dual role of ras and rho proteins: at the cutting edge of life and death

Review Article

Dual role of Ras and Rho proteins: At the cutting edge

of life and death

JAVIER GOÂ MEZ, CARLOS MARTIÂ NEZ-A, ANA GONZAÂ LEZ andANGELITA REBOLLO

Department of Immunology and Oncology, Centro Nacional de BiotecnologõÂa, Campus Cantoblanco, UniversidadAutoÂnoma de Madrid, Madrid, Spain

Summary Small GTP-binding proteins of the Ras superfamily are master controllers of the cell physiology. The

range of processes in which these proteins are involved include cell cycle progression, cell division, regulation ofcell morphology and motility and intracellular tra�cking of molecules and organelles. The study of apoptosis, thephysiological form of cell suicide, is progressively linking the functions of small G proteins to the control of the

mechanisms that trigger the genetic programmes of cell death. To date, isoforms of the Ras and Rho groups havebeen related to both promotion and suppression of apoptosis. Further, signalling pathways driven by theseproteins have been associated with the function and/or expression of molecules that regulate apoptotic responses.

Thus, all available evidence points to a critical role for Ras and Rho proteins as major gatekeepers of the decisionbetween cellular life and death.

Key words: adhesion, apoptosis, cell di�erentiation, cell proliferation, cytoskeleton, GTP-binding proteins, Ras,

Rho, transformation.

Introduction

Programmed cell death or apoptosis is a process during

which di�erent stimuli activate a genetic programme thatculminates in the death of the cell.1 Apoptosis is charac-terized by changes in the nuclear membrane and chromatin

condensation, followed by DNA fragmentation at thelinker regions between nucleosomes, apparently throughactivation of an intracellular nuclease.2,3 Withdrawal of

growth factors from growth factor-dependent cells and celllines is one of the signals that induces apoptosis. Otherstimuli that induce apoptosis include genotoxic damage,UV or ionizing irradiation, stress and the presence of spe-

ci®c death factors such as Fas±Fas ligand or TNF-a.4,5

Through the cell cycle, cells alternate DNA synthesis andmitosis in a highly regulated manner by a process which is

controlled by the existence of checkpoints capable ofsensing the progress of each cell cycle phase and, only uponits completion, allowing progression into the next. Dys-

function of checkpoints can prove fatal for the a�ectedcells. Regulatory information from growth factors is inte-grated by the cell during the late G1 phase.6 Cellular pro-

liferation proceeds and orderly progression is controlled byprotein complexes which are composed of cyclins in asso-

ciation with their catalytic subunits, the cyclin-dependentkinases (cdk)7 and regulated by the cyclin-dependent kinase

inhibitors (CKI).The Ras superfamily comprises a large number of mol-

ecules involved in cell proliferation, di�erentiation, cyto-

skeletal rearrangement, apoptosis, nuclear import of

proteins and vesicular tra�cking.8±10 The Ras family

comprises H-Ras, K-Ras, N-Ras and other homologous

proteins such as R-Ras, TC21, Rap and Ral. They are in-

volved in cell proliferation, di�erentiation and apoptosis.

Ras protein function is controlled by a GTP-GDP cycle

that is regulated by at least two distinct classes of regulatory

proteins.11±13 First, GTPase-activating proteins (p120 GAP

and neuro®bromin/NF1 GAP) recognize the active, GTP-

bound protein and stimulate the intrinsic GTPase activity

of Ras to form the inactive, GDP-bound protein.14 Second,guanine nucleotide exchange factors (GEF) promote the

dissociation of bound nucleotide to promote the formation

of the active, GTP-bound state.15 Several Ras GEF have

been cloned and characterized. They include Cdc25, Sos1

and Sos2 proteins.16,17

Although guanine nucleotide binding is essential for Rasfunction, its physiological role is also critically dependenton its localization in the plasma membrane.18,19 Ras

membrane association takes place through three types ofpost-translational modi®cations, namely prenylation, pro-teolysis and methylation18±20 that are signalled by the

C-terminal CAAX motif which is present in all Ras pro-teins. In addition to the C-terminal CAAX motif, thestructure of a typical Ras/Rho protein shows four regions:G1, G3, G4 and G5, which mediate binding and hydrolysis

Correspondence: Dr Angelita Rebollo, Centro Nacional de

BiotecnologõÂ a, Campus de Cantoblanco, Universidad AutoÂ noma

de Madrid, 28049 Madrid, Spain.

