins c-Src/Vav2/Crk [105]
Bombesin Crk/CrkL [106]
TIMP2 Crk [81]
Figure 4 Differentiated human monocytes express higher levels
of C3G protein
Two human monocytic cell lines U937 and HL-60 were induced to dif-
ferentiate to a macrophage lineage by treating with 10 ng of PMA for
48 h or 1 % DMSO for 24 and 48 h respectively. Whole-cell lysates
were prepared along with UT (untreated) cells and subjected to Western
blotting by using indicated antibodies. Hck was used as a marker of
differentiation and Cdk2 as a protein loading control.
C3G protein levels also increase on differentiation of NB (neur-
oblastoma) cells [23]. Similarly, enhancement in C3G protein
was seen on differentiation of human monocytic cells to a mac-
rophage lineage (Figure 4). A several-fold increase in C3G gene
expression was observed on keratinocyte growth factor treatment
of human airway epithelia, indicating that C3G expression may
be regulated transcriptionally [24]. No information is available
on the promoter of C3G. In silico analysis carried out by us
using web-based software, Promo 3.0 and BKL TRANSFAC,
has shown binding sites for multiple transcription factors in the
upstream regulatory region, but they require experimental valida-
tion. Decreased C3G expression was found in cervical squamous
cell carcinomas due to hypermethylation of upstream regulat-
ory sequences [25]. There appears to be an inter-relationship in
the expression of some GEFs. Knocking down of DOCK-180,
a GEF for Rac resulted in an increase in C3G levels leading to
changes in many cellular properties such as reduced proliferation
and attenuated migration in ovarian carcinoma cells [26].
Most of the Rap GEFs are multidomain proteins and their
activation is regulated by protein–lipid interaction, binding of
second messengers, post-translational modification and subcel-
lular localization. C3G activation has been shown to be regulated
by tyrosine phosphorylation at Y504 and membrane targeting, en-
abled through its interaction with the adaptor protein Crk [27]. c-
Src, Hck, Fyn and c-Abl are kinases known to phosphorylate C3G
at Y504 [7,28–30]. The sequence surrounding Y504 of human
C3G is not totally conserved in rat and mouse, indicating species-
specific differences in C3G regulation. In addition to Y504, C3G
is phosphorylated on other tyrosine residues, but their contri-
bution to C3G regulation has not been studied [30]. The SH2
domain in Crk enables translocation of the Crk–C3G complex
to tyrosine-phosphorylated molecules [such as receptor tyrosine
kinases, p130Cas, Cbl, ARMS (ankyrin repeat-rich membrane
spanning), IRS-1 (insulin receptor substrate-1) and paxillin] in
response to extracellular stimuli [31–34]. Complex formation
between Crk and C3G is influenced by Crk phosphorylation
and the tyrosine phosphatase PTP1B regulates this modification
[6,35–37].
C3G regulation to activate specific GTPases may be complex.
C3G shows constitutive membrane binding upon v-Crk trans-
formation [38]. C3G expression enhances JNK activation and
transformation in v-Crk NIH 3T3 cells. In this case, localization
to the plasma membrane was not sufficient for JNK activation.
The catalytic domain was required but was independent of Rap1
indicating that, under these conditions, C3G targeted other GT-
Pases. Constitutive association of C3G with Crk has been de-
scribed. This interaction seems to vary in an adhesion-dependent
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6. V. Radha and others
Figure 5 C3G localizes to filopodia tips
Left panel: HeLa cells transfected with C3G expression vector were
stained for C3G and F-actin. C3G expressing cells show stable filopodial
extensions with C3G localized to their tips. Right panel: HeLa cells
treated with 50 mM PV, for 20 min show filopodia extensions. Staining
for p-C3G and F-actin showed pC3G localized predominantly at the Golgi
and filopodia tips. Images were captured using a confocal microscope.
