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  1. 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 ' & $ % 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). www.bioscirep.org / Volume 31 (4) / Pages 231–244 231 BioscienceReportswww.bioscirep.org
  2. 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 ................................................................... ............................................................ .................................................................. ............................................................. ........................................................ .................................................... 232 C The Authors Journal compilation C 2011 Biochemical Society
  3. 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) ...................................................................... .......................................................... .................................................................... ........................................................... ........................................................... ................................................ www.bioscirep.org / Volume 31 (4) / Pages 231–244 233
  4. 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]. ................................................................... ............................................................ .................................................................. ............................................................. ........................................................ .................................................... 234 C The Authors Journal compilation C 2011 Biochemical Society
  5. 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 ...................................................................... .......................................................... .................................................................... ........................................................... ........................................................... ................................................ www.bioscirep.org / Volume 31 (4) / Pages 231–244 235
  6. 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, ................................................................... ............................................................ .................................................................. ............................................................. ........................................................ .................................................... 236 C The Authors Journal compilation C 2011 Biochemical Society
  7. 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 ...................................................................... .......................................................... .................................................................... ........................................................... ........................................................... ................................................ www.bioscirep.org / Volume 31 (4) / Pages 231–244 237
  8. 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 ................................................................... ............................................................ .................................................................. ............................................................. ........................................................ .................................................... 238 C The Authors Journal compilation C 2011 Biochemical Society
  9. 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 ...................................................................... .......................................................... .................................................................... ........................................................... ........................................................... ................................................ www.bioscirep.org / Volume 31 (4) / Pages 231–244 239
  10. 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 ................................................................... ............................................................ .................................................................. ............................................................. ........................................................ .................................................... 240 C The Authors Journal compilation C 2011 Biochemical Society
  11. 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. REFERENCES 1 Bos, J. L., Rehmann, H. and Wittinghofer, A. (2007) GEFs and GAPs: critical elements in the control of small G proteins. 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