Email: <[email protected]>

Received 26 September 1997; accepted 21 November 1997.

Immunology and Cell Biology (1998) 76, 125±134

of the guanine nucleotide (Fig. 1) being the G2 region im-plicated in the interaction with e�ector molecules.

Upon activation by ligand-stimulated receptors, the Ras-GTP form recruits the serine/threonine kinase Raf to the

plasma membrane21±23 allowing its activation. Once acti-vated, Raf phosphorylates and activates the dual kinasesMEK1 and MEK2, whose substrates are the extracellular

signal-regulated kinases ERK1 and ERK2.24±28 The acti-vated ERK phosphorylate speci®c cytoplasmic substratesand translocate to the nucleus, where they phosphorylate

transcription factors leading to immediate-early gene in-duction.29

Activation of the Ras pathway occurs in response to a

variety of ligands, including those that use receptors of thecytokine receptor superfamily. IL-3 and Epo activate theRas pathway through their ability to induce tyrosinephosphorylation of Shc, and their association with Brb2

and Sos, followed by activation of the MAP kinase path-way and induction of immediate early genes. Carboxi-truncations of the IL-2Rb chain do not retain the ability to

activate the Ras pathway, although the ability to activateJak2 and to induce a mitogenic response is retained. Jak2activation per se is not su�cient for Shc phosphorylation,

activation of Ras, Raf and MAP kinase. Activation of thisRas pathway may require another tyrosine kinase whichassociates with the membrane-distal region of the cyto-plasmic domain of the b-chain of granulocyte/macrophage

colony stimulating factor (GM-CSF).30,31

Other proposed Ras e�ectors include phosphatidylino-sitol 3 kinase (PI3K),32 the zeta isoform of protein kinase C

(PKC),33 the Ras-dependent extracellular signal regulatedkinase kinase stimulator (REKS),34 the Ral protein gua-nine-nucleotide dissociation stimulator (Ral GDS),35 the

protein RGL, the protein Rin,36 the MEK kinase 137 andthe kinase suppressor of Ras (KSR) or ceramide-activatedprotein kinase (CAPK).36,38±40 It has been shown recently

that KSR, the dimeric protein 14-3-3 and Raf form anoligomeric signalling complex that positively regulates theRas signalling pathway.41

Ras activation was also shown to play a critical role in

activation of c-Jun N-terminal kinases (JNK; also known asStress-Activated Protein Kinases, SAPK).42 Like the ERK,activated JNK translocate to the nucleus, where they

phosphorylate transcription factors such as c-Jun.43

Ras and Rho proteins have been involved in variousbiological processes such as control of signalling pathways,

cellular proliferation and apoptosis.44±47 In addition, Rasmay promote either proliferation or di�erentiation of hae-

matopoietic cells, depending upon cell type and/or state ofmaturation.48

The Rho family of small guanine nucleotide-bindingproteins comprises several members: RhoA, RhoB, RhoC,RhoG, RhoE, Rac1, Rac2, Cdc42Hs, and TC10.10 Like

Ras, Rho proteins switch between an inactive GDP-boundstate and an active GTP-bound state. Transition betweenthese two forms is regulated by a complex set of molecules:

the GAP group is represented by Rho-GAP, p190A,10

p190B,49 and p115C1,50 whereas the guanine nucleotideexchange factors include Dbl, Ost, Abr, Lbc, Tiam1, Bcr,Rho-GDI, and Ly-GDI.51

Members of this family have been implicated in theregulation of cell morphology,52 cell motility, cell-mediatedcytotoxicity,53 vesicular transport in Golgi,54 platelet ag-

gregation, metastasis, cell proliferation and apopto-sis.44,46,55,56

Several putative Rho e�ectors have also been described,

such as PI3K,57 phospholipase D (PLD),58 a genistein-sensitive tyrosine kinase,59 the molecule citron, serine/threonine protein kinase N (PKN),60 the PKN-relatedmolecule rhophilin61 and the serine/threonine kinase

RhoA-binding kinase.62 The latter protein has been shownto mediate the control of the actin cytoskeleton.63