manner and in response to other stimuli [36,39]. The ability of
molecules such as Cbl to alter CrkL–C3G interaction affects
C3G activation [40]. Cbl-b plays a negative role since Cbl− / −
T-cells show better interaction and higher Rap1 activation. In
response to insulin receptor signalling in skeletal muscle cells,
translocation of C3G to lipid rafts regulates its activation, and
disruption of flotillin-based membrane domains prevents C3G
activation [41]. In neutrophils, the bacterial chemoattractant pro-
tein fMLP (fMet-Leu-Phe) causes membrane targeting of C3G
dependent on function of the cytoskeletal regulator protein VASP
(vasodilator-stimulated phosphoprotein) [42]. Expression of pro-
teins like Bcr-Abl reduces the interaction of C3G and CrkL and
inhibits tyrosine phosphorylation of C3G upon cell spreading
and attachment of NIH 3T3 cells [43]. Bcr-Abl has been found
in a complex containing C3G dependent on CrkL [44].
C3G is also subject to autoregulation. It is known that C3G
enzyme activity is regulated negatively by its non-catalytic se-
quence since deletion of non-catalytic residues results in con-
stitutive catalytic activity [27]. The activation of C3G in the cells
may also be regulated through targeting to specific intracellu-
lar domains [45,46]. All studies so far have shown that C3G
localizes to the cytoplasmic compartment. In epithelial cells,
overexpressed C3G induces filopodia and localizes to filopodia
tips (Figure 5). PV (pervanadate)-induced filopodia show pC3G
(Y504-phosphorylated C3G) localized to their tips indicating a
role for C3G in filopodia functions (Figure 5). C3G, upon being
phosphorylated by SFKs (Src family kinases) or c-Abl, has been
shown to localize to the subcortical actin cytoskeleton, Golgi and
retracting lamellipodia of cells undergoing apoptosis [23,28,30].
Multimolecular complex formation involving C3G in response
to stimuli is a major means of activating C3G. Several proteins
that are capable of interacting with C3G directly or indirectly
have been identified and their involvement in pathways leading
to specific functions are shown in Figure 6. Components of mul-
timolecular complexes containing C3G also vary depending on
the stimulus [39,47]. Stimulation of PC12 cells by EGF (epi-
dermal growth factor), results in the formation of a short-lived
complex containing Crk, C3G, Rap1 and B-Raf. NGF (nerve
growth factor) stimulation causes formation of a stable complex
containing FRS2 (fibroblast growth factor receptor substrate 2),
Crk, C3G, Rap1 and B-Raf leading to prolonged MAPK activa-
tion [48]. In response to cell adhesion, Cas association with C3G
brings it into proximity of Src and focal adhesion kinase at focal
adhesions leading to the activation of JNK by integrins in fibro-
blasts [49]. In response to the activation of Fcγ R1 of myeloid
cells, complex formation is seen with the cytoskeletal protein
Hef-1, Crk, Cbl and C3G [50]. In Ba/F3 haematopoietic cells,
CrkL was found in a complex with C3G, Sos (Son of seven-
less) and c-Abl, but upon Bcr-Abl expression this complex is
disrupted [51]. In NIH 3T3 cells, PDGF (platelet-derived growth
factor) induces formation of complexes containing Necl-5, Integ-
rin α1βIII, PDGF-R (PDGF receptor), Rap1, Crk, C3G and Ral
GDS that enable cell movement [52].
FUNCTIONS
Role in embryonic development
The in vivo function of mammalian C3G has been studied by de-
veloping mice lacking C3G expression (knockout) or having very
low expression from a hypomorphic allele. C3G− / −
homozyg-
ous mice died before embryonic day 7.5, suggesting a significant
role for C3G during mammalian development [53]. The lethality
was rescued by expression of the human C3G transgene. Em-
bryonic fibroblasts from C3G knockout mouse embryos showed
impaired cell adhesion, delayed cell spreading and accelerated
cell migration. These effects were suppressed by expression
of active Rap1, Rap2 or R-Ras. This suggested the requirement of
C3G-dependent activation of GTPase targets for adhesion and
spreading of embryonic fibroblasts and for early embryogenesis
[53]. The fact that other Rap GEFs do not compensate for em-
bryonic lethality indicated that spatial and temporal functions
of C3G other than Rap1 activation may be required during em-
bryonic development.
To help study the role of C3G in other tissues and at later devel-
opmental stages, a mouse strain carrying a hypomorphic C3G al-
lele, C3Ggt
, was developed. Lysates of primary embryonic fibro-
blasts from C3Ggt/gt
mice showed less than 5% protein seen in
cells from wild-type animals, but they survived up to embryonic
day 14.5 [54]. C3Ggt/gt
mutant embryos die due to a blood ves-
sel maturation defect caused by inappropriate development of
vascular supporting cells. C3G-deficient fibroblasts responded to
PDGF-BB abnormally, exhibited cell adhesion defects and lacked
paxillin and integrin-β1-positive cell adhesions. This study elu-
cidated the requirement of C3G for vascular myogenesis, cell
adhesion and response to PDGF, necessary for vascular myogen-
esis [54].
C3Ggt/gt
mice also showed over proliferation of the cortical
neuroepithelium [55]. Neuroepithelial cells from these animals
failed to exit the cell cycle in vivo. C3G mutant neural pre-
cursor cells failed to activate Rap1, exhibited Akt/PKB activation,
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7. C3G signals to actin remodelling
Figure 6 Interacting partners of C3G and their involvement in pathways leading to specific functions
These members interact with the proline-rich Crk-binding region of C3G through their SH3 domain, except for some
members such as E-cadherin. A direct interaction has been characterized only in case of some members like Crk, Cas,
Hck and Abl. IL3, interleukin 3.
Gsk3β inhibition and β-catenin accumulation, when exposed to
growth factors, in vitro. These findings indicated that the size of
the cortical neural precursor population is controlled by C3G-
mediated inhibition of the Ras signalling pathway [55]. Mutant
embryos also exhibited a cortical neuron migration defect leading
to a failure of preplate splitting into marginal zone and subplate
and a failure to form a cortical plate. The basement membrane
was disrupted and radial glial processes were disorganized indic-
ating the requirement of C3G in neuronal migration and radial
glial attachment during cerebral cortex development [56].
A role for C3G in the development of invertebrates is also
known. During Drosophila eye and wing development, overex-
pression of membrane targeted full-length C3G phenotypically
mimics activation of the Ras-MAPK pathway, suggesting that
DC3G (Drosophila C3G) is involved in MAPK activation in vivo
[57]. The effects of C3G overactivity can be suppressed by re-
ducing the gene dose of components of the Ras-MAPK pathway
and of Rap1. DC3G is likely to stimulate both Ras1 and Rap1 dir-
ectly, which in turn leads to a convergent activation of the MAPK
pathway [57]. Deletion of C3G caused semi-lethality [58]. It is
an accessory component of the Drosophila musculature, essen-
tial for the proper localization of integrins at muscle–muscle and
muscle–epidermis attachment sites and important for maintain-
ing muscle integrity during larval stages.
Cellular functions
Various cellular functions regulated by C3G are mediated either
through changes in gene expression or through signalling to actin
cytoskeletal reorganization. Expression of constitutively active
C3G, or knocking down endogenous C3G have been used to
understand these functions. Changes in gene expression have
been seen under conditions of C3G overexpression as well as
repression [25].