Role of Ras in cell proliferation, di�erentiation

and transformation

The relevance of Ras proteins in cell proliferation is bestexempli®ed by the high frequency of mutated Ras genes in

human neoplasia. Ras acts at several distinct phases of thecell cycle including early G1 phase, the G1/S boundary64

and at G2/M.65 Ras activity is required soon after the re-

lease of cells from quiescence.64 Recent studies have sug-gested functional interactions between Ras and cyclin D1.Thus, cyclin D1 collaborates with Ras in ®broblast trans-

formation66 and it has been shown that cyclin D1 is one ofthe major G1/S transition control factors whose level ismodulated by the Ras signalling pathway.67 Moreover,activation of the cyclin D1 promoter by the Ras signalling

pathway. This activation provides evidence for cross-talkbetween Ras and cell cycle regulatory pathways.68 Ras-in-duced cyclin D1 up-regulation is accompanied by increased

expression of retinoblastoma (Rb) tumour suppressorprotein.69 It has also been shown that Rb, by acting in apathway with Ras, is important for adequate growth reg-

ulation during mitogenic Ras-dependent signalling, sug-gesting that Rb links Ras signalling to the cell cycle.70

Ras down-regulates expression of the cdk inhibitorp27KIP1,71 and Myc and Ras collaborate in inducing ac-

cumulation of active cyclinE/cdk2 and E2F.72 Activation ofcdk2, which also phosphorylates Rb, together with shuttingdown a cdk inhibitor, may represent two additional steps

that complement cyclin D up-regulation for triggering theG1-S transition as a consequence of the mid-G1 Ras acti-vation peak.

Once the restriction point has been overtaken, the cellcycle progresses even without further mitogenic stimulus.However, the ability of Raf to activate the cell cycle

phosphatase cdc25A suggests that the Ras pathway might

Figure 1 Scheme of a typical small GTP-binding protein

showing the GTP-binding sites, which mediate binding and

hydrolysis of the guanine nucleotide, the e�ector region, which

interacts with e�ector molecules, and the C-terminal cysteine

motif. The additional insertion of nine to 12 residues in Rho

proteins is indicated.

126 J GoÂmez et al.

also modulate cell cycle progression at the G2-M transi-tion.73 This raises two possibilities; either Raf activity is

sustained throughout G2 even if Ras activation has de-creased, or Ras becomes active at this stage without growthfactor stimulation, as occurs in the mid-G1 peak of Ras

activity. It is interesting also to note that Ras is also capableof exerting anti-proliferative e�ects. Activated Raf inducesexpression of p21CIP1 and the subsequent G1 arrest.74 In

the same direction, oncogenic Ras causes growth arrest andpremature cellular senescence associated with up-regulationof p53 and p16INK4.75 Still, these results have been ob-tained using oncogenic variants that are active throughout

the cell cycle, and it is not quite clear if these e�ects wouldalso be observed using endogenously activated wild-typeproteins. Thus, the reported e�ects might re¯ect a protec-

tive or stress response of the cell to a Ras signal at aninappropriate cycle stage,76 a sort of an emergency brakethat will stop the cell when Ras activity is out of growth

factor control.Also, Ras mediates the signalling pathway responsible

for phosphorylation and activation of the Cdc25phosphotyrosine phosphatase,77 involved in dephosphory-

lation of the cyclin B/cd2 complex that regulates cell pro-gression along G2/M transition.7 Recent reports suggestthat activation of Ras during mid-G1 phase appears to

di�er from its rapid activation by growth factors, suggest-ing a novel mechanism of regulation that may be intrinsicto cell cycle progression.78 Cellular functions of Ras have

been frequently assessed by the use of dominant negativemutants, such as Ras N17 and Ras A15, which exhibit areduced binding a�nity for GTP. Using this approach, it

has been shown that ®broblast cell lines require active Rasfor proliferation.79,80