Actin remodelling
Initial evidence that C3G is involved in signalling pathways lead-
ing to actin rearrangement came from studies which showed that
C3G expression resulted in filopodia formation in epithelial cell
lines dependent on an intact actin cytoskeleton [8]. C3G was also
required for c-Abl-induced filopodia formation. It was shown that
C3G could signal to actin by engaging N-Wasp, but independent
of Cdc42, a Rho family GTPase whose activation has generally
been associated with filopodia formation. C3G expressing cells
showed loss of stress fibres suggesting that C3G can alter actin
dynamics in these cells. In response to PV treatment, which is
known to cause filopodia formation [59], pC3G localized to the
subcortical actin cytoskeleton and to the tips of filopodia (Fig-
ure 5). The unique morphology of neuronal cells is achieved and
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8. V. Radha and others
maintained through extensive changes in microfilaments and mi-
crotubules. Neurite extension is also dependent on filopodia at
the growth cone. C3G expression in human NB cells resulted
in their morphological differentiation to neurons and Cdc42 and
N-Wasp-dependent signalling was involved [23]. The ability of
C3G to suppress transformation was dependent on its localiza-
tion at the subcortical actin cytoskeleton and its association with
protein phosphatase 2A [45]. It was indicated that C3G could
also directly interact with actin in a yeast two-hybrid assay.
In c-Abl-induced cell death, C3G was phosphorylated select-
ively in actin-rich cellular domains dependent on F-actin-binding
domain of c-Abl [30]. Localized phosphorylation of C3G re-
quired intact actin cytoskeleton, but was not affected by micro-
tubule disruption. Phosphorylation of C3G enhanced its ability
to associate with cytoskeletal structures. Previously it was shown
that C3G phosphorylation on tyrosine in response to adhesion of
NIH 3T3 cells was dependent on an intact cytoskeleton [43]. In
T-cells, it was seen that the actin remodelling protein WAVE-2
was required for C3G phosphorylation on Y504 [29]. In re-
sponse to mechanical signals such as cytoskeletal stretch, C3G
was found associated with Triton-insoluble structures to locally
activate Rap1 [60]. In v-Abl-transformed cells, cytoskeletal re-
arrangement is dependent on the CrkL–C3G complex, Rap1 and
Rac1 [61]. A link between the actin-regulating protein VASP and
C3G has been shown in human polymorphonuclear neutrophils,
with VASP serving to regulate C3G activation [42].
Vesicle traffic which is dependent on actin dynamics is also
regulated by C3G, through its target, TC10. Insulin-stimulated
GLUT4 (glucose transporter type 4) translocation is dependent
on C3G and an intact actin cytoskeleton [17]. TC10 binds COP1
in the Golgi and aids actin polymerization on membrane trans-
port vesicles [62]. Vesicular trafficking of E-cadherin is regulated
by C3G during the formation and breakdown of adherens junc-
tions. Interaction between E-cadherin and C3G is induced on cell
junction disassembly and activation of Rap1 and Rab11 positive
recycling endosomes [63]. In Drosophila, C3G could rescue the
NSF2 (N-ethylmaleimide-sensitive factor 2) phenotype which
shows defects in vesicular trafficking [64].
Targets of C3G also function in actin regulation. Rap1 func-
tions to regulate actin remodelling by engaging diverse effectors
[65]. C3G-Rap1-dependent Rac and Cdc42 activation through
their GEFs, Vav2 and FRG respectively are seen in response to
nectin engagement [66]. C3G-induced morphological changes
associated with neurons are achieved through Cdc42-mediated
signalling to actin [23]. TC10 activity regulates F-actin dynam-
ics and neurite growth [62,67,68]. Membrane protrusion is caused
by interaction between Exo70 and TC10 [69]. R-Ras regulates
cell migration of melanoma cells through association with the
actin-binding scaffold protein Filamin A [70]. R-Ras signals
to cause membrane protrusions through PLC (phospholipase C)
activity [71]. R-Ras also engages Rho and Rac GTPases to cause
morphological changes in epithelial and myeloid cells [72,73].