In addition, smooth muscle cell proliferation mediatedby platelet derived growth factor, epidermal growth factor

or ®broblast growth factor is inhibited by the expression ofdominant negative Ras mutants, suggesting that Ras isessential for smooth muscle mitogenic signalling path-

ways.81 Ras activation is also involved in neuronal di�er-entiation of phaeochromocytoma cell line PC12, as anactive Ras mutant can mimic the action of NGF and

dominant negative Ras mutant can block the di�erentiationof PC12 cells induced by NGF.82 These results suggest thatRas protein functions as a transducer of di�erentiationsignals in PC12 cells. Ras activation is also implicated in

proliferation and di�erentiation of haematopoietic cells.Accumulation of active Ras can be detected after stimula-tion of T cells.83,84 When T, B and mast cell lines are

stimulated with IL-2, IL-3, IL-5, GM-CSF, M-CSF or steelfactor, an accumulation of Ras-GTP is observed.84,85

However, under similar conditions, no increase of Ras-

GTP is detected after stimulation with IL-4.85 Similarly,using the murine T cell line TS1ab, which proliferates inresponse to IL-2, IL-4 or IL-9, we have shown that Ras

activation plays a critical role in proliferation and preven-tion of apoptosis mediated by IL-2, as expression of adominant negative Ras mutant inhibits proliferation andinduces apoptosis.44 In the same cellular model, IL-4 in-

duces proliferation and inhibits apoptosis by Ras-inde-pendent signals.44 In addition, Rho and Rac play animportant role in cell transformation by oncogenic Ras86±88

and ®nally, it has been shown that Ras activation is nec-essary for integrin-mediated activation of ERK2 and

phospholipase A.89

Interaction of Ras and PKC is required in a number ofmitogenic or activation signalling pathways, suggesting an

interactive regulation of cell growth or apoptosis involvingat least three molecules: Ras, PKC and Bcl-2.90 In hae-matopoietic cells, IL-3 and GM-CSF induce DNA syn-

thesis and suppression of apoptosis by activating multiplesignalling cascades. The activation of Ras/Raf/MAPK byIL-3 and GM-CSF in these cells appears to be more im-portant for the prevention of apoptosis than for the in-

duction of DNA synthesis, suggesting that activation of theRas pathway induces expression of genes that are necessaryfor cell survival.91

Finally, e�cient transformation by Ras requires activa-tion of other direct e�ectors in addition to Raf and it isinhibited by inactivation of the PI3 kinase pathway. The

ability of activated Ras to stimulate PI3 kinase is thereforeimportant in Ras transformation of mammalian cells andessential in Ras-induced cytoskeletal reorganization.92

Moreover, survival of Ras-transformed epithelial cells is

mediated by PI3 kinase, acting through PKB/Akt.93

Role of Rho in cell proliferation, adhesion

and cytoskeleton organization

The actin cytoskeleton is critical for many aspects of cellbehaviour. In addition to maintaining cell morphology, it isrequired for cell motility, cell division and intracellular

transport.94 Actin organization is also induced by manyextracellular factors and/or by adhesion to the extracellularmatrix.95 Rho controls stress ®bre formation10 and is in-

volved in the assembly of focal adhesion complexes.96

Many actin-binding proteins have been characterized thatcan regulate the rate of actin polymerization or the type of

actin structure formed and are likely targets of any signal-ling pathway leading to actin organization.97 The formationof actin stress ®bres in quiescent ®broblasts can be inducedby the addition of lysophosphatidic acid (LPA), the active

constituent of serum.98 Lysophosphatidic acid is known tostimulate a number of signalling pathways including acti-vation of Ras, PKC, calcium mobilization and reduction in

cAMP levels.99

We have established a functional relationship betweenRho, PI3K, fPKC and the organization of actin cytoskel-

eton in IL-2-stimulated TS1ab cells. We suggest that thispathway transduces signals that are necessary for themaintenance of actin structures during cell proliferation.Furthermore, this signalling process is IL-2 dependent, as

cells growing in IL-4 do not require the Rho-mediatedpathway for proliferation. The requirement of Rho func-tion in IL-2-stimulated cells has been proved by the use of

Rho mutants and the toxin B from Clostridium di�cile.Pretreatment of TS1ab cells with toxin B inhibits IL-2-in-duced proliferation and collapses the actin cytoskeleton.47

Rho itself regulates a variety of signal transduction ele-ments that may be involved in the control of cell prolifer-ation, including PI3K, PI5K, focal adhesion kinase (FAK),

ERK and PLD.100,101 Rho is also required for progressionthrough the cell cycle, as microinjection of Rho into qui-