RgL3, a Ral GDS (guanine nucleotide dissociation stimulator)-
related protein serves to mediate interaction between Rap family
members and profilin, an important activator of actin polymeriz-
ation [74]. Rap2 engages TNIK (TRAF2/Nck-interacting kinase)
to cause changes in the cytoskeleton of cultured mammalian cells
[75]. Rap activation is required for phorbol-ester-induced actin
polymerization and morphological changes in B-cells [65]. Rap1
localizes to cell junctions and is a key regulator of junction form-
ation and disruption [76]. Evidence that C3G signals to actin is
also strengthened by the fact that most of the molecules that in-
teract with C3G such as Crk, Hck, Src, c-Abl etc. are known to
have roles in actin remodelling. Therefore reciprocal regulation
seems to exist between actin dynamics and C3G. On one hand,
polymerized actin serves as a platform for C3G activation and on
the other hand, activated C3G leads to target activation to achieve
changes in actin dynamics. These changes in turn are responsible
for a multitude of cellular functions as described below.
Adhesion and migration
C3G, being a regulator of Rap GTPase, plays an important role
in integrin signalling, adhesion and migration. C3G is phos-
phorylated in response to adhesion to fibronectin and overex-
pression in Ba/F3 haematopoietic cells enhances migration [43].
Expression of membrane-targeted C3G in HeLa cells also induces
extensive cell spreading [77]. Overexpression of C3G in 32D
cells increased adhesion to fibronectin through the activation of
VLA-4 and VLA-5, mediated by R-Ras [78]. Overexpression
of C3G increases adhesion of NIH 3T3 cells to laminin [79]. C3G
localizes to the focal adhesions in v-Crk transformed cells causing
abnormal activation of MAPK and JNK [80]. In TIMP2 (tissue
inhibitor of metalloproteinases 2)-treated human microvascular
endothelial cells, C3G induced RECK expression and reduced
cell migration [81]. SFK-dependent regulation of cell adhesion
also engages C3G.
Cell proliferation
Constitutive activation of C3G by expression of a membrane-
targeted variant in Drosophila resulted in enhanced Ras-MAPK
signalling and overproliferation and cell fate changes [57].
In haematopoietic progenitor cells, expression of membrane-
targeted C3G resulted in expression of double-positive T-cells,
associated with lethal T-cell acute lymphoblastic leukaemia. This
is achieved through enhanced expression of Notch 1 and 3 and
its target genes like Hes1 and c-Myc [82]. SIHA cells expressing
siRNA (small interfering RNA) targeting C3G showed enhanced
proliferation [25]. In NB cells, in addition to causing morpholo-
gical changes of differentiation, C3G induced p21, an inhibitor
of cell proliferation [23]. This is also reflected in vivo in a mouse
model where C3G neuroepithelial cells are retained in the cell
cycle without arresting and differentiating [55].
Differentiation
C3G is induced during neuronal differentiation and regulates
survival and differentiation of human NB cells [23]. Human NB
cells, IMR-32 induced to differentiate by serum starvation or by
treatment with NGF or forskolin showed enhanced C3G pro-
tein levels. Transient overexpression of C3G stimulated neurite
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238 C The Authors Journal compilation C 2011 Biochemical Society

9. C3G signals to actin remodelling
growth and also increased responsiveness to NGF and serum
deprivation induced differentiation. Forskolin and NGF treat-
ment resulted in phosphorylation of C3G at Tyr504 predom-
inantly in the Golgi. The activation of the C3G/Rap1 pathway
results in neurite outgrowth of mouse pheochromocytoma cells,
PC12, which is inhibited by either overexpression of Rap1GAP
or siRNA-mediated knockdown of Rap1 or the GEF C3G [83].
Dephosphorylation of Crk and association with C3G was re-
quired for adipocyte differentiation [84]. C3G protein levels also
increased during differentiation of monocytes to macrophage
lineage (Figure 4). The phenotype shown by mice expressing
a hyphomorphic allele was also indicative of a requirement of
C3G for differentiation of a variety of cells [55].