Ras and Rho in proliferation and apoptosis 127

escent ®broblasts induces cell cycle progression through G1and subsequent DNA synthesis.102

Rho B and Rho G accumulation is periodic through thecell cycle. First detected at the G1/S phase transition, thelevel is maximal during S phase and declines at the S/G2-M

transition, suggesting that they play a role in the G1/Sphase transition and/or in the S phase of the cell cycle.99 Arole for Rho in the control of cell proliferation is also

suggested by the rapidly expanding number of oncogenesassigned to the Dbl family.49 Members of this family con-tain a common domain (DH) which possesses guaninenucleotide exchange activity for Rho.103,104

The observation that both substrate adhesion and acti-vated Rho stimulate PI5K suggests that Rho might be animportant element in the signal transduction pathway that

limits proliferation to adherent cells, controlling anchoragedependence. Rho regulates the myosin light chain phos-phatase and controls the synthesis of phosphatidylinositol

4,5-biphosphate, two activities that might help to explainthe e�ects of Rho on the actin cytoskeleton.105 Rac, Cdc42and Rho activate the JNK pathway and also activate SRF-dependent transcription.106 In addition, RhoA, B and C

can induce activation of JNK. This activation does notinvolve Pak1, suggesting a novel Pak-independent signal-ling route communicating Rho proteins to the JNK path-

way.107 Kinase-de®cient Pak1 mutants inhibit transcrip-tional activation and cell transformation mediated by theRas pathway108,109 (Fig. 2). In addition, Rho has been

proposed to drive Ras-induced cellular transformation58,88

and to possess, by itself, a weak transforming potential.110

Inactivation of Rho proteins by C3 exoenzyme of Clos-

tridium botulinum induces multinuclear cell formation,which is likely to be an e�ect caused by transient mal-

function of a G2/M checkpoint or an e�ect of blockedcytokinesis caused by the dissolution of the actin micro®l-

ament. Nevertheless, cell cycle progression continues tooccur despite the inhibition of cytokinesis.111

A critical role for Rho proteins has been proposed in

coupling G-protein-linked chemoattractant receptors tointegrin-mediated adhesion in leucocytes, suggesting thatRhoproteins can be an important target for pharmacological

modulation of adhesive functions of lymphocytes in patho-logical in¯ammation.112 In addition, recent results supportthe view that Rho is a functionally important mediator ofintegrin signalling.113 It appears that the assembly of large

integrin complexes is not required for actin organization orcell morphology changes induced by Rho activation.114

Role of Ras and Rho in apoptosis

An increasing number of proteins have been shown to beinvolved in opposite biological processes such as cell sur-

vival and apoptosis. Among these proteins we can includeRas and Rho.

As described earlier, Ras participates in proliferation

and also has been identi®ed as a mediator of the signallingpathway triggered through the cell death receptor Fas.115

Inhibition of Ras by antibodies or a dominant negative

mutant suppressed apoptosis. In this case, initiation of theRas pathway was assigned to the ceramide production bythe acidid sphingomyelinase. The JNK family of kinases

has also been involved in Fas signalling.116 Opposing ef-fects of ERK and JNK kinases in proliferation and apop-tosis have been shown,117 suggesting a switch at the level ofsmall G-proteins that drives the signal either to prolifera-

tion or apoptosis.A number of experimental systems linking Ras to the

induction of apoptosis were found using the phaeo-

chromocytoma cell line PC12. PC12 cells are rescued fromwithdrawal-induced cell death upon transfection of adominant negative Ras mutant.118 Implication of Ras ac-

tivation in apoptosis was also found in p120-RasGAP-de-®cient mice, which show high basal Ras activity and su�erfrom neuronal apoptosis.119 Constitutive activation ofR-Ras enhances apoptosis in IL-3-deprived cells120 and an

active form of H-Ras confers susceptibility to apoptosiswhen expressed in murine ®broblasts.121 Thyroid cellstransformed with K-Ras undergo apoptosis upon serum

deprivation.122 In the same direction, embryonic ®broblaststransformed with H-Ras undergo apoptosis after inhibitionof cell proliferation.123