Transformation
C3G expression increases the growth rate, anchorage-
independent growth and JNK activation in v-Crk transformed
NIH 3T3 cells. The catalytic domain of C3G is essential for
this activity. Rap1 does not act as a C3G substrate in this con-
text. Dominant-negative C3G can reverse the transformed phen-
otype suggesting that C3G is essential for v-Crk-induced trans-
formation of NIH 3T3 cells [38]. C3G-dependent Rap1 activ-
ation also contributes to RET/PTC (rearranged during trans-
fection/papillary thyroid carcinomas) oncogene-mediated trans-
formation of thyroid follicular cells [85].
C3G, like Rap1, is capable of down-regulating the trans-
forming ability of Ras and Sis oncogenes [86]. However, the
transformation suppression activity of C3G is higher than that
of Rap1A. Through its ability to activate Rap1, C3G has been
shown to counteract signalling through the Ras/MAPK pathway
and has also been shown to transmit signals through the stress
kinase JNK pathway [15]. Moreover, C3G can also inhibit v-Raf-
and dbl-induced transformation of NIH 3T3 cells. The catalytic
domain of C3G is not required for this transformation suppression
activity, rather the proline-rich motifs of C3G are essential and
sufficient for this. C3G inhibits Ras-induced ERK activation, cyc-
lin A expression and anchorage-independent growth [79]. Farne-
sylated C3G, which localizes to the membrane, causes signific-
antly higher morphological reversion of transformed phenotype
of v-ki-Ras-transformed NIH 3T3 cells than normal C3G.
Apoptosis and cell survival
Co-expression of Hck with C3G induced a high level of apoptosis
in many cell lines and this property was not dependent on Y-504
phosphorylation or the catalytic domain of C3G but required the
catalytic activity of Hck. This indicated that C3G co-expression
could alter Hck activity towards select targets leading to apoptosis
[7]. c-Abl expression-induced cell death was dependent on C3G
and its phosphorylation in distinct actin-rich retracting lamellipo-
dia was associated with apoptosis. Oxidative-stress-induced cell
death mediated through c-Abl activation was dependent on C3G
phosphorylation [30].
By negatively regulating p38α MAPK, C3G plays a dual role
in regulating cell death in MEFs (mouse embryonic fibroblasts)
depending on the stimulus. C3G mediates cell death in response
to oxidative stress, whereas it induces cell survival upon serum
starvation. On serum deprivation, C3G induces survival through
inhibition of p38α MAPK activity, which mediates apoptosis;
whereas, in response to oxidative stress, C3G behaves as a
proapoptotic molecule, as its knockdown or knockout enhances
survival through upregulation of p38α activity, which plays an
antiapoptotic role under these conditions [87]. C3G acts to signal
to apoptosis and cell survival in response to the c-Abl inhibitor,
ST1-571 [88]. Differentiation of NB cells involves activation of
survival pathways along with induction of cell cycle arrest. C3G
is required for cell survival during differentiation as its knock-
down caused enhanced cell death in response to serum starvation
[23].
Filopodia formation and cell junction integrity
Work from our laboratory has shown that C3G plays a role in cyto-
skeletal reorganization and filopodia formation [8]. Knockdown
of C3G inhibited c-Abl-induced filopodia during cell spreading
on fibronectin. C3G expression induces actin cytoskeletal reor-
ganization and promotes filopodia formation independent of its
catalytic activity. It showed enrichment at filopodia tips charac-
teristic of molecules involved in filopodial dynamics (Figure 5).
AJs (adherens junctions) responsible for the integrity of epi-
thelial monolayers are formed by linking actin networks of neigh-
bouring cells. C3G directly interacts with E-cadherin, a primary
component of epithelial AJs, and excludes binding of β-catenin
to E-cadherin [9]. C3G’s function has therefore been implicated
in recruitment of E-cadherin to the junctions. E-cadherin-rich
filopodia extensions function as adhesion zippers to interlock
neighbouring cells before mature junction formation. E-cadherin
internalization on junctional breakdown also depends on C3G
binding to intracellular E-cadherin to activate Rap1 [89]. Nectins
(Ig-like transmembrane molecules) which aid in AJ formation
also signal by recruiting C3G to activate Rap1 [90].