TNF-resistant cells transfected with H-Ras show ex-treme sensitivity to TNF-induced apoptosis.124 Moreover,induction of apoptosis in H-Ras transfected Jurkat cells

occurs only upon PKC inhibition and can be blocked by thesurvival-promoting Bcl-2 protein.90 Similarly, expression ofK-Ras in murine ®broblasts induces apoptosis duringsuppression of PKC activity.90,125 Ras activation is also

essential for the induction of apoptosis in IL-2-deprivedTS1ab cells, as suppression of Ras activity by a dominantnegative Ras mutant inhibits apoptosis.46 Interestingly, Ras

activation in collaboration with apoptotic stimuli fails toinduce apoptosis in IRF-1-de®cient embryonic ®bro-blasts.123

Figure 2 Diagram illustrating the relationships between re-

ceptors, GTP-binding proteins and signalling molecules. Ras

activates Cdc42Hs, which in turn activates Rac, which in turn

activates Rho. Ras, Cdc42Hs, Rac and Rho can also be acti-

vated by growth factors and themselves activate several path-

ways, including JNK, p38, ERK and Rho-kinase.


The Ras-related protein R-Ras has been frequently as-sociated to the promotion of apoptosis,126 but it interacts

with Raf in a GTP-dependent manner in the same way asclassical Ras isoforms do.127 If this is the case, we coulddeduce that Ras signals are modulated by other simulta-

neous cellular events that determine the fate of the cell. Inparallel with the model proposed for the product of theprotoncogen c-myc,128 it could be possible that Ras triggers

both proliferative and apoptotic signals, the latter beingaborted by other simultaneous growth factor-inducedpathways. Alternatively, the Ras pathway could be pro-apoptotic unless another signal precludes this e�ect and

forces the cell to a mitogenic response. It can also beimagined that Ras might use di�erent e�ectors for drivingproliferation or cell death. The question would then be how

is regulation achieved at the step of Ras±e�ector interac-tion. An obvious possibility is the functional discriminationbetween di�erent Ras isoforms, which seems not to be the

case, or between Ras pools with distinct subcellular local-izations or post-translational modi®cations.

Several reports have demonstrated association betweenRas and Bcl-2 proteins and this association is coincident

with Bcl-2 phosphorylation.125,129 The Ras signallingpathway can also up-regulate Bcl-2 and Bcl-XL expres-sion91 and Ras-promoted cell death can be suppressed by

Bcl-2 expression.46,90,120,121 It has also been shown that Rasactivation of PI3 kinase suppresses c-Myc-induced apop-tosis through the activation of Akt/PKB but not p70s6k. In

addition, Ras is an e�ective promoter of apoptosis throughthe raf pathway.130

There is little evidence to link Rho with the control of

cell survival and apoptosis. Inhibition of Rho in EL4 Tlymphoma cells is a potent inducer of apoptosis, which maybe the result of impaired signalling in a survival pathway.Conversely, overexpression of RhoA induces tumours with

a high apoptotic index in nude mice,106 suggesting an im-plication of Rho in promotion of apoptosis.56 Indeed,overexpression of Rho wild type in NIH 3T3 cells induces

apoptosis when deprived of serum and in response to cy-totoxic agents.55,56 In contrast, transfection of cells with aconstitutively active Rho mutant is less potent as a stimu-

lator of apoptosis. Thus, Rho may play a role in regulationof apoptosis. In addition, transgenic mice lacking Rhofunction in the thymus show proliferative and cell survivaldefects during T cell development that impair the genera-

tion of thymocytes and mature T cells. These results de®neRho as a critical integrator of proliferation and cell survivalsignals.131

We have described a Rho-mediated cell death suppres-sion pathway driven by Bcl-2 expression that is su�cient tomaintain survival of IL-4-deprived TS1ab cells. Transient

transfection with a constitutively active RhoA mutant res-cues IL-4-deprived TS1ab cells from apoptosis. Cell sur-vival in these conditions parallels the triggering of Bcl-2

expression, silent in untransfected IL-4-stimulated cells.132

Role of Ras and Rho in the activation triggered by IL-2

The IL-2 receptor signal transduction system is probablyone of the best characterized signalling complexes in

mammalian cells. Current models consider at least threeindependent signalling pathways induced upon IL-2 stim-

ulation:133±135 a tyrosine kinase/Ras/c-fos/c-jun inductionpathway (channel 1); a Syk/c-myc expression pathway(channel 2) and a pathway leading to Bcl-2 expression