Association with human disease
In malignant transformation associated with human cancers,
changes in C3G expression is tissue-specific. C3G overex-
pression was found in several samples of primary NSCLCs
(non-small-cell lung cancers) compared with corresponding
non-cancerous tissues. Six of seven NSC cell lines also showed
higher levels of C3G [91]. In contrast, cervical squamous cell
carcinomas were associated with decreased C3G levels. This
was attributed to frequent hypermethylation of upstream regu-
latory gene sequence [25]. Gene expression profiling of chronic
lymphocytic leukaemia samples showed downregulation of C3G
during disease progression [92]. Expression of an alternately
spliced form of C3G, p87, lacking N-terminal 305 residues in
CML cell lines and Ph+
[Philadelphia chromosomal transloca-
tion t(9;22)(q34;q11) positive] patients has been suggested to
play a role in the pathogenesis of CML [21,93].
Single-nucleotide polymorphisms in the C3G gene have shown
association with T2D (Type 2 diabetes), but the molecular basis
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10. V. Radha and others
is not clear. In a Finnish population, SNP rs4740283, located
4kb downstream of the C3G gene showed positive association
with T2D [94]. The GG phenotype of the polymorphism at
rs11243444, located in intron 13, had a protective effect on the
development of T2D in a Korean population [95]. In an experi-
mental model of glomerular nephritis, C3G and R-Ras-dependent
signalling has been implicated [96]. Disease-associated deletions
are known in the 9q34.3 chromosomal location that harbours the
C3G gene [97]. It is to be determined whether lack of C3G con-
tributes to the disease phenotype. It has also been predicted that
C3G deregulation may be associated with human disorders show-
ing defective leucocyte adhesion to the endothelium [42]. Mice
lacking C3G show cortical neuronal migration defects resulting
in failure to split preplate into marginal zone and subplate [56].
In humans, defective neuronal migration during development
leads to disorders like lissencephaly. It would therefore be in-
teresting to check for defects in C3G in lissencephaly patients.
CONCLUSIONS AND PERSPECTIVES
Multiple lines of evidence exist to show that many of the cellular
functions regulated by C3G involve reorganization of the actin
cytoskeleton. Through its ability to signal to actin reorganization,
C3G is involved in regulation of both structural and functional
processes in the cell. Morphogenesis is primarily dependent on
adhesive and migratory behaviour of cells and these functions of
C3G may be essential during embryonic development. The fact
that C3G is engaged in response to diverse signals indicates its
role in multiple tissue types and also explains the early embryonic
lethality due to defective development of multiple organ systems.
Requirement of C3G for mammalian development leads us to
ask whether C3G mutations could be associated with human
developmental defects. Examining aborted foetuses for mutations
or expression changes in C3G may help in determining whether
it plays a role in human embryonic development.
C3G being a member of a family constituting a large number
of proteins, it was surprising to note that other Rap GEFs do not
compensate C3G function under several situations.
Action of C3G in a spatial and temporal manner appears to be
essential during embryonic development, which may be one of
the reasons as to why its function is not complemented by other
GEFs. There is need to understand much more about the regula-
tion of C3G both in terms of its expression as well as activation.
Isoform-specific functions of C3G need to be elucidated. Iden-
tification of the C3G promoter and the regulatory transcription
factors and their response elements is warranted. There is good
reason to think that transcription factors that regulate differenti-
ation and migratory behaviour of cells may regulate C3G expres-
sion. Other modifications of C3G in addition to phosphorylation
on Y504 need to be investigated and studied. C3G has multiple
proline tracts but it is not clear as to whether it can interact directly
with more than one protein to form a multimolecular complex and
serve as a scaffold. One question that has not been addressed is
whether there is mutual exclusion of interacting partners enabling
the activation of only a subset of downstream effector pathways.
It is also possible that two or more protein binding motifs in C3G
function in a co-operative manner.
Suppression of transformation is an important role played by
C3G, which is a property independent of its catalytic activity.
C3G expression resulted in upregulation of the cell cycle inhibitor
p21 and suppression of cyclin A expression. Understanding how
C3G signals to changes in expression of genes regulating the
cell cycle will be important to understand its role as a tumour
suppressor. In some cell types, C3G also functions to enhance cell
proliferation and therefore its role in enhancing or suppressing
proliferation is context-dependent. At present, it is not clear as
to how C3G activates specific GTPases belonging to either Ras
or Rho family in a stimulus-dependent manner. Further studies
need to be carried out to determine whether C3G can regulate the
activity of other GTPases directly or indirectly.
On the basis of existing evidence, we propose that C3G may
be a master regulator of the differentiated phenotype in multiple
tissues. Differentiation removes cells from the proliferative mode
without affecting their integrity. Differentiation pathways are rel-
evant for tumour suppression in the light of continuous tissue
regeneration and therefore understanding them in various tissue
types has been important. In cells that have defects in apoptotic
pathways, inducing irreversible arrest through differentiation is a
good alternative in cancer therapy. The function of C3G as a reg-
ulator of differentiation in multiple tissues may be an important
property that could be utilized for achieving tumour suppression.
The 3D (three-dimensional) structure of C3G (either of the
whole molecule or its subdomains) has not been elucidated. Ana-
lysis of the 3D structure of C3G will help in understanding its
properties better. A 3D homology model constructed by using
SWISS-MODEL software indicated considerable structural ho-
mology between the catalytic sequence of C3G and the GEF do-
main (Cdc25 homology domain) of Sos, a Ras family GEF whose
crystal structure has been studied [107]. The GEFs interact with
their respective GTPases by using the same overall interface but
different specific interactions provide target specificity [108].
Targeting GEFs for either activation or inhibition for therapy
has been shown to be possible in principle [1]. Small-molecule
inhibitors have been developed for some GEFs and selective ag-
onists used for activation in other instances. C3G being a ubiquit-
ously expressed molecule with a role in pathways triggered by
a variety of signals, any attempt at therapeutic intervention must
aim at achieving selectivity in specific cell types. Some sugges-
ted approaches for activation of C3G are: (1) enabling membrane
targeting; (2) inhibition of tyrosine phosphatases or activation of
kinases that specifically regulate C3G; (3) introduction of pep-
tides that bind negative regulatory sequences; and (4) treatment
with agents that cause increase in C3G levels in specific cell types.
Just as in the case of Rho GEFs, C3G activity can be inhibited
by finding small molecule inhibitors that target its GEF domain.
Since C3G has functions dependent on catalytic activity as well
as protein interaction leading to different cellular functions, it
should be possible to target specific pathways selectively.
Other major questions that remain to be answered are ‘how are
developmental processes co-ordinated by C3G at the molecular
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240 C The Authors Journal compilation C 2011 Biochemical Society

11. C3G signals to actin remodelling
level?’ and ‘how does C3G regulate actin dynamics?’ Although
one straight answer would be that these functions are carried out
through activation of GTPases, there appears to be more com-
plexity. Association of C3G directly with actin indicates multiple
mechanisms that could be involved. Although we have high-
lighted a role for C3G in regulating actin dynamics, it is possible
that C3G signals to cytoskeletal changes by also affecting mi-
crotubule dynamics. A detailed knowledge of the regulation and
function of C3G at the cellular and molecular level will hopefully
provide us with means to selectively target it in specific tissues
where its deregulation is associated with pathology.
ACKNOWLEDGEMENTS
We thank Dr Ghanshyam Swarup for a critical reading of the manu-
script prior to submission.
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Received 17 August 2010/11 October 2010; accepted 13 October 2010
Published on the Internet 2 March 2011, doi 10.1042/BSR20100094
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244 C The Authors Journal compilation C 2011 Biochemical Society