(channel 3). A combination of any two of these pathways isrequired for proliferation of BAF/B03 cells, while all threeare necessary for IL-2-dependent proliferation of TS1abcells124 (Fig. 3). Activation of the Ras pathway in TS1abcells plus any of the other two pathways promotes cellsurvival.44,45,135 Ras is also activated in the absence of IL-4in TS1ab cells and this event induces apoptosis.46 The role

of IL-2 in this system may be to supply additional signalscapable of preventing Ras-triggered apoptosis. This is inagreement with the hypothesis that considers cell prolifer-

ation as a consequence of the activation of a minimalnumber of pathways and that incomplete signals may resultin cell death.

The relevance of the ERK/MAPK pathway in the IL-2-induced signalling system is questionable. Raf is activatedupon Ras stimulation by IL-2,136±138 but subsequent ERKactivation is surprisingly transient in comparison with the

time course of Ras activation139,140 or it is even undetect-able.132 A possible explanation for this is the triggering ofan early response gene that encodes a dual speci®city ty-

rosine/threonine MAPK phosphatase referred to as PAC-1,which dephosphorylates ERK proteins and thus terminatesthe signal through this pathway.141 Interestingly, activation

of the PAC-1 gene is ERK dependent,142 suggesting thatthe shutting o� of the IL-2-induced, ERK-mediated signalin T cells is a negative feedback regulation mechanism, al-

though its functional meaning is still unsolved.

Figure 3 Hypothetical three-channel model of the IL-2 re-

ceptor signalling system. While some cell lines need only any

two active channels to proliferate, TS1ab cells require all three

channels. In this model, two signals provide protection from

apoptosis, one of them being channel 1, whereas three signals

promote proliferation.


We have identi®ed an IL-2-induced pathway that con-trols actin organization by sequential activation of Rho,

PI3K and fPKC in TS1ab cells. In contrast, IL-4 induces aRho-independent PI3K activation of fPKC and this path-way has no implications in cytoskeleton organization.45 In

addition, we have described a Rho-mediated cell deathsuppression pathway that proceeds through Bcl-2 expres-sion and involves PI3K and fPKC activation. Finally,

TS1ab cells express Bcl-2 upon IL-2 stimulation, whereasIL-4-mediated growth of this cell line proceeds in the ab-sence of Bcl-2 expression. The IL-2-induced Bcl-2 expres-sion in this case may be mediated through the triggering of

nuclear transcription factor NF-AT (Fig. 3; J GoÂ mez et al.unpubl. data, 1997).

Concluding remarks

Commitment to the process of cell death is far less under-stood than the process itself. The latter is, in part, charac-

terized by the up-regulation of expression and theactivation of cysteine proteases or caspases, capable ofinducing cell death. A di�erent situation arises when death

comes upon cell cycle progression; that is, aberrant or in-complete development of the cycle renders the cell out ofcontrol and potentially dangerous. The importance of a ®ne

regulation of commitment to apoptosis during the cell cycleis critical for the prevention of the tumorigenic state. Thus,it is mandatory that regulation of the triggering of the ap-

optotic programme at this stage comes from the samemechanisms that sense and control cell cycle function. Thisis when caspases are submitted to complex regulation,primarily by proteins of the Bcl-2 family. Although the role

of Bcl-2 family proteins are proposed to include a certainmodulation of the cell cycle, it seems clear that the bulk ofthe regulatory task must rely on cellular components driv-

ing mitogenic signalling pathways. The functional spectrumof the Ras superfamily covers almost every cellular process,and recent data suggest that they are functionally inter-

connected. Also, several reports point out close relation-ships between small G-proteins and Bcl-2 family members.Thus, a major part of the choice between cell life and deathmay be assumed by Ras superfamily proteins. The nature of

the signals they transmit may be modulated by other si-multaneously triggered parallel signalling pathways that, atthe level of nuclear integration, may result in the ®nal order

that either allows the cell to keep on living or switches onthe death execution mechanisms.

Acknowledgements

The Department of Immunology and Oncology wasfounded and is supported by the Consejo Superior de In-

vestigaciones Cienti®cas and Pharmacia & Upjohn.

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dual role of ras and rho proteins: at the cutting edge of life and death